Pharmaceutical composition for preventing or treating cognitive dysfunction or degenerative brain disease, comprising somatostatin or derivative thereof and hesperidin or derivative thereof

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cognitive dysfunction or degenerative brain disease, the composition comprising: somatostatin or a somatostatin derivative or a pharmaceutically acceptable salt thereof as a first active ingredient; and hesperidin or a hesperidin derivative or a pharmaceutically acceptable salt thereof as a second active ingredient. The composition comprising somatostatin or a somatostatin derivative and hesperidin or a hesperidin derivative as active ingredients according to one specific embodiment of the present invention not only improves behavior related to reduced cognitive ability and degenerative brain diseases, but also has a neuroprotective effect and the effect of alleviating cognitive dysfunction or degenerative brain diseases through decreased production of inflammatory transcription factors and increased production of anti-inflammatory transcription factors, and can thus be effectively used as a therapeutic agent for cognitive dysfunction or degenerative brain diseases.

Description

Pharmaceutical composition for preventing or treating cognitive dysfunction or degenerative brain disease comprising somatostatin or a derivative thereof and hesperidin or a derivative thereof

[0001] The present invention relates to a pharmaceutical composition for preventing or treating cognitive dysfunction or degenerative brain disease, comprising somatostatin or a derivative thereof and hesperidin or a derivative thereof.

[0002] Recently, as the elderly population has rapidly increased, neurological diseases caused by irreversible damage to brain function during the aging process, such as functional degeneration of synapses and loss of nerve cells, have become the main cause of death among the elderly and middle-aged. Among dementias classified as degenerative brain diseases, Alzheimer's disease (hereinafter referred to as "AD") is a disease that accounts for more than 70% of the total dementia prevalence (Ministry of Health & Welfare, 2012).

[0003] Inflammation is a key mechanism that contributes to neurodegenerative diseases. The central nervous system (CNS) is home to microglia, which form a natural immune response and a defense line against bacterial invasion and injury. Microglia, a type of macrophage in the CNS, play a crucial role in neuroinflammation. They can be activated by various exogenous and endogenous substances. Activated microglia produce and release various proinflammatory mediators, including the inflammatory cytokines TNF-α and IL-1, nitric oxide, prostaglandins, and superoxide dismutase. While the production of these substances may induce an immune response in the short term, excessive or sustained production can induce the death of nearby neurons, ultimately leading to neurodegeneration. Meanwhile, these inflammatory mediators are regulated by mitogen-activated protein kinases (MAPK), which consist of three major signaling cascades: p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). MAPK is characterized by regulating cellular functions including gene expression, differentiation, and proliferation as well as cellular responses to stimuli such as stress, mitogens, or proinflammatory cytokines. In particular, NF-κ, a transcription factor called the "Master switch," is known to induce high production of inflammatory cytokines in the central nervous system where neuroinflammation has occurred.

[0004] Currently, the only drugs approved by the FDA and being developed and used as dementia treatments are acetylcholinesterase inhibitors, but they only slow the progression of the disease and are not very effective in direct treatment. In addition, they have a limited therapeutic range in the early stages of the disease, so efforts have been made to develop drugs that treat the fundamental cause of Alzheimer's dementia.

[0005] Against this backdrop, the inventors of the present invention have completed the present invention to develop a therapeutic agent for preventing and treating cognitive dysfunction and degenerative brain diseases with a safe and potent effect by repositioning somatostatin or its derivatives, which are human-derived substances and have already been approved and used as a treatment for acromegaly, and using hesperidin or its derivatives, which are natural substances.

[0006] One aspect is to provide a pharmaceutical composition for preventing or treating cognitive dysfunction or degenerative brain disease, comprising as a first active ingredient somatostatin, a somatostatin derivative or a pharmaceutically acceptable salt thereof; and as a second active ingredient hesperidin, a hesperidin derivative or a pharmaceutically acceptable salt thereof.

[0007] Another aspect is to provide a health functional food composition for preventing or improving cognitive dysfunction, comprising as a first active ingredient somatostatin, a somatostatin derivative or a pharmaceutically acceptable salt thereof; and as a second active ingredient hesperidin, a hesperidin derivative or a pharmaceutically acceptable salt thereof.

[0008] Another aspect is to provide a food composition for preventing or improving cognitive dysfunction, comprising as a first active ingredient somatostatin, a somatostatin derivative or a pharmaceutically acceptable salt thereof; and as a second active ingredient hesperidin, a hesperidin derivative or a pharmaceutically acceptable salt thereof.

[0009] Another aspect comprises a first active ingredient comprising somatostatin, a somatostatin derivative or a pharmaceutically acceptable salt thereof; or a second active ingredient comprising hesperidin, a hesperidin derivative or a pharmaceutically acceptable salt thereof;

[0010] The present invention provides a pharmaceutical composition for preventing or treating cognitive dysfunction or degenerative brain disease, wherein the first effective ingredient is administered in combination with a second effective ingredient, or the second effective ingredient is administered in combination with the first effective ingredient.

[0011] Another aspect provides the use of somatostatin, a somatostatin derivative or a pharmaceutically acceptable salt thereof; and hesperidin, a hesperidin derivative or a pharmaceutically acceptable salt thereof, for use in the manufacture of a medicament for combination administration for the prevention or treatment of cognitive dysfunction or degenerative brain diseases.

[0012] Another aspect provides a method for preventing or treating cognitive dysfunction or degenerative brain disease, comprising the steps of: administering to a subject in need thereof an effective amount of somatostatin, a somatostatin derivative, or a pharmaceutically acceptable salt thereof; and administering to a subject in need thereof an effective amount of hesperidin, a hesperidin derivative, or a pharmaceutically acceptable salt thereof.

[0013] One aspect provides a pharmaceutical composition for preventing or treating cognitive dysfunction or degenerative brain disease, comprising as a first active ingredient somatostatin, a somatostatin derivative or a pharmaceutically acceptable salt thereof; and as a second active ingredient hesperidin, a hesperidin derivative or a pharmaceutically acceptable salt thereof.

[0014] The term "somatostatin" used herein refers to a peptide hormone that plays an important role in the nervous and endocrine systems, primarily suppressing hormone secretion. The somatostatin, a peptide composed of 14 amino acids, was extracted from the hypothalamus of a sheep by German in the United States in 1973. Somatostatin is a neuromodulator and neurotransmitter that is widely distributed and functions throughout the central nervous system, and studies have shown that its amount is significantly reduced in the cerebral cortex and cerebrospinal fluid of Alzheimer's disease patients.

[0015] The term "somatostatin derivative" as used herein refers to a synthetic peptide chemically modified from naturally occurring somatostatin, primarily mimicking or enhancing the hormone secretion-inhibiting function of somatostatin. The somatostatin derivative can bind to all five subtypes of somatostatin receptors and activate their signaling in the same manner.

[0016] In one specific example, the somatostatin derivative may be any one selected from the group consisting of cortistatin, octreotide, lanreotide, and pasireotide, but is not limited thereto.

[0017] The term “hesperidin” used herein refers to a flavonoid glycoside abundantly contained in the peel and pulp of citrus fruits (oranges, lemons, limes, etc.). After oral ingestion, the hesperidin is not hydrolyzed by glucosidase in the small intestine, but moves to the colon, where it is hydrolyzed by colonic microflora (Bifidobacterium pseudocatenulatum) into hesperetin, an aglycone of hesperidin (a major flavonoid having three hydroxy groups at the 3', 5', and 7' positions and a methoxy substituent at the 4' position), releasing the rutinose moiety and hesperetin, which are then absorbed into colonic enterocytes.

[0018] Specifically, the hesperidin may be a glycosyl compound including a hesperetin flavanone nucleus (3',5',5-trihydroxy-4'-methoxyflavanone), which is a glucoside moiety covalently bonded to rutinose (L-rhamnosyl-(α1→6)-glucose) that is bonded to a hydroxyl group present at carbon at position 7 of hesperetin. More specifically, the hesperidin is a compound (S)-7-[[6-0-(6-deoxy-α-L-mannopyranosyl)-β-D-glucopyranosyl]oxy]-2,3-dihydro-5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one.

[0019] The term “hesperidin derivative” as used herein refers to a compound having a similar physiological activity to hesperidin, which is a synthetic or natural substance based on the chemical structure of hesperidin but in which the structure is partially modified or replaced. The hesperidin derivative can be selected from its aglycone form, its chalcone form, its glycosyl form, its methylated form, and its sulfate or glucuronide form found as a metabolic product in the blood circulation. The hesperidin can be passively absorbed directly into the enterocytes of the small intestine. Hesperetin is metabolized at the 3' and 7' positions by uridine 5'-diphospho-glucuronosyltransferase and sulfotransferase in the colon, small intestine, and liver.

[0020] The above hesperidin derivatives can be obtained by various methods known to those skilled in the art, for example, by enzymatic treatment, or alternatively, by synthesis. For example, glucose-7-hesperetin can be prepared by treatment with rhamnosidase or hesperidinase.

[0021] In one specific example, the hesperidin derivative may be any one selected from the group consisting of, but is not limited to, (S)-2,3-dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one, 3',5,7-trihydroxy-4'-methoxyflavanone, α-glucosyl-hesperidin comprising a chain of 1 to 20 glucose residues linked together via 1,4 linkages, 3-methyl-7-(rhamnosyl-2-methylglucosyl)hesperidin, 3-methylhesperidin, hesperetin, a conjugate of hesperetin and a sulfate or glucuronide, and neohesperidin dihydrochalcone.

[0022] The term “degenerative brain disease” as used herein refers to a chronic and progressive disease caused by gradual damage and degeneration of brain cells (neurons) and nervous tissue, and includes various neurological and cognitive disorders due to the loss and dysfunction of nerve cells.

[0023] In one specific example, the degenerative brain disease may be any one selected from the group consisting of vascular cognitive impairment, dementia, Alzheimer's disease, Parkinson's disease, and Huntington's disease, but is not limited thereto.

[0024] In one specific example, the composition may have one or more of the following characteristics:

[0025] (a) Neuroprotective effect;

[0026] (b) Inhibitory effect on inflammatory transcription factor activation; and

[0027] (c) Increased activation of anti-inflammatory transcription factors.

[0028] The above neuroprotective effect refers to the effect of protecting neurons from damage or restoring or maintaining the function of already damaged neurons. Specifically, it can mean protecting neurons from the accumulation of beta-amyloid, oxidative stress, inflammation, and toxic substances.

[0029] The above-mentioned inflammatory transcription factor activation inhibitory effect refers to a physiological effect that alleviates neuroinflammatory responses by reducing excessive activation of major inflammatory signaling pathways in central nervous system cells, thereby suppressing the expression of inflammatory mediators (e.g., TNF-α, IL-6, IL-1β), thereby preventing or delaying damage to nerve cells and contributing to reducing the risk of developing degenerative brain diseases (e.g., Alzheimer's disease, Parkinson's disease).

[0030] For example, the inflammatory transcription factors may include NF-κB (Nuclear Factor kappa-light-chain-enhancer of activated B cells), AP-1 (Activator Protein 1), STAT3 (Signal Transducer and Activator of Transcription 3)-HIF-1α (Hypoxia-Inducible Factor 1-alpha).

[0031] The above-mentioned effect of increasing the activation of anti-inflammatory transcription factors means that by promoting the expression and activity of anti-inflammatory transcription factors in nervous system cells, the expression of inflammatory mediators (TNF-α, IL-6, IL-1β, etc.) is suppressed, and the expression of anti-inflammatory cytokines (IL-10, TGF-β, etc.) and protective proteins is increased to regulate the neuroinflammatory response, thereby preventing damage to nerve cells and alleviating the onset or progression of degenerative brain diseases.

[0032] For example, the anti-inflammatory transcription factors may include Nrf2 (Nuclear Factor Erythroid 2-Related Factor 2), PPAR-γ (Peroxisome Proliferator-Activated Receptor Gamma), STAT6 (Signal Transducer and Activator of Transcription 6), FoxP3 (Forkhead Box P3), CREB (cAMP Response Element-Binding Protein), etc.

[0033] In one specific example, the somatostatin, somatostatin derivative or pharmaceutically acceptable salt thereof can be administered orally, sublingually, intramuscularly, intravenously, intranasally, intrathecally, etc., and is preferably administered nasally.

[0034] In one specific example, the hesperidin, hesperidin derivative or pharmaceutically acceptable salt thereof can be administered orally, sublingually, intramuscularly, intravenously, intranasally, intrathecally, etc., and is preferably administered orally.

[0035] In one specific example, the daily adult dose of the somatostatin, somatostatin derivative or pharmaceutically acceptable salt thereof is 0.5-12 mg, preferably 1-8 mg, and the daily adult dose of the hesperidin, hesperidin derivative or pharmaceutically acceptable salt thereof is 10-2000 mg, preferably 20-1200 mg.

[0036] In one specific example, the pharmaceutical composition may contain 0.1-2 parts by weight of the second effective ingredient based on 100 parts by weight of the first effective ingredient. For example, the pharmaceutical composition may contain 0.1-2 parts by weight of somatostatin or a somatostatin derivative based on 100 parts by weight of the hesperidin or hesperidin derivative.

[0037] The pharmaceutical composition according to the present invention may contain 0.1-2 parts by weight of somatostatin or a somatostatin derivative based on 100 parts by weight of the hesperidin or hesperidin derivative, preferably 0.1-1 parts by weight of somatostatin or a somatostatin derivative based on 100 parts by weight of the hesperidin or hesperidin derivative, more preferably 0.2-0.6 parts by weight of somatostatin or a somatostatin derivative based on 100 parts by weight of the hesperidin or hesperidin derivative, and even more preferably 0.3-0.5 parts by weight of somatostatin or a somatostatin derivative based on 100 parts by weight of the hesperidin or hesperidin derivative.

[0038]

[0039] Another aspect provides a health functional food composition for preventing or improving cognitive dysfunction, comprising as a first active ingredient somatostatin, a somatostatin derivative or a pharmaceutically acceptable salt thereof; and as a second active ingredient hesperidin, a hesperidin derivative or a pharmaceutically acceptable salt thereof.

[0040] The term “health functional food” used in this specification refers to food manufactured or processed using raw materials or ingredients that have functionality useful to the human body.

[0041] The term "prevention" as used herein refers to a method of partially or completely delaying or preventing the onset or recurrence of a disease, disorder, or its attendant symptoms, preventing the acquisition or reacquisition of a disease or disorder, or reducing the risk of acquiring a disease or disorder.

[0042] In one specific example, the somatostatin derivative may be any one selected from the group consisting of cortistatin, octreotide, lanreotide, and pasireotide, but is not limited thereto.

[0043] In one specific example, the hesperidin derivative may be any one selected from the group consisting of, but is not limited to, (S)-2,3-dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one, 3',5,7-trihydroxy-4'-methoxyflavanone, α-glucosyl-hesperidin comprising a chain of 1 to 20 glucose residues linked together via 1,4 linkages, 3-methyl-7-(rhamnosyl-2-methylglucosyl)hesperidin, 3-methylhesperidin, hesperetin, conjugates of hesperetin and sulfate or glucuronide, and neohesperidin dihydrochalcone.

[0044] The term “cognitive impairment” used in this specification refers to a state in which memory, attention, language ability, visuospatial ability, judgment, etc. are impaired.

[0045] In one specific example, the daily adult dose of the somatostatin, somatostatin derivative or pharmaceutically acceptable salt thereof is 0.5-12 mg, preferably 1-8 mg, and the daily adult dose of the hesperidin, hesperidin derivative or pharmaceutically acceptable salt thereof is 10-2000 mg, preferably 20-1200 mg.

[0046]

[0047] Another aspect provides a food composition for preventing or improving cognitive dysfunction, comprising as a first active ingredient somatostatin, a somatostatin derivative or a pharmaceutically acceptable salt thereof; and as a second active ingredient hesperidin, a hesperidin derivative or a pharmaceutically acceptable salt thereof.

[0048] In one specific example, the daily adult dose of the somatostatin, somatostatin derivative or pharmaceutically acceptable salt thereof is 0.5-12 mg, preferably 1-8 mg, and the daily adult dose of the hesperidin, hesperidin derivative or pharmaceutically acceptable salt thereof is 10-2000 mg, preferably 20-1200 mg.

[0049]

[0050] Another aspect provides a pharmaceutical composition for preventing or treating cognitive dysfunction or degenerative brain disease, comprising a first active ingredient comprising somatostatin, a somatostatin derivative, or a pharmaceutically acceptable salt thereof; or a second active ingredient comprising hesperidin, a hesperidin derivative, or a pharmaceutically acceptable salt thereof; wherein the first active ingredient is administered in combination with the second active ingredient, or the second active ingredient is administered in combination with the first active ingredient.

[0051] In the pharmaceutical composition according to the present invention, the combined administration may be administered simultaneously or separately at different times. The time difference may range from 1 second to 7 days, preferably from 1 second to 12 hours, and more preferably from 1 second to 6 hours.

[0052] In one specific example, the somatostatin, somatostatin derivative or pharmaceutically acceptable salt thereof can be administered orally, sublingually, intramuscularly, intravenously, intranasally, intrathecally, etc., and is preferably administered nasally.

[0053] In one specific example, the hesperidin, hesperidin derivative or pharmaceutically acceptable salt thereof can be administered orally, sublingually, intramuscularly, intravenously, intranasally, intrathecally, etc., and is preferably administered orally.

[0054]

[0055] Another aspect provides the use of somatostatin, a somatostatin derivative or a pharmaceutically acceptable salt thereof; and hesperidin, a hesperidin derivative or a pharmaceutically acceptable salt thereof, for use in the manufacture of a medicament for combination administration for the prevention or treatment of cognitive dysfunction or degenerative brain diseases.

[0056]

[0057] Another aspect provides a method for preventing or treating cognitive dysfunction or a degenerative brain disease, comprising the steps of: administering to a subject in need thereof an effective amount of somatostatin, a somatostatin derivative, or a pharmaceutically acceptable salt thereof; and administering to a subject in need thereof an effective amount of hesperidin, a hesperidin derivative, or a pharmaceutically acceptable salt thereof.

[0058]

[0059] The terms and methods described for the above inventions apply equally to each invention.

[0060] According to one specific example of the present invention, a composition comprising as active ingredients somatostatin, a somatostatin derivative or a pharmaceutically acceptable salt thereof as a first active ingredient; and hesperidin, a hesperidin derivative or a pharmaceutically acceptable salt thereof as a second active ingredient not only improves reduced cognitive ability and behaviors related to degenerative brain diseases, but also has an effect of preventing, improving or treating cognitive dysfunction or degenerative brain diseases through a neuronal cell protection effect, an inflammatory transcription factor activation inhibition effect and an anti-inflammatory transcription factor activation increase effect.

[0061] Figure 1 is a graphical representation of the results of the Morris water maze experiment conducted on mice modeling Alzheimer's disease after daily administration of hesperidin (HSP; 100 mg / kg), somatostatin (SST; 400 μg / kg), and hesperidin (100 mg / kg) + somatostatin (HSP+SST; 400 μg / kg) for 8 weeks (+P<0.05, +++P<0.001 vs. MT_HSP, MT_SST alone, ***P<0.001 vs. MT_control).

[0062] Figure 2 is a graphical representation of the results of the passive avoidance test after administering hesperidin (100 mg / kg), somatostatin (400 μg / kg), and hesperidin (100 mg / kg) + somatostatin (400 μg / kg) daily to Alzheimer's disease model mice for 8 weeks (+P<0.05, ++P<0.01 vs. MT_HSP, MT_SST alone, **P<0.01, ***P<0.001 vs. control).

[0063] Figure 3 is a graphical representation of the results of a visual cognitive behavioral test conducted on Alzheimer's disease model mice daily for 8 weeks with hesperidin (100 mg / kg), somatostatin (400 μg / kg), and hesperidin (100 mg / kg) + somatostatin (400 μg / kg) (++P<0.01, +++P<0.001 vs. MT_HSP, MT_SST alone, ***P<0.001 vs. MT_control).

[0064] Figure 4a is a graph depicting the neuroprotective effect of the culture medium obtained by treating BV2 microglial cells, an in vitro model of Alzheimer's disease, with hesperidin (200 μM), somatostatin (10 μM), and hesperidin (200 μM) + somatostatin (10 μM), respectively, and stimulating them with Amyloid beta (5 μM), and then treating them on N2a neurons for 24 hours (+P < 0.05, ++P < 0.01 vs. HSP, SST alone, ***P < 0.001 vs. control).

[0065] Figure 4b is a graph depicting the neuroprotective effect of the culture medium obtained by treating BV2 microglial cells, an in vitro model of Alzheimer's disease, with hesperidin (200 μM), somatostatin (10 μM), lanreotide (10 μM), pasireotide (10 μM), octreotide (10 μM), cortistatin-14 (10 μM), and cortistatin-17 (10 μM) alone or in combination and stimulating with Amyloid beta (5 μM) and treating N2a neurons for 24 hours (+P < 0.05, ++P < 0.01 vs. HSP, SST alone, ***P < 0.001 vs. control).

[0066] Figure 4c is a graph depicting the neuroprotective effect of the culture medium obtained by treating BV2 microglial cells, an in vitro model of Alzheimer's disease, with hesperetin (200 μM), somatostatin (10 μM), lanreotide (10 μM), pasireotide (10 μM), octreotide (10 μM), cortistatin-14 (10 μM), and cortistatin-17 (10 μM) alone or in combination and stimulating with Amyloid beta (5 μM) and then treating N2a neurons for 24 hours (+P < 0.05, ++P < 0.01 vs. HSP, SST alone, ***P < 0.001 vs. control).

[0067] Figure 5a is a graph depicting the inhibitory effect of hesperidin (200 μM), somatostatin (10 μM), and hesperidin (200 μM) + somatostatin (10 μM) on the activation of inflammatory transcription factors in BV2 microglial cells, an in vitro model of Alzheimer's disease, after stimulating with Amyloid beta (5 μM) and obtaining nuclear extracts 1 hour later (++P < 0.01 vs. HSP, SST alone, **P < 0.01, ***P < 0.001 vs. control).

[0068] Figure 5b is a graph depicting the inhibitory effect of hesperidin (200 μM), somatostatin (10 μM), lanreotide (10 μM), pasireotide (10 μM), octreotide (10 μM), cortistatin-14 (10 μM), and cortistatin-17 (10 μM) alone or in combination on BV2 microglial cells, an in vitro experimental model of Alzheimer's disease, and stimulation with Amyloid beta (5 μM). Nuclear extracts were obtained 1 hour later and the inhibitory effect on NF-κB inflammatory transcription factor activation in BV2 microglial cells was depicted (++P < 0.01 vs. HSP, SST alone, **P < 0.01, ***P < 0.001 vs. control).

[0069] Figure 5c is a graph depicting the inhibitory effect of hesperetin (200 μM), somatostatin (10 μM), lanreotide (10 μM), pasireotide (10 μM), octreotide (10 μM), cortistatin-14 (10 μM), and cortistatin-17 (10 μM) alone or in combination on BV2 microglial cells, an in vitro experimental model of Alzheimer's disease, and stimulation with amyloid beta (5 μM). Nuclear extracts were obtained 1 hour later and the inhibitory effect on NF-κB inflammatory transcription factor activation in BV2 microglial cells was depicted (++P < 0.01 vs. HSP, SST alone, **P < 0.01, ***P < 0.001 vs. control).

[0070] Figure 6a is a graph depicting the effect of increasing the activation of the NRF2 anti-inflammatory transcription factor in BV2 microglial cells, an in vitro experimental model of Alzheimer's disease, when hesperidin (200 μM), somatostatin (10 μM), lanreotide (10 μM), pasireotide (10 μM), octreotide (10 μM), cortistatin-14 (10 μM), and cortistatin-17 (10 μM) were treated alone or in combination, and stimulated with Amyloid beta (5 μM). Nuclear extracts were obtained 1 hour later. (++P < 0.01, +++P < 0.001 vs. SST derivative alone, **P < 0.01, ***P < 0.001 vs. control)

[0071] Figure 6b is a graph depicting the effect of increasing the activation of the NRF2 anti-inflammatory transcription factor in BV2 microglial cells, an in vitro experimental model of Alzheimer's disease, when hesperetin (200 uM), somatostatin (10 uM), lanreotide (10 uM), pasireotide (10 uM), octreotide (10 uM), cortistatin-14 (10 uM), and cortistatin-17 (10 uM) were treated alone or in combination, stimulated with Amyloid beta (5 uM), and then nuclear extracts were obtained 1 hour later (††P<0.01, †††P<0.001 vs. SST derivative alone, **P<0.01, ***P<0.001 vs. control).

[0072] Hereinafter, the present invention will be described in detail.

[0073] Pharmaceutically acceptable salts

[0074] The active substance of the present invention can be used in the form of a pharmaceutically acceptable salt, and as a salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. The term "pharmaceutically acceptable salt" means any organic or inorganic addition salt of a base compound of the active substance, which has an effective effect at a concentration that is relatively non-toxic and harmless to the patient, and in which side effects due to the salt do not diminish the beneficial effects of the base compound of the active substance. These salts can use inorganic acids and organic acids as free acids, and inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, perchloric acid, phosphoric acid, etc. can be used, and organic acids such as citric acid, acetic acid, lactic acid, maleic acid, fumaric acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, tartaric acid, galacturonic acid, embonic acid, glutamic acid, aspartic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, 4-toluenesulfonic acid, salicylic acid, citric acid, benzoic acid, or malonic acid can be used. In addition, these salts include alkali metal salts (sodium salts, potassium salts, etc.) and alkaline earth metal salts (calcium salts, magnesium salts, etc.).For example, acid addition salts include acetate, aspartate, benzate, besylate, bicarbonate / carbonate, bisulfate / sulfate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulfate, naphthylate, 2-naphthylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate / dihydrogen phosphate, saccharate, stearate, succinate, It may contain tartrate, tosylate, trifluoroacetate, aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, zinc salts, etc., of which hydrochloride or trifluoroacetate is preferred.

[0075] The acid addition salt according to the present invention can be prepared by a conventional method, for example, dissolving the active substance in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile, etc., adding an organic acid or inorganic acid, filtering and drying the resulting precipitate, or by distilling the solvent and an excess acid under reduced pressure and then drying or crystallizing the same in an organic solvent.

[0076] Additionally, pharmaceutically acceptable metal salts can be prepared using bases. Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving a compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering out the undissolved compound salt, and evaporating and drying the filtrate. In this case, sodium, potassium, or calcium salts are pharmaceutically suitable as metal salts. Furthermore, the corresponding silver salts are obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

[0077] Furthermore, the present invention encompasses not only the active substance and its pharmaceutically acceptable salts, but also all possible solvates, hydrates, isomers, optical isomers, etc. that can be prepared therefrom.

[0078]

[0079] pharmaceutical composition

[0080] The active substance of the present invention can be administered in various oral and parenteral dosage forms during clinical administration, and when formulated, it is manufactured using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0081] Solid preparations for oral administration include tablets, tablets, powders, granules, capsules, troches, etc., and these solid preparations are prepared by mixing one or more active substances of the present invention with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, or syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included.

[0082] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin, glycerol, and gelatin.

[0083] In addition, the effective dosage for the human body of the active substance of the present invention may vary depending on the patient's age, body weight, sex, dosage form, health condition, and disease severity, and is generally about 0.001-100 mg / kg / day, and preferably 0.01-35 mg / kg / day. For an adult patient weighing 70 kg, the dosage is generally 0.07-7000 mg / day, and preferably 0.7-2500 mg / day, and may be administered once or several times a day at regular intervals depending on the judgment of a doctor or pharmacist.

[0084] In the pharmaceutical composition according to the present invention, the somatostatin (or derivative) can be administered in a total of 0.5-12 mg per day, preferably 1-8 mg, based on an average adult body weight of 60 kg.

[0085] In the pharmaceutical composition according to the present invention, the hesperidin (or derivative) can be administered in a total of 10-2000 mg per day, preferably 20-1200 mg, based on an average adult body weight of 60 kg.

[0086]

[0087] Food and health functional food compositions

[0088] There are no special restrictions on the type of food, and it includes both food in the normal sense and health functional foods.

[0089] Examples of foods include drinks, meat, sausages, bread, candy, snacks, noodles, ice cream, dairy products, soups, sports drinks, soft drinks, alcoholic beverages, gum, and tea.

[0090] Examples of health functional foods include health functional foods in the form of tablets, capsules, pills, and liquids, and may also include inner beauty products.

[0091]

[0092] The food and health functional food compositions containing the active ingredient according to the present invention can be added directly to foods or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. The amount of the active ingredient mixed can be appropriately determined depending on its intended use (prevention or improvement). Generally, the amount of the composition in foods and health functional foods can be added in an amount of 0.1 to 90 parts by weight based on the total food weight. However, in the case of long-term intake for the purpose of maintaining or regulating health, the amount can be below the above range, and since there is no problem in terms of safety, the active ingredient can also be used in an amount exceeding the above range.

[0093] The food and health functional food compositions of the present invention contain the active substance of the present invention as an essential ingredient in the indicated proportions, and there are no particular limitations on other ingredients, and may contain various flavoring agents or natural carbohydrates as additional ingredients, like conventional beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, etc.; and polysaccharides such as dextrin, cyclodextrin, etc., and conventional sugars, and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As flavoring agents other than those described above, natural flavoring agents (thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of the above-mentioned natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g, per 100 g of the health functional food composition of the present invention.

[0094] In addition to the above, the food and health functional food composition containing the effective substance of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), 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. In addition, the food and health functional food composition of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks.

[0095] These ingredients can be used independently or in combination. The proportion of these additives is not particularly critical, but is typically selected within the range of 0.1 to about 20 parts by weight per 100 parts by weight of the food and health functional food composition containing the active substance of the present invention.

[0096]

[0097] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following examples.

[0098]

[0099] Experimental method

[0100] Preparation of the composition

[0101] Hesperidin (HSP) and hesperetin (HST) were dissolved in DMSO at high concentrations and then dissolved in saline according to the concentration at the time of use. Somatostatin (SST), lanreotide (LAT), pasireotide (PAT), and octreotide (OCT) were dissolved in saline, and cortistatin-14 (CST-14) and cortistatin-17 (CST-17) were dissolved in DMSO at high concentrations and then dissolved in saline according to the concentration at the time of use. All samples were aliquoted and stored in a -80℃ sample freezer, and were taken out when necessary and discarded after a single use.

[0102]

[0103] Administration of the composition

[0104] Wild type control (normal group) and mutant type control (Alzheimer's disease-induced group) of Alzheimer's disease model mice were administered each vehicle orally and intranasally. The hesperidin (HSP; Sigma Chemical Company, St Louis, MO, USA) group was administered orally at 100 mg / kg dissolved in 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% saline daily for 8 weeks, and the somatostatin (SST; Sigma Chemical Company) group was administered intranasally at 400 μg / kg dissolved in saline daily for 8 weeks. The hesperidin + somatostatin group was administered at the same dose and method as the single group.

[0105]

[0106] Alzheimer's disease mouse model

[0107] Four-month-old 5xFAD mice (25-30 g) were obtained by crossing B6 / SJL obtained by crossing C57BL / 6J and SJL / J mice supplied by Jackson laboratory (Bar Harbor, Maine, USA) with MT_5xFAD. The animals were provided with sufficient solid feed (antibiotic-free, Samyang Feed Co.) and water, and were acclimated for 1 week under an environment of 22±2℃, 55±15% humidity, and 12-hour light-dark cycle before being used in the experiment.

[0108]

[0109] Morris water maze test

[0110] The Morris water maze experiment is a method for screening long-term memory, as it is an experiment for improving hippocampal-dependent spatial learning and cognitive abilities. For the water maze experiment, a circular tank (diameter 90 cm, height 45 cm) was filled with water to a depth of 30 cm (23±2°C). A 6 cm diameter escape platform was placed 1 cm below the surface of the water in one of the four quadrants of the tank. Non-toxic, water-soluble white dye was dissolved to conceal the platform. On the first day of the experiment, the animals were allowed to swim freely for 60 seconds in the tank without the platform. For four days, cognitive adaptation training was performed by repeatedly swimming twice a day in a different quadrant each day. Upon reaching the platform, the animals were instructed to remain there for 10 seconds. If they failed to find the platform within 60 seconds, they were instructed to remain there for another 10 seconds to ensure they remembered the platform. On the fifth day of the experiment, a probe test was conducted to measure working memory, removing the escape platform and recording the time it took to find the platform (escape latency). The total measurement time was 60 seconds, and all experiments were recorded and measured using video tracking software (SMART 3.0, Panlab, Spain).

[0111]

[0112] Passive avoidance test

[0113] The passive avoidance test is a widely used experiment to measure learning and memory. The avoidance learning box (each compartment 20 × 20 × 20 cm) is divided into a dark room and a bright room. When a laboratory animal is placed in the bright room, it tends to prefer the dark and moves to the dark room. At that moment, the guillotine door is closed and a 0.5 mA electric shock is delivered for 5 seconds. When the experimental animal is returned to the bright room the day after the electric shock, it remembers the electric shock in the dark room and remains in the bright room. The time it takes for this to occur (step-through latency) is measured to assess memory. The total measurement time was 180 seconds, and all experiments were recorded and measured using video tracking software (SMART 3.0, Panlab, Spain).

[0114]

[0115] Visual cognitive behavior test

[0116] For visual cognitive behavioral experiments, the avoidance learning box (each compartment 20 × 20 × 20 cm) was divided into crosshatch and square rooms. On the first day of the experiment, the experimental animals were placed in the crosshatch room with the guillotine door open and allowed to acclimate for 10 minutes. On the second day of the experiment, the animals were placed in the crosshatch room with the guillotine door closed and received 10 electric shocks (1 shock; 0.6 mA for 1 second) over a total of 10 minutes. The day after administering the electric shock, the animals were placed in the square room with the guillotine door closed for 10 minutes to make them perceive it as safe. On the fourth day of the experiment, the animals were placed in the crosshatch room with the guillotine door open. The time it took for the animals to remember the electric shock in the crosshatch room and stay in the square room (step-through latency) was measured to evaluate their memory. The total measurement time was 10 minutes, and all experiments were recorded and measured using video tracking software (SMART 3.0, Panlab, Spain).

[0117]

[0118] cell culture

[0119] BV2 and N2a cells used in this experiment were obtained from ATCC (Manassas, USA). BV2 and N2a cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 U / mL of penicillin, and 100 μg / mL of streptomycin (Gibco) at 37°C in a 5% CO2 incubator.

[0120]

[0121] Measurement of neuronal survival rate

[0122] To measure the neuroprotective effect of the composition using an in vitro model of Alzheimer's disease, the WST cytotoxicity evaluation test was performed. BV2 microglial cells, a mouse microglial cell line, were injected (2 × 10 5 / ml) were dispensed into 96-well plates and cultured for 24 hours. Then, hesperidin (200 μM), hesperetin (200 μM), somatostatin (10 μM), lanreotide (10 μM), pasireotide (10 μM), octreotide (10 μM), cortistatin-14 (10 μM), and cortistatin-17 (10 μM) were treated alone or in combination for 3 hours, and then stimulated with Amyloid beta (5 μM). After 6 hours, the medium was replaced with fresh medium, and the culture medium cultured for 12 hours was treated to N2a neural cells, a mouse neural cell line, for 24 hours. After reacting the culture medium with EZ-CYTOX (WST; DoGenBio Co., Ltd., Seoul, Korea) reagent for 1 hour, the absorbance was measured at a wavelength of 405 nm.

[0123]

[0124] Measurement of NF-κB inflammatory transcription factor production in microglia

[0125] To measure the inhibitory effect of the composition on NF-κB inflammatory transcription factor activation using an in vitro model of Alzheimer's disease, a western blot test was performed. BV2 microglial cells, a mouse microglial cell line, were used (2 × 10 5 / ml) were dispensed into 60π dishes and cultured for 24 hours, then treated with hesperidin (200 μM), hesperetin (200 μM), somatostatin (10 μM), lanreotide (10 μM), pasireotide (10 μM), octreotide (10 μM), cortistatin-14 (10 μM), and cortistatin-17 (10 μM) alone or in combination for 3 hours, and then stimulated with Amyloid beta (5 μM) for 2 hours. After obtaining cells and extracting nuclear extracts, western blot experiments were performed, and the resulting membranes were blocked, then reacted with NF-κB and TBP primary antibodies at 4°C for one day, and the secondary antibodies were reacted for 1 hour at room temperature the next day. After evenly spreading the membrane using ECL Detection solution, ChemiDoc TM XRS + (Bio-Rad, Richmond, CA) and analyzed.

[0126]

[0127] Measurement of the production of the anti-inflammatory transcription factor NRF2 in microglia

[0128] To measure the effect of the composition on increasing the activation of the NRF2 anti-inflammatory transcription factor, an in vitro experimental model of Alzheimer's disease was used, using a western blot assay.

[0129] BV2 microglial cells, a mouse microglial cell line, were seeded (2×105 / ml) in a 60π dish and cultured for 24 hours. Then, they were treated with hesperidin (200uM), hesperetin (200uM), somatostatin (10uM), lanreotide (10uM), pasireotide (10uM), octreotide (10uM), cortistatin-14 (10uM), and cortistatin-17 (10uM) alone or in combination for 3 hours, and then stimulated with Amyloid beta (5uM) for 2 hours. After obtaining the cells, nuclear extracts were extracted, and western blot experiments were performed. The obtained membranes were blocked and then reacted with NRF2 and TBP primary antibodies at 4℃ for one day, and secondary antibodies were reacted for 1 hour at room temperature the next day. After evenly spreading the ECL Detection solution on the membrane, it was developed and analyzed using ChemiDocTMXRS+ (Bio-Rad, Richmond, CA).

[0130]

[0131] statistical processing

[0132] All data are expressed as mean ± standard error (mean ± SD). The results of each experimental group were statistically processed using a statistical program (one-way ANOVA; post hoc Tukey tests), and significance tests were performed at a level of P < 0.05 or less (*p < 0.05, **p < 0.01, ***p < 0.001).

[0133]

[0134] <Comparative Example 1> Hesperidin monotherapy

[0135] Hesperidin was dissolved in DMSO at a high concentration and then dissolved in saline solution at the appropriate concentration for use. Samples were aliquoted and stored in a -80℃ sample freezer. When necessary, they were removed and discarded after a single use.

[0136] The hesperidin (HSP; Sigma Chemical Company, St Louis, MO, USA) group was administered orally at 100 mg / kg dissolved in 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% saline solution daily for 8 weeks.

[0137]

[0138] <Comparative Example 2> Somatostatin monotherapy

[0139] Somatostatin was dissolved in saline at a high concentration and then re-dissolved in saline at the appropriate concentration for use. Samples were aliquoted and stored in a -80℃ sample freezer. When necessary, they were removed and discarded after a single use.

[0140] The somatostatin (SST; Sigma Chemical Company) group was administered intranasally daily for 8 weeks, dissolved in saline at 400 μg / kg.

[0141]

[0142] <Comparative Example 3> Administration of hesperidin derivative alone

[0143] Hesperetin (HST) was dissolved in DMSO at a high concentration and then dissolved in saline solution at the appropriate concentration for use. The sample was aliquoted and stored in a -80℃ sample freezer. When necessary, it was removed and discarded after a single use.

[0144]

[0145] <Comparative Example 4> Somatostatin derivative monotherapy

[0146] Lanreotide (LAT), pasireotide (PAT), and octreotide (OCT) were dissolved in saline at high concentrations and then dissolved in saline according to the concentration at the time of use. The samples were aliquoted and stored in a -80℃ sample freezer, and were removed when necessary, used once, and then discarded.

[0147] Cortistatin-14 (CST-14; Cortistatin 14) and cortistatin-17 (CST-17; Cortistatin 17) were dissolved in DMSO at high concentrations and then dissolved in saline solution according to the concentration at the time of use. The samples were aliquoted and stored in a -80℃ sample freezer, and were removed when necessary, used once, and then discarded.

[0148]

[0149] <Example 1> Co-administration of hesperidin and somatostatin

[0150] The hesperidin and somatostatin combination group was administered in the same dosage and manner as the single-administration group (Comparative Examples 1 and 2).

[0151] An experiment was conducted to determine the effect of a composition containing somatostatin and hesperidin on cognitive dysfunction or degenerative brain disease.

[0152] According to the above experimental method, hesperidin (HSP; Sigma Chemical Company, St Louis, MO, USA) was orally administered at 100 mg / kg daily for 8 weeks to 5xFAD Alzheimer's disease model mice, and somatostatin (SST; Sigma Chemical Company) was intranasally administered at 400 μg / kg daily for 8 weeks. After the experiment, changes in cognitive dysfunction and behavioral patterns related to degenerative brain disease were examined.

[0153] In addition, according to the above experimental method, the neuroprotective effect of the composition was measured using an Alzheimer's disease in vitro experimental model, and the inhibitory effect on the activation of NF-κB inflammatory transcription factor in microglial cells was measured.

[0154]

[0155] <Example 2> Co-administration of hesperidin and somatostatin derivatives

[0156] The hesperidin and somatostatin derivative combination group was administered in the same dosage and manner as the single-administration group (Comparative Examples 1 and 4).

[0157] An experiment was conducted to determine the effect of a composition containing hesperidin and somatostatin derivatives on cognitive dysfunction or degenerative brain diseases.

[0158] According to the above experimental method, the neuroprotective effect of the composition was measured using an Alzheimer's disease in vitro experimental model, the inhibitory effect on the activation of NF-κB inflammatory transcription factor in microglial cells was measured, and the effect on increasing the activation of NRF2 anti-inflammatory transcription factor was measured.

[0159]

[0160] <Example 3> Co-administration of hesperidin derivatives and somatostatin

[0161] The hesperidin derivative and somatostatin combination administration group was administered in the same dosage and manner as the single administration group (Comparative Example 3 and Comparative Example 2).

[0162] An experiment was conducted to determine the effect of a composition containing a hesperidin derivative and somatostatin on cognitive dysfunction or degenerative brain disease.

[0163] According to the above experimental method, the neuroprotective effect of the composition was measured using an Alzheimer's disease in vitro experimental model, the inhibitory effect on the activation of NF-κB inflammatory transcription factor in microglial cells was measured, and the effect on increasing the activation of NRF2 anti-inflammatory transcription factor was measured.

[0164]

[0165] <Example 4> Co-administration of hesperidin derivatives and somatostatin derivatives

[0166] The hesperidin derivative and somatostatin derivative combination group was administered in the same dosage and manner as the single administration group (Comparative Examples 3 and 4).

[0167] An experiment was conducted to determine the effect of a composition containing a hesperidin derivative and a somatostatin derivative on cognitive dysfunction or degenerative brain disease.

[0168] According to the above experimental method, the neuroprotective effect of the composition was measured using an Alzheimer's disease in vitro experimental model, the inhibitory effect on the activation of NF-κB inflammatory transcription factor in microglial cells was measured, and the effect on increasing the activation of NRF2 anti-inflammatory transcription factor was measured.

[0169]

[0170] <Experimental Example 1> Morris Water Maze Experiment Using Alzheimer's Disease Model Mice

[0171] According to the above experimental method, the Morris water maze test was conducted on 5xFAD Alzheimer's disease model mice.

[0172] Figure 1 is a graphical representation of the results of the Morris water maze experiment conducted on Alzheimer's disease model mice that were administered hesperidin (HSP; 100 mg / kg), somatostatin (SST; 400 μg / kg), and hesperidin (100 mg / kg) + somatostatin (HSP+SST; 400 μg / kg) daily for 8 weeks (+P< 0.05, +++P< 0.001 vs. MT_HSP, MT_SST alone, ***P < 0.001 vs. MT_control).

[0173] As shown in Fig. 1, in the case of MT_control (Alzheimer's disease-induced group), the time to find the platform was significantly increased compared to the WT_control (normal group), and compared to the MT_control, the time to find the platform was decreased in both the hesperidin (HSP), somatostatin (SST) monotherapy group and the hesperidin + somatostatin (HSP + SST) combination therapy group, but it was confirmed that the HSP + SST combination therapy group showed a significantly greater reduction effect compared to the HSP, SST monotherapy group.

[0174]

[0175] <Experimental Example 2> Passive avoidance experiment using Alzheimer's disease model mice

[0176] According to the above experimental method, a passive avoidance test was conducted on 5xFAD Alzheimer's disease model mice.

[0177] Figure 2 is a graphical representation of the results of the passive avoidance test in which hesperidin (100 mg / kg), somatostatin (400 μg / kg), and hesperidin (100 mg / kg) + somatostatin (400 μg / kg) were administered daily to Alzheimer's disease model mice for 8 weeks (+P< 0.05, ++P< 0.01 vs. MT_HSP, MT_SST alone, **P< 0.01, ***P< 0.001 vs. control).

[0178] As shown in Fig. 2, the MT_control (Alzheimer's disease-induced group) showed a decrease in the time spent in the bright room compared to the WT_control (normal group), and compared to the MT_control, the time spent in the bright room increased in both the hesperidin (HSP), somatostatin (SST) monotherapy group and the hesperidin + somatostatin (HSP + SST) combination therapy group. However, it was confirmed that the HSP + SST combination therapy group showed a significantly greater increasing effect compared to the HSP, SST monotherapy group.

[0179]

[0180] <Experimental Example 3> Visual cognitive behavioral experiment using Alzheimer's disease model mice

[0181] According to the above experimental method, a visual cognitive behavioral test was conducted on 5xFAD Alzheimer's disease model mice.

[0182] Figure 3 is a graphical representation of the results of a visual cognitive behavioral test conducted on Alzheimer's disease model mice daily administered hesperidin (100 mg / kg), somatostatin (400 μg / kg), and hesperidin (100 mg / kg) + somatostatin (400 μg / kg) for 8 weeks (++P< 0.01, +++P< 0.001 vs. MT_HSP, MT_SST alone, ***P < 0.001 vs. MT_control).

[0183] As shown in Figure 3, the MT_control (Alzheimer's disease-induced group) showed a decrease in the time spent in the vertically slashed room compared to the WT_control (normal group), and compared to the MT_control, the time spent in the vertically slashed room increased in both the somatostatin (SST) monotherapy group and the hesperidin + somatostatin (HSP + SST) combination therapy group, but it was confirmed that the HSP + SST combination therapy group showed a significantly greater increase than the HSP and SST monotherapy groups.

[0184]

[0185] <Experimental Example 4> Neuroprotective Effects Using an In Vitro Experimental Model of Alzheimer's Disease

[0186] 4.1 Confirmation of the neuroprotective effect of combined administration of hesperidin and somatostatin

[0187] According to the above experimental method, an experiment was conducted to investigate the neuroprotective effect on an in vitro model of Alzheimer's disease.

[0188] Figure 4a is a graph depicting the neuroprotective effect of the culture medium obtained by treating BV2 microglial cells, an in vitro model of Alzheimer's disease, with hesperidin (200 μM), somatostatin (10 μM), and hesperidin (200 μM) + somatostatin (10 μM), respectively, and stimulating them with Amyloid beta (5 μM), and then treating them on N2a neurons for 24 hours (+P < 0.05, ++P < 0.01 vs. HSP, SST alone, ***P < 0.001 vs. control).

[0189] As shown in Fig. 4a, the cell viability was found to decrease in the control group compared to the untreated group, and compared to the control group, the cell viability increased in the hesperidin (HSP), somatostatin (SST) single administration group and the hesperidin + somatostatin (HSP + SST) combined administration group, but it was confirmed that the HSP + SST combined administration group showed a significantly greater increase effect compared to the HSP, SST single administration group.

[0190]

[0191] 4.2 Confirmation of the neuroprotective effect of combined administration of hesperidin and somatostatin derivatives

[0192] According to the above experimental method, an experiment was conducted to investigate the neuroprotective effect on an in vitro model of Alzheimer's disease.

[0193] Figure 4b is a graph depicting the neuroprotective effect of the culture medium obtained by treating BV2 microglial cells, an in vitro model of Alzheimer's disease, with hesperidin (200 μM), somatostatin (10 μM), lanreotide (10 μM), pasireotide (10 μM), octreotide (10 μM), cortistatin-14 (10 μM), and cortistatin-17 (10 μM) alone or in combination and stimulating with Amyloid beta (5 μM) and treating N2a neurons for 24 hours (+P < 0.05, ++P < 0.01 vs. HSP, SST alone, ***P < 0.001 vs. control).

[0194] As shown in Fig. 4b, the control group showed a decrease in cell viability compared to the untreated group (Normal), and compared to the control group, the cell viability increased in all groups administered alone or in combination with hesperidin (200uM), somatostatin (10uM), lanreotide (10uM), pasireotide (10uM), octreotide (10uM), cortistatin-14 (10uM), and cortistatin-17 (10uM), but it was confirmed that the combined administration group showed a significantly greater increase compared to the single administration group.

[0195]

[0196] 4.3 Confirmation of neuroprotective effects of combined administration of hesperidin derivatives and somatostatin or somatostatin derivatives

[0197] According to the above experimental method, an experiment was conducted to investigate the neuroprotective effect on an in vitro model of Alzheimer's disease.

[0198] Figure 4c is a graph depicting the neuroprotective effect of the culture medium obtained by treating BV2 microglial cells, an in vitro model of Alzheimer's disease, with hesperetin (200 μM), somatostatin (10 μM), lanreotide (10 μM), pasireotide (10 μM), octreotide (10 μM), cortistatin-14 (10 μM), and cortistatin-17 (10 μM) alone or in combination and stimulating with Amyloid beta (5 μM) and then treating N2a neurons for 24 hours (+P < 0.05, ++P < 0.01 vs. HSP, SST alone, ***P < 0.001 vs. control).

[0199] As shown in Fig. 4c, the control group showed a decrease in cell viability compared to the untreated group (Normal), and compared to the control group, the cell viability increased in all groups administered alone or in combination with hesperetin (200uM), somatostatin (10uM), lanreotide (10uM), pasireotide (10uM), octreotide (10uM), cortistatin-14 (10uM), and cortistatin-17 (10uM), but the combined administration group showed a significantly greater increase compared to the single administration group.

[0200]

[0201] <Experimental Example 5> Inhibitory effect of NF-κB inflammatory transcription factor activation using an in vitro experimental model of Alzheimer's disease

[0202] 5.1 Confirmation of the inhibitory effect of NF-κB inflammatory transcription factor activation by combined administration of hesperidin and somatostatin

[0203] According to the above experimental method, an experiment was conducted to investigate the inhibitory effect of NF-κB inflammatory transcription factor activation on an in vitro model of Alzheimer's disease.

[0204] Figure 5a is a graph depicting the inhibitory effect of hesperidin (200 μM), somatostatin (10 μM), and hesperidin (200 μM) + somatostatin (10 μM) on the activation of inflammatory transcription factors in BV2 microglial cells, an in vitro model of Alzheimer's disease, after stimulating BV2 microglial cells with Amyloid beta (5 μM) and obtaining nuclear extracts 1 hour later (++P < 0.01 vs. HSP, SST alone, **P < 0.01, ***P < 0.001 vs. control).

[0205] As shown in Fig. 5a, the control group showed an increase in NF-κB production compared to the untreated group, and the hesperidin (HSP), somatostatin (SST) monotherapy group and the hesperidin + somatostatin (HSP + SST) combination group all showed a decrease in NF-κB production compared to the control group. However, the HSP + SST combination therapy group showed a significantly greater reduction effect compared to the HSP and SST monotherapy groups.

[0206]

[0207] 5.2 Confirmation of the inhibitory effect of NF-κB inflammatory transcription factor activation by combined administration of hesperidin and somatostatin or somatostatin derivatives

[0208] According to the above experimental method, an experiment was conducted to investigate the inhibitory effect of NF-κB inflammatory transcription factor activation on an in vitro model of Alzheimer's disease.

[0209] Figure 5b is a graph depicting the inhibitory effect of hesperidin (200 μM), somatostatin (10 μM), lanreotide (10 μM), pasireotide (10 μM), octreotide (10 μM), cortistatin-14 (10 μM), and cortistatin-17 (10 μM) alone or in combination on BV2 microglial cells, an in vitro experimental model of Alzheimer's disease, and stimulation with amyloid beta (5 μM). Nuclear extracts were obtained 1 hour later and the results are shown in the graph (++P < 0.01 vs. HSP, SST alone, **P < 0.01, ***P < 0.001 vs. control).

[0210] As shown in Fig. 5b, the control group showed an increase in NF-κB production compared to the untreated group (Normal), and compared to the control group, the NF-κB production decreased in all groups administered alone or in combination with hesperidin (200uM), somatostatin (10uM), lanreotide (10uM), pasireotide (10uM), octreotide (10uM), cortistatin-14 (10uM), and cortistatin-17 (10uM), but it was confirmed that the combined administration group showed a significantly greater reduction effect compared to the single administration group.

[0211]

[0212] 5.3 Hesperidin derivatives and somatostatin or somatostatin derivatives; Confirmation of the inhibitory effect on NF-κB inflammatory transcription factor activation following co-administration

[0213] According to the above experimental method, an experiment was conducted to investigate the inhibitory effect of NF-κB inflammatory transcription factor activation on an in vitro model of Alzheimer's disease.

[0214] Figure 5c is a graph depicting the inhibitory effect of hesperetin (200 μM), somatostatin (10 μM), lanreotide (10 μM), pasireotide (10 μM), octreotide (10 μM), cortistatin-14 (10 μM), and cortistatin-17 (10 μM) alone or in combination on BV2 microglial cells, an in vitro experimental model of Alzheimer's disease, and stimulation with amyloid beta (5 μM). Nuclear extracts were obtained 1 hour later and the inhibitory effect on NF-κB inflammatory transcription factor activation in BV2 microglial cells was depicted (++P < 0.01 vs. HSP, SST alone, **P < 0.01, ***P < 0.001 vs. control).

[0215] As shown in Fig. 5c, the control group showed an increase in NF-κB production compared to the untreated group, and compared to the control group, the NF-κB production decreased in all groups administered hesperetin (200uM), somatostatin (10uM), lanreotide (10uM), pasireotide (10uM), octreotide (10uM), cortistatin-14 (10uM), and cortistatin-17 (10uM) alone or in a combination, but it was confirmed that the combination administration group showed a significantly greater reduction effect than the single administration group.

[0216]

[0217] <Experimental Example 6> Effect of increasing the activation of the NRF2 anti-inflammatory transcription factor using an in vitro experimental model of Alzheimer's disease.

[0218] 6.1 Confirmation of the effect of increased activation of the NRF2 anti-inflammatory transcription factor following combined administration of hesperidin and somatostatin derivatives.

[0219] According to the above experimental method, an experiment was conducted to investigate the effect of increasing the activation of the NRF2 anti-inflammatory transcription factor in an in vitro model of Alzheimer's disease.

[0220] Figure 6a is a graph depicting the effect of increasing the activation of the NRF2 anti-inflammatory transcription factor in BV2 microglial cells, an in vitro experimental model of Alzheimer's disease, when hesperidin (200 uM), somatostatin (10 uM), lanreotide (10 uM), pasireotide (10 uM), octreotide (10 uM), cortistatin-14 (10 uM), and cortistatin-17 (10 uM) were treated alone or in combination, stimulated with Amyloid beta (5 uM), and then nuclear extracts were obtained 1 hour later (++P<0.01 +++P<0.001 vs. SST derivative alone, **P<0.01, ***P<0.001 vs. control).

[0221] As shown in Fig. 6a, the control group showed a slight increase in NRF2 production compared to the untreated group (Normal), and compared to the control group, the NRF2 production increased in all groups administered alone or in combination with hesperidin (200uM), somatostatin (10uM), lanreotide (10uM), pasireotide (10uM), octreotide (10uM), cortistatin-14 (10uM), and cortistatin-17 (10uM), but the combined administration group showed a significantly greater increase compared to the single administration group.

[0222]

[0223] 6.2 Confirmation of the effect of increasing the activation of the NRF2 anti-inflammatory transcription factor by co-administration of hesperidin derivatives and somatostatin or somatostatin derivatives

[0224] According to the above experimental method, an experiment was conducted to investigate the effect of increasing the activation of the NRF2 anti-inflammatory transcription factor in an in vitro model of Alzheimer's disease.

[0225] Figure 6b is a graph depicting the effect of increasing the activation of the NRF2 anti-inflammatory transcription factor in BV2 microglial cells, an in vitro experimental model of Alzheimer's disease, when hesperetin (200 uM), somatostatin (10 uM), lanreotide (10 uM), pasireotide (10 uM), octreotide (10 uM), cortistatin-14 (10 uM), and cortistatin-17 (10 uM) were treated alone or in combination, stimulated with Amyloid beta (5 uM), and then nuclear extracts were obtained 1 hour later (††P<0.01, †††P<0.001 vs. SST derivative alone, **P<0.01, ***P<0.001 vs. control).

[0226] As shown in Fig. 6b, the control group showed a slight increase in NRF2 production compared to the untreated group (Normal), and compared to the control group, the NRF2 production increased in all groups administered alone or in combination with hesperetin (200uM), somatostatin (10uM), lanreotide (10uM), pasireotide (10uM), octreotide (10uM), cortistatin-14 (10uM), and cortistatin-17 (10uM), but it was confirmed that the combined administration group showed a significantly greater increasing effect compared to the single administration group.

[0227]

[0228] As described above, when a composition containing somatostatin and hesperidin was administered to Alzheimer's disease model mice, it was confirmed that reduced cognitive ability and behaviors associated with degenerative brain diseases were improved. Furthermore, when a composition containing somatostatin and hesperidin; a hesperidin derivative and somatostatin; a hesperidin and somatostatin derivative; or a hesperidin derivative and a somatostatin derivative; was administered to an in vitro Alzheimer's disease model, it was confirmed to be effective in alleviating cognitive dysfunction or degenerative brain diseases through neuroprotective effects, a reduction in the production of inflammatory transcription factors, and an increase in the production of anti-inflammatory transcription factors. Through these results, the inventors concluded that the composition can be applied as a preventive and therapeutic agent for various cognitive dysfunctions or degenerative brain diseases.

[0229]

[0230] Example of manufacturing a drug

[0231] The active substance according to the present invention can be formulated in various forms depending on the intended purpose. The following are examples of formulation methods containing the active substance according to the present invention as an active ingredient, but the present invention is not limited thereto.

[0232]

[0233] <Pharmaceutical Manufacturing Example 1> Manufacturing of powder

[0234] 2 g of active ingredient

[0235] 1 g lactose

[0236] After mixing the above ingredients, the powder was prepared by filling it into a sealed bag.

[0237]

[0238] <Pharmaceutical Manufacturing Example 2> Manufacturing of tablets

[0239] 100 mg of active ingredient

[0240] 100 mg of corn starch

[0241] 100 mg of lactose

[0242] Magnesium stearate 2 mg

[0243] After mixing the above ingredients, tablets were manufactured by pressing them according to a conventional tablet manufacturing method.

[0244]

[0245] <Pharmaceutical Manufacturing Example 3> Manufacturing of capsules

[0246] 100 mg of active ingredient

[0247] 100 mg of corn starch

[0248] 100 mg of lactose

[0249] Magnesium stearate 2 mg

[0250] After mixing the above ingredients, the mixture was filled into a gelatin capsule according to a conventional capsule manufacturing method to produce a capsule.

[0251]

[0252] <Pharmaceutical Manufacturing Example 4> Manufacturing of Injectables

[0253] 10 μg / ml of active substance

[0254] Dilute hydrochloric acid BP until pH 3.5

[0255] Sodium chloride for injection BP up to 1 ml

[0256] The active ingredient according to the present invention was dissolved in an appropriate volume of sodium chloride BP for injection, the pH of the resulting solution was adjusted to pH 3.5 using diluted hydrochloric acid BP, the volume was adjusted using sodium chloride BP for injection, and the mixture was thoroughly mixed. The solution was filled into a 5 ml Type I ampoule made of transparent glass, sealed under an upper grid of air by dissolving the glass, and sterilized by autoclaving at 120°C for more than 15 minutes to prepare an injection solution.

[0257]

[0258] <Pharmaceutical Manufacturing Example 5> Manufacturing of nasal spray

[0259] Active ingredient 1.0 g

[0260] 0.3 g of sodium acetate

[0261] 0.1 g of methylparaben

[0262] Propylparaben 0.02 g

[0263] Sodium chloride appropriate amount

[0264] Appropriate amount of HCl or NaOH for pH adjustment

[0265] Appropriate amount of purified water

[0266] According to the manufacturing method of a conventional absorbent, 3 mg of the active substance was prepared per 1 mL of saline solution (0.9% NaCl, w / v, solvent: purified water), filled into an opaque spray container, and sterilized to prepare a absorbent.

[0267]

[0268] <Pharmaceutical Manufacturing Example 6> Preparation of liquid

[0269] 100 mg of active ingredient

[0270] 10 g of isoflavonoids

[0271] 5 g of mannitol

[0272] Appropriate amount of purified water

[0273] According to the usual method of manufacturing a liquid, each ingredient was dissolved in purified water, lemon scent was added, the above ingredients were mixed, purified water was added, the total volume was adjusted to 100 mL, and the liquid was filled into a brown bottle and sterilized to manufacture a liquid.

[0274]

[0275] Manufacturing examples of health foods

[0276] The active ingredient according to the present invention can be manufactured into various health foods, depending on the intended purpose. The following are examples of manufacturing methods for several health foods containing the active ingredient according to the present invention, but the present invention is not limited thereto.

[0277]

[0278] <Health food manufacturing example 1> Manufacturing of dairy products

[0279] 0.01-1 part by weight of the active substance of the present invention was added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

[0280]

[0281] <Health Food Manufacturing Example 2> Manufacturing of snacks

[0282] Brown rice, barley, glutinous rice, and Job's tears were alpha-treated and dried using a known method, and then roasted and ground into powder with a grinder with a particle size of 60 mesh. Black beans, black sesame seeds, and perilla seeds were also steamed and dried using a known method, roasted, and ground into powder with a particle size of 60 mesh using a grinder. The effective substance of the present invention was concentrated under reduced pressure in a vacuum concentrator to obtain a dry powder. The grains, seeds, and dry powders of the effective substance prepared above were mixed in the following ratios to produce a composition.

[0283] Grains (34 parts by weight of brown rice, 19 parts by weight of Job's tears, 20 parts by weight of barley),

[0284] Seeds (7 parts by weight of perilla seeds, 8 parts by weight of black beans, 7 parts by weight of black sesame seeds),

[0285] Active ingredient (2 parts by weight),

[0286] Reishi (1.5 parts by weight), and

[0287] Rehmannia glutinosa (1.5 parts by weight).

[0288]

[0289] Manufacturing examples of health functional foods

[0290] The active substance according to the present invention can be manufactured into various types of health functional foods, depending on the intended purpose. The following are examples of manufacturing methods for several health functional foods containing the active substance according to the present invention as an active ingredient, but the present invention is not limited thereto.

[0291]

[0292] <Health Functional Food Manufacturing Example 1> Manufacturing of Health Functional Food

[0293] 100 mg of active ingredient

[0294] Vitamin mixture appropriate amount

[0295] Vitamin A acetate 70 μg

[0296] Vitamin E 1.0 mg

[0297] Vitamin B1 0.13 mg

[0298] Vitamin B2 0.15 mg

[0299] Vitamin B6 0.5 mg

[0300] Vitamin B12 0.2 μg

[0301] Vitamin C 10 mg

[0302] 10 μg of biotin

[0303] 1.7 mg of nicotinamide

[0304] 50 μg of folic acid

[0305] Calcium pantothenate 0.5 mg

[0306] Appropriate amount of mineral mixture

[0307] 1.75 mg of ferrous sulfate

[0308] 0.82 mg of zinc oxide

[0309] Magnesium carbonate 25.3 mg

[0310] 15 mg of monobasic potassium phosphate

[0311] 55 mg of dibasic calcium phosphate

[0312] 90 mg of potassium citrate

[0313] 100 mg of calcium carbonate

[0314] Magnesium chloride 24.8 mg

[0315] The composition ratio of the above vitamin and mineral mixture is a preferred example of a mixture of ingredients relatively suitable for health functional foods, but the mixing ratio may be arbitrarily modified, and the above ingredients may be mixed according to a conventional health functional food manufacturing method, and then granules may be manufactured and used to manufacture a health functional food composition according to a conventional method.

[0316]

[0317] <Health Functional Food Manufacturing Example 2> Manufacturing of Health Functional Beverage

[0318] 100 mg of active ingredient

[0319] 100 mg of citric acid

[0320] 100 mg of oligosaccharides

[0321] 2 mg of plum concentrate

[0322] 100 mg of taurine

[0323] Add purified water to make a total of 500 mL

[0324] The above ingredients are mixed according to a conventional health beverage manufacturing method, then stirred and heated at 85°C for about 1 hour, the resulting solution is filtered, placed in a sterilized container, sealed and sterilized, and then stored in a refrigerator before being used to manufacture the health beverage composition of the present invention. The above composition ratio is a preferred example of a mixture of ingredients relatively suitable for a preferred beverage, but the mixing ratio may be arbitrarily modified according to regional and ethnic preferences such as the demand class, demand country, and intended use.

[0325]

[0326] The present invention has been described above, focusing on preferred embodiments thereof. Those skilled in the art will appreciate that the present invention can be implemented in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered illustrative rather than restrictive. The scope of the present invention is set forth in the claims, not the foregoing description, and all differences within the scope equivalent thereto should be construed as being encompassed by the present invention.

Claims

1. As the first effective ingredient, somatostatin, a somatostatin derivative or a pharmaceutically acceptable salt thereof; and A pharmaceutical composition for preventing or treating cognitive dysfunction or degenerative brain disease, comprising hesperidin, a hesperidin derivative or a pharmaceutically acceptable salt thereof as a second effective ingredient.

2. In claim 1, A pharmaceutical composition, wherein the somatostatin derivative is any one selected from the group consisting of cortistatin, octreotide, lanreotide, and pasireotide.

3. In claim 1, A pharmaceutical composition, wherein the hesperidin derivative is any one selected from the group consisting of (S)-2,3-dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one, 3',5,7-trihydroxy-4'-methoxyflavanone, α-glucosyl-hesperidin comprising a chain of 1 to 20 glucose residues linked together via 1,4 bonds, 3-methyl-7-(rhamnosyl-2-methylglucosyl)hesperidin, 3-methylhesperidin, hesperetin, a conjugate of hesperetin and sulfate or glucuronide, and neohesperidin dihydrochalcone.

4. In claim 1, A pharmaceutical composition, wherein the above degenerative brain disease is any one selected from the group consisting of vascular cognitive impairment, dementia, Alzheimer's disease, Parkinson's disease, and Huntington's disease.

5. In claim 1, A pharmaceutical composition, wherein the composition has one or more of the following characteristics: (a) Neuroprotective effect; (b) Inhibitory effect on activation of inflammatory transcription factors; and (c) Increased activation of anti-inflammatory transcription factors.

6. In claim 1, A pharmaceutical composition, wherein the above pharmaceutical composition contains 0.1-2 parts by weight of a second effective ingredient based on 100 parts by weight of the first effective ingredient.

7. As the first effective ingredient, somatostatin, a somatostatin derivative or a pharmaceutically acceptable salt thereof; and A health functional food composition for preventing or improving cognitive dysfunction, comprising hesperidin, a hesperidin derivative or a pharmaceutically acceptable salt thereof as a second effective ingredient.

8. In claim 7, A pharmaceutical composition, wherein the somatostatin derivative is any one selected from the group consisting of cortistatin, octreotide, lanreotide, and pasireotide.

9. In claim 7, A pharmaceutical composition, wherein the hesperidin derivative is any one selected from the group consisting of (S)-2,3-dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one, 3',5,7-trihydroxy-4'-methoxyflavanone, α-glucosyl-hesperidin comprising a chain of 1 to 20 glucose residues linked together via 1,4 bonds, 3-methyl-7-(rhamnosyl-2-methylglucosyl)hesperidin, 3-methylhesperidin, hesperetin, a conjugate of hesperetin and sulfate or glucuronide, and neohesperidin dihydrochalcone.

10. As the first effective ingredient, somatostatin, a somatostatin derivative or a pharmaceutically acceptable salt thereof; and A food composition for preventing or improving cognitive dysfunction, comprising hesperidin, a hesperidin derivative or a pharmaceutically acceptable salt thereof as a second effective ingredient.

11. A first active ingredient comprising somatostatin, a somatostatin derivative or a pharmaceutically acceptable salt thereof; or A second active ingredient comprising hesperidin, a hesperidin derivative or a pharmaceutically acceptable salt thereof; The above first effective ingredient is administered in combination with the second effective ingredient, or A pharmaceutical composition for preventing or treating cognitive dysfunction or degenerative brain disease, wherein the second effective ingredient is administered in combination with the first effective ingredient.

12. For use in the manufacture of a combination drug for the prevention or treatment of cognitive dysfunction or degenerative brain disease. Use of somatostatin, a somatostatin derivative or a pharmaceutically acceptable salt thereof; and hesperidin, a hesperidin derivative or a pharmaceutically acceptable salt thereof.

13. A step of administering an effective amount of somatostatin, a somatostatin derivative or a pharmaceutically acceptable salt thereof to a subject in need thereof; and A method for preventing or treating cognitive dysfunction or degenerative brain disease, comprising the step of administering an effective amount of hesperidin, a hesperidin derivative or a pharmaceutically acceptable salt thereof to a subject in need thereof.

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