WO2025127845A1 - Pharmaceutical composition for preventing or treating cognitive dysfunction or degenerative brain disease, comprising somatostatin or derivative thereof and hesperidin or derivative thereof - Google Patents

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

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.

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

One of the main mechanisms that causes degenerative neurological diseases is inflammation. The central nervous system of the brain is home to microglia, which form a unique immune response and a defense line against bacterial invasion and injury. Microglia are a type of macrophage in the central nervous system and play an important role in neuroinflammation. They can be activated by various exogenous and endogenous substances, and activated microglia produce and release various proinflammatory mediators such as inflammatory cytokines TNF-α and IL-1, nitric oxide, prostaglandins, and superoxide. The production of these substances induces an immune response in the short term, but their excessive or continuous production induces the death of nearby nerve cells, ultimately causing 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 has the characteristic of 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 "Master switch," is known to induce high production of inflammatory cytokines in the central nervous system where neuroinflammation has occurred.

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.

Against this backdrop, the inventors of the present invention 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 a derivative thereof, which is a human-derived substance and has already been approved and used as a treatment for acromegaly, and using it together with hesperidin or a derivative thereof, which is a natural product.

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

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

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

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;

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.

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.

Another aspect is to provide 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.

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.

The term “somatostatin” in this specification is a peptide hormone that plays an important role in the nervous system and endocrine system, and mainly performs the function of inhibiting hormone secretion. The somatostatin is a peptide composed of 14 amino acids and was extracted from the hypothalamus of a sheep by German in the United States in 1973. Somatostatin is a neuromodulator and neurotransmitter that is evenly distributed and functions in the central nervous system, and it has been studied that its amount is significantly reduced in the cerebral cortex tissue and cerebrospinal fluid of Alzheimer’s patients.

The term “somatostatin derivative” as used herein refers to a synthetic peptide chemically modified based on naturally occurring somatostatin, which primarily mimics or enhances the hormone secretion inhibitory function of somatostatin. The somatostatin derivative can bind to all five subtypes of somatostatin receptors and activate their signaling in the same manner.

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.

The term “hesperidin” used herein is 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 (a major flavonoid having three hydroxy groups at the 3', 5', and 7' positions and a methoxy substituent at the 4' position), the aglycone of hesperidin, releasing the rutinose moiety and hesperetin, which are then absorbed into colonic enterocytes.

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) bound 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.

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, its sulfate or glucuronide form which are found as metabolic products in the blood circulation. The hesperetin 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.

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.

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.

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 loss and dysfunction of nerve cells.

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

In one specific embodiment, the composition may have 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.

The above neuroprotective effect refers to the effect of protecting neurons from damage, or restoring or maintaining the function of neurons that have already been damaged. Specifically, it can refer to protecting neurons from accumulation of beta-amyloid, oxidative stress, inflammation, toxic substances, etc.

The above-mentioned effect of inhibiting activation of inflammatory transcription factors refers to a physiological effect that alleviates neuroinflammatory responses by reducing excessive activation of major inflammatory signaling pathways in central nervous system cells, thereby inhibiting 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 (Alzheimer's disease, Parkinson's disease, etc.).

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).

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

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.

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 intranasally.

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

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

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 the hesperidin derivative.

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 the 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 the 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 the 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 the hesperidin derivative.

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

The term “health functional food” as used herein refers to food manufactured or processed using raw materials or ingredients that have functionality useful to the human body.

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 accompanying symptoms, preventing the acquisition or reacquisition of a disease or disorder, or reducing the risk of acquiring a disease or disorder.

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.

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.

The term “cognitive impairment” as used herein refers to a state in which memory, attention, language ability, visuospatial ability, judgment, etc. are impaired.

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

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

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

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.

In the pharmaceutical composition according to the present invention, the combined administration may be administered simultaneously or separately with a time difference. The time difference may be in the range of 1 second to 7 days, preferably 1 second to 12 hours, and more preferably 1 second to 6 hours.

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 intranasally.

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

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.

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.

The terms and methods described for the above inventions are equally applicable to each invention.

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; and hesperidin, a hesperidin derivative or a pharmaceutically acceptable salt thereof as a second effective ingredient not only improves reduced cognitive ability and behavior related to degenerative brain diseases, but also has the effect of preventing, improving or treating cognitive dysfunction or degenerative brain diseases through the effects of protecting neuronal cells, inhibiting inflammatory transcription factor activation and increasing anti-inflammatory transcription factor activation.

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

Figure 2 is a graphical representation of the results of the passive avoidance experiment 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).

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).

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

Figure 4b is a graph depicting the neuroprotective effect of the culture medium obtained by treating BV2 microglial cells, an in vitro experimental 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 h (+P < 0.05, ++P < 0.01 vs. HSP, SST alone, ***P < 0.001 vs. control).

Figure 4c is a graph depicting the neuroprotective effect of cultured BV2 microglial cells, an in vitro experimental model of Alzheimer's disease, treated 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 stimulated with amyloid beta (5 μM), and then treated on N2a neurons for 24 h (+P < 0.05, ++P < 0.01 vs. HSP, SST alone, ***P < 0.001 vs. control).

Figure 5a is a graphical representation of 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 experimental model of Alzheimer's disease, after stimulation 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).

Figure 5b is a graphic representation of 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 in 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).

Figure 5c is a graphic representation of 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 in 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).

Figure 6a is a graphical representation of the effect of increasing the activation of the anti-inflammatory transcription factor NRF2 in BV2 microglial cells after 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 in BV2 microglial cells, which were stimulated with Amyloid beta (5 μM), 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).

Figure 6b is a graphical representation of the effect of increasing the activation of the anti-inflammatory transcription factor NRF2 in BV2 microglial cells after treatment 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 stimulation with amyloid beta (5 μM) for 1 hour. (††P < 0.01, †††P < 0.001 vs. SST derivative alone, **P < 0.01, ***P < 0.001 vs. control).

Hereinafter, the present invention will be described in detail.

pharmaceutically acceptable salts

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 expression pharmaceutically acceptable salt means any organic or inorganic addition salt of a base compound of an active substance, which has an effective effect that is relatively nontoxic and harmless to a patient, and the side effects caused by the salt do not reduce the beneficial efficacy 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, methyl sulfate, 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.

The acid addition salt according to the present invention can be prepared by a conventional method, for example, dissolving an effective substance in an organic solvent, such as methanol, ethanol, acetone, methylene chloride, acetonitrile, etc., adding an organic acid or inorganic acid, and 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 the presence of an organic solvent.

In addition, a pharmaceutically acceptable metal salt can be prepared using a base. An alkali metal or alkaline earth metal salt is obtained, for example, by dissolving a compound in an excess of an alkali metal hydroxide or an alkaline earth metal hydroxide solution, filtering off the undissolved compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare a sodium, potassium or calcium salt as the metal salt. In addition, the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

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

Pharmaceutical composition

The active substance of the present invention can be administered in various oral and parenteral formulations 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.

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.

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

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. When based on an adult patient weighing 70 kg, it 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.

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.

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.

Food and health functional food compositions

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

Examples of foods include drinks, meats, sausages, bread, candy, snacks, noodles, ice cream, dairy products, soups, sports drinks, soft drinks, alcoholic beverages, gum, tea, etc.

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.

The food and health functional food composition containing the effective substance according to the present invention can be added to food as it is or used together with other foods or food ingredients, and can be used appropriately according to a conventional method. The amount of the effective substance mixed can be appropriately determined depending on its purpose of use (prevention or improvement). In general, the amount of the composition in the food and health functional food can be added in an amount of 0.1 to 90 parts by weight of the total food weight. However, in the case of long-term intake for the purpose of maintaining health or regulating health, the amount can be below the above range, and since there is no problem in terms of safety, the effective ingredient can also be used in an amount above the above range.

The food and health functional food composition of the present invention contains the effective substance of the present invention as an essential ingredient in the indicated ratio, and has no particular limitations on other ingredients, and may contain various flavoring agents or natural carbohydrates as additional ingredients, like a typical beverage. Examples of the above-mentioned natural carbohydrates are monosaccharides, for example, glucose, fructose, etc.; disaccharides, for example, maltose, sucrose, etc.; and polysaccharides, for example, dextrin, cyclodextrin, etc., and typical sugars, and sugar alcohols, for example, xylitol, sorbitol, erythritol, etc. As flavoring agents other than those described above, natural flavoring agents (thaumatin, stevia extracts (for example, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio of the above-mentioned natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 of the health functional food composition of the present invention.

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 thickeners (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, 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 and fruit juice drinks and vegetable drinks.

These components can be used independently or in combination. The proportion of these additives is not so important, but is generally selected in 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 effective substance of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only intended to illustrate the present invention, and the content of the present invention is not limited to the following examples.

Experimental method

Preparation of the composition

Hesperidin (HSP) and hesperetin (HST) were dissolved in DMSO at high concentrations and then dissolved in saline solution at the appropriate concentration when used. Somatostatin (SST), lanreotide (LAT), pasireotide (PAT), and octreotide (OCT) were dissolved in saline solution, and cortistatin-14 (CST-14) and cortistatin-17 (CST-17) were dissolved in DMSO at high concentrations and then dissolved in saline solution at the appropriate concentration when used. All samples were aliquoted and stored in a -80℃ sample freezer, and were taken out when necessary, used once, and then discarded.

Administration of the composition

Wild type control and mutant type control 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 solution daily for 8 weeks. The somatostatin (SST; Sigma Chemical Company) group was administered intranasally at 400 μg/kg dissolved in saline solution daily for 8 weeks. The hesperidin + somatostatin group was administered at the same dose and by the same method as the single group.

Alzheimer's disease mouse model

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 the Jackson laboratory (Bar Harbor, Maine, USA) with MT_5xFAD. The animals were supplied with sufficient solid feed (antibiotic-free, Samyang Feed Co.) and water, and were acclimated for 1 week in an environment maintained at 22±2℃, humidity 55±15%, and 12-h light-dark cycle before being used in the experiment.

Morris water maze test

The Morris water maze experiment is an experiment on hippocampus-dependent spatial learning and cognitive improvement, and is known as a screening method for long-term memory. In the water maze experiment, a circular tank (diameter 90 cm, height 45 cm) was filled with water to a height of 30 cm (23±2℃). An escape platform with a diameter of 6 cm was placed 1 cm below the water surface in one of the four quadrants of the tank, and nontoxic, water-soluble white paint was dissolved so that the base was not visible. On the first day of the experiment, the experimental animals were allowed to swim freely in the tank for 60 seconds without the escape platform, and cognitive adaptation training was performed twice a day for four days by changing the quadrant they entered each day. When the experimental animals reached the escape platform, they were allowed to stay on the platform for 10 seconds, and if they failed to find the escape platform within 60 seconds, they were allowed to stay on the platform for 10 seconds to memorize the escape platform. On the fifth day of the experiment, the escape platform was removed to measure working memory, and a probe test was conducted to record the time it took to find the escape 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).

Passive avoidance test

The passive avoidance test is a widely used experiment to measure learning and memory. The avoidance learning box (each compartment 20×20×20cm) is divided into a dark room and a bright room. When a laboratory animal is placed in the bright room, it moves to the dark room due to its preference for dark places. At that moment, the guillotine door is closed and an electric shock of 0.5 mA is applied for 5 seconds. When the laboratory animal is placed back in the bright room the day after the electric shock, it remembers the electric shock in the dark room and stays in the bright room. The time it spends there (step-through latency) is measured to evaluate memory. The total measurement time was 180 seconds, and all experiments were recorded and measured using video tracking software (SMART 3.0, Panlab, Spain).

Visual cognitive behavior test

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

Cell culture

BV2 cells and N2a cells used in this experiment were obtained from ATCC (Manassas, USA). BV2 cells 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/㎖ of penicillin, and 100 ㎍/㎖ of streptomycin (Gibco) at 37℃ in a 5% CO ₂ incubator.

Measuring neuronal survival rate

To measure the neuroprotective effect of the composition using an in vitro experimental model of Alzheimer's disease, the WST cytotoxicity evaluation test method was performed. BV2 microglial cells, a mouse microglial cell line, were seeded (2×10 ⁵ /ml) in a 96-well plate and cultured for 24 hours. Then, they were 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). After replacing with a new medium after 6 hours, the culture medium was cultured for 12 hours and treated with 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.

Measurement of NF-κB inflammatory transcription factor production in microglia

To measure the inhibitory effect of the composition on NF- κB inflammatory transcription factor activation using an in vitro experimental model of Alzheimer's disease, a western blot assay was performed. BV2 microglial cells, a mouse microglial cell line, were seeded (2×10 ⁵ /ml) in 60π dishes and cultured for 24 hours. Then, they were 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, the membranes obtained by performing western blot experiments were blocked and reacted with NF- κB and TBP primary antibodies at 4°C for one day, and then reacted with secondary antibodies at room temperature for 1 hour the next day. The membranes were evenly distributed using ECL Detection solution, developed with ChemiDoc ^TM XRS ⁺ (Bio-Rad, Richmond, CA), and analyzed.

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

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

BV2 microglial cells, a mouse microglial cell line, were seeded in a 60π dish at a density of (2×105/ml) 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 the secondary antibodies were reacted at room temperature for 1 hour the next day. After evenly spreading the ECL Detection solution on the membrane, it was developed and analyzed using ChemiDocTMXRS+ (Bio-Rad, Richmond, CA).

statistical processing

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

<Comparative Example 1> Hesperidin alone

Hesperidin was dissolved in DMSO at a high concentration and then dissolved in saline solution at the appropriate concentration when used. The sample was aliquoted and stored in a -80℃ sample freezer, and when necessary, taken out and discarded after a single use.

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.

<Comparative Example 2> Somatostatin monotherapy

Somatostatin was dissolved in saline at a high concentration and then dissolved in saline at the appropriate concentration when used. The sample was aliquoted and stored in a -80℃ sample freezer, and when necessary, taken out and discarded after a single use.

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

<Comparative Example 3> Administration of hesperidin derivative alone

Hesperetin (HST) was dissolved in DMSO at a high concentration and then dissolved in saline solution at the appropriate concentration when used. The sample was dispensed and stored in a -80℃ sample freezer, and when necessary, taken out and discarded after a single use.

<Comparative Example 4> Somatostatin derivative monotherapy

Lanreotide (LAT), pasireotide (PAT), and octreotide (OCT) were dissolved in saline at high concentrations and then dissolved in saline at the appropriate concentration when used. The samples were aliquoted and stored in a -80℃ sample freezer, and when necessary, taken out and discarded after a single use.

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 at the appropriate concentration when used. The samples were aliquoted and stored in a -80℃ sample freezer, and when necessary, taken out and discarded after a single use.

<Example 1> Co-administration of hesperidin and somatostatin

The hesperidin and somatostatin combination group was administered the same dosage and method as the single-administration group (Comparative Example 1 and Comparative Example 2).

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

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, cognitive dysfunction and behavioral changes related to degenerative brain diseases were examined.

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

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

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

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

According to the above experimental method, the neuroprotective effect of the composition was measured using an in vitro experimental model of Alzheimer's disease, 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.

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

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

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

According to the above experimental method, the neuroprotective effect of the composition was measured using an in vitro experimental model of Alzheimer's disease, 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.

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

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).

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.

According to the above experimental method, the neuroprotective effect of the composition was measured using an in vitro experimental model of Alzheimer's disease, 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.

<Experimental Example 1> Morris water maze experiment using Alzheimer's disease model mice

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

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

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. However, the HSP + SST combination therapy group showed a significantly greater reduction effect compared to the HSP, SST monotherapy group.

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

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

Figure 2 is a graphical representation of the results of the passive avoidance experiment 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).

As shown in Fig. 2, in the case of MT_control (Alzheimer's disease-induced group), the time spent in the bright room decreased 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, the HSP + SST combination therapy group showed a significantly greater increasing effect compared to the HSP, SST monotherapy group.

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

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

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).

As shown in Figure 3, the MT_control (Alzheimer's disease-induced group) showed a decrease in the time spent in the vertically cross-hatched room compared to the WT_control (normal group), and compared to the MT_control, the time spent in the vertically cross-hatched room increased in both the somatostatin (SST) monotherapy group and the hesperidin + somatostatin (HSP + SST) combination therapy group. However, the HSP + SST combination therapy group showed a significantly greater increasing effect compared to the HSP and SST monotherapy groups.

<Experimental Example 4> Alzheimer's disease in vitro Neuroprotective effects using experimental models

4.1 Confirmation of neuroprotective effect by combined administration of hesperidin and somatostatin

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

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

As shown in Fig. 4a, the cell viability of the control group was found to decrease compared to the untreated group, and compared to the control group, the cell viability increased in the hesperidin (HSP), somatostatin (SST) monotherapy group and the hesperidin + somatostatin (HSP + SST) combination therapy group. However, the HSP + SST combination therapy group showed a significantly greater increase compared to the HSP and SST monotherapy groups.

4.2 Confirmation of neuroprotective effect by combined administration of hesperidin and somatostatin derivatives

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

Figure 4b is a graph depicting the neuroprotective effect of the culture medium obtained by treating BV2 microglial cells, an in vitro experimental 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 h (+P < 0.05, ++P < 0.01 vs. HSP, SST alone, ***P < 0.001 vs. control).

As shown in Fig. 4b, the cell viability of the control group was found to decrease compared to the untreated group (Normal), and compared to the control group, the cell viability increased in all groups administered hesperidin (200uM), somatostatin (10uM), lanreotide (10uM), pasireotide (10uM), octreotide (10uM), cortistatin-14 (10uM), and cortistatin-17 (10uM) alone or in a mixture, but it was confirmed that the mixed administration group showed a significantly greater increasing effect compared to the single administration group.

4.3 Confirmation of neuroprotective effect by combined administration of hesperidin derivatives and somatostatin or somatostatin derivatives

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

Figure 4c is a graph depicting the neuroprotective effect of cultured BV2 microglial cells, an in vitro experimental model of Alzheimer's disease, treated 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 stimulated with amyloid beta (5 μM), and then treated on N2a neurons for 24 h (+P < 0.05, ++P < 0.01 vs. HSP, SST alone, ***P < 0.001 vs. control).

As shown in Fig. 4c, the cell viability of the control group was found to decrease compared to the untreated group (Normal), and compared to the control group, the cell viability increased in all of the groups administered hesperetin (200uM), somatostatin (10uM), lanreotide (10uM), pasireotide (10uM), octreotide (10uM), cortistatin-14 (10uM), and cortistatin-17 (10uM) alone or in a mixture, but it was confirmed that the mixed administration group showed a significantly greater increasing effect compared to the single administration group.

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

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

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

Figure 5a is a graphical representation of 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 experimental model of Alzheimer's disease, after stimulation 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).

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 group showed a significantly greater reduction effect compared to the HSP and SST monotherapy groups.

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

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

Figure 5b is a graphical representation of 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 in 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 of 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).

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 hesperidin (200uM), somatostatin (10uM), lanreotide (10uM), pasireotide (10uM), octreotide (10uM), cortistatin-14 (10uM), and cortistatin-17 (10uM) alone or in a mixture, but it was confirmed that the mixture administration group showed a significantly greater reduction effect compared to the single administration group.

5.3 Hesperidin derivatives and somatostatin or somatostatin derivatives; Confirmation of the inhibitory effect on NF-κB inflammatory transcription factor activation by combined administration

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

Figure 5c is a graphical representation of the inhibitory effect on the activation of NF-κB inflammatory transcription factor in BV2 microglial cells after treatment 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 in BV2 microglial cells, stimulated with amyloid beta (5 μM), and then nuclear extracts were obtained 1 hour later (++P<0.01). vs. HSP, SST alone, **P < 0.01, ***P < 0.001 vs. control).

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 (200 uM), somatostatin (10 uM), lanreotide (10 uM), pasireotide (10 uM), octreotide (10 uM), cortistatin-14 (10 uM), and cortistatin-17 (10 uM) alone or in a mixture, but it was confirmed that the mixture administration group showed a significantly greater reduction effect compared to the single administration group.

<Experimental Example 6> Alzheimer's disease in vitro Increased activation of NRF2 anti-inflammatory transcription factor using experimental models

6.1 Confirmation of the effect of increasing the activation of NRF2 anti-inflammatory transcription factor by combined administration of hesperidin and somatostatin derivatives

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 on an in vitro model of Alzheimer's disease.

Figure 6a is a graphical representation of the effect of increasing the activation of the anti-inflammatory transcription factor NRF2 in BV2 microglial cells after 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 in BV2 microglial cells, stimulated with Amyloid beta (5 μM), 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).

As shown in Fig. 6a, the control group showed a slight increase in NRF2 production compared to the untreated group (Normal). Compared to the control group, all of the groups administered hesperidin (200uM), somatostatin (10uM), lanreotide (10uM), pasireotide (10uM), octreotide (10uM), cortistatin-14 (10uM), and cortistatin-17 (10uM) alone or in combination showed an increase in NRF2 production. However, the mixed administration group showed a significantly greater increasing effect compared to the single administration group.

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

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 on an in vitro model of Alzheimer's disease.

Figure 6b is a graphical representation of the effect of increasing the activation of the anti-inflammatory transcription factor NRF2 in BV2 microglial cells after treatment 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 stimulation with amyloid beta (5 μM) for 1 hour. (††P < 0.01, †††P < 0.001 vs. SST derivative alone, **P < 0.01, ***P < 0.001 vs. control).

As shown in Fig. 6b, the control group showed a slight increase in NRF2 production compared to the untreated group (Normal). Compared to the control group, all of the groups administered hesperetin (200uM), somatostatin (10uM), lanreotide (10uM), pasireotide (10uM), octreotide (10uM), cortistatin-14 (10uM), and cortistatin-17 (10uM) alone or in combination showed an increase in NRF2 production. However, the mixed administration group showed a significantly greater increasing effect compared to the single administration group.

As described above, when a composition containing somatostatin and hesperidin was administered to Alzheimer's disease model mice, it was confirmed that the reduced cognitive ability and behaviors related to degenerative brain diseases were improved. In addition, 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 Alzheimer's disease in vitro model, it was confirmed that it was effective in alleviating cognitive dysfunction or degenerative brain diseases through a neuronal cell protection effect, a decrease in the amount of inflammatory transcription factors produced, and an increase in the amount of anti-inflammatory transcription factors produced. Through this, the inventors of the present invention concluded that it can be applied as a preventive and therapeutic agent for various cognitive dysfunctions or degenerative brain diseases.

Example of manufacturing a drug

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

<Pharmaceutical Manufacturing Example 1> Manufacturing of a powder

2 g of active substance

1 g lactose

After mixing the above ingredients, the powder was filled into a sealed bag to prepare a powder.

<Pharmaceutical Manufacturing Example 2> Manufacturing of tablets

100 mg of active ingredient

Corn starch 100 mg

100 mg of lactose

Magnesium stearate 2 mg

After mixing the above ingredients, tablets were manufactured by pressing them according to the usual method for manufacturing tablets.

<Pharmaceutical Manufacturing Example 3> Manufacturing of capsules

100 mg of active ingredient

Corn starch 100 mg

100 mg of lactose

Magnesium stearate 2 mg

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

<Pharmaceutical Manufacturing Example 4> Manufacturing of injection

Effective substance 10 ㎍/㎖

until the diluted hydrochloric acid BP pH becomes 3.5.

Sodium Chloride BP for Injection Up to 1 ㎖

The effective substance 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 15 minutes or more to prepare an injection solution.

<Pharmaceutical Manufacturing Example 5> Manufacturing of nasal spray

Active ingredient 1.0 g

Sodium acetate 0.3 g

0.1 g of methylparaben

Propylparaben 0.02 g

Sodium chloride appropriate amount

HCl or NaOH pH adjustment appropriate amount

Appropriate amount of purified water

According to the manufacturing method of a conventional absorbent, 3 mg of the effective substance was contained per 1 mL of saline (0.9% NaCl, w/v, purified water as the solvent), filled into an opaque spray container, and sterilized to manufacture a absorbent.

<Pharmaceutical Manufacturing Example 6> Preparation of liquid

Active ingredient 100 mg

10 g of isoflavone

Mannitol 5 g

Appropriate amount of purified water

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

Manufacturing examples of health foods

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

<Health food manufacturing example 1> Manufacturing of dairy products

0.01-1 weight part of the effective substance of the present invention was added to milk, and various dairy products such as butter and ice cream were manufactured using the milk.

<Health food manufacturing example 2> Manufacturing of snacks

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 having a particle size of 60 mesh using a grinder. Black beans, black sesame seeds, and perilla seeds were also steamed and dried using a known method, roasted, and ground into powder having 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 manufactured above were mixed in the following ratio to manufacture a composition.

Grains (34 parts by weight of brown rice, 19 parts by weight of barley, 20 parts by weight of barley),

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

Active ingredient (2 parts by weight),

Reishi (1.5 parts by weight), and

Rehmannia glutinosa (1.5 parts by weight).

Manufacturing example of health functional food

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

<Health functional food manufacturing example 1> Manufacturing of health functional food

Active ingredient 100 mg

Vitamin mixture appropriate amount

Vitamin A acetate 70 μg

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

Vitamin B12 0.2 μg

Vitamin C 10 mg

Biotin 10 μg

Nicotinamide 1.7 mg

Folic acid 50 μg

Calcium pantothenate 0.5 mg

Appropriate amount of mineral mixture

Ferrous sulfate 1.75 mg

Zinc oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium phosphate monobasic 15 mg

55 mg of dibasic calcium phosphate

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

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.

<Health functional food manufacturing example 2> Manufacturing of health functional beverage

Active ingredient 100 mg

100 mg citric acid

100 mg of oligosaccharide

Plum concentrate 2 mg

Taurine 100 mg

Add purified water to make a total of 500 mL

According to a conventional health beverage manufacturing method, the above ingredients are mixed, stirred and heated at 85℃ for about 1 hour, the resulting solution is filtered, placed in a sterilized container, sealed and sterilized, and then stored in a refrigerator, and then used in manufacturing the health beverage composition of the present invention. The above composition ratio is a preferred example of mixing ingredients relatively suitable for a favorite 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.

The present invention has been described with reference to preferred embodiments thereof. Those skilled in the art will appreciate that the present invention may be implemented in modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is not set forth in the foregoing description, but rather in the claims, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (13)

As a first active 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. In claim 1, A pharmaceutical composition, wherein the somatostatin derivative is any one selected from the group consisting of cortistatin, octreotide, lanreotide, and pasireotide. 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. 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. 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. 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. As a first active 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. In claim 7, A pharmaceutical composition, wherein the somatostatin derivative is any one selected from the group consisting of cortistatin, octreotide, lanreotide, and pasireotide. 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. As a first active 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. 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. For use in the manufacture of a pharmaceutical composition for combination administration for the prevention or treatment of cognitive dysfunction or degenerative brain diseases. Use of somatostatin, a somatostatin derivative or a pharmaceutically acceptable salt thereof; and hesperidin, a hesperidin derivative or a pharmaceutically acceptable salt thereof. 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.

Images