Pharmaceutical composition comprising hl-neuropion (complex of pinus densiflora extract and alpinia officinarum extract) as active ingredient for improving cognitive function and treating dementia through excellent Anti-inflammatory effect, inhibition of neurotransmitter degradation, and inhibition of amyloid beta aggregation

Abstract

The present invention relates to: a health functional food, food product, or therapeutic pharmaceutical composition for preventing or ameliorating dementia and cognitive decline, wherein a complex of a Pinus densiflora extract and an Alpinia officinarum extract is included as an active ingredient; and a method for producing same. The complex provided by the present invention was not only found to have excellent acetylcholinesterase inhibitory activity and nitric oxide production inhibitory activity, but was also found to have an activity of improving cognitive decline induced by beta amyloid administration in an animal model, and can thus be used as a health functional food and pharmaceutical for preventing, ameliorating, or treating dementia and cognitive decline.

Description

Pharmaceutical composition for improving cognitive function or treating dementia by containing HL-NEUROPION (a complex of pine needle extract and galangal extract) as an active ingredient, with excellent anti-inflammatory effects, inhibition of neurotransmitter decomposition, and inhibition of beta-amyloid aggregation.

[0001] This application claims the benefit of priority from Korean Patent Application No. 10-2024-0012539, filed on January 26, 2024, the entire contents of which are incorporated herein by reference. The present invention relates to a pharmaceutical composition for preventing, improving or treating dementia and cognitive decline, and more particularly, to a pharmaceutical composition, health functional food or a method for preparing the same, which has an effect of preventing, improving or treating dementia and cognitive decline by simultaneously imparting excellent nitric oxide production inhibition and acetylcholinesterase inhibition effects, anti-inflammatory effects, neurotransmitter decomposition inhibition effects and beta-amyloid aggregation inhibition effects, including a complex of pine needle extract and galangal extract as an active ingredient.

[0002]

[0003] As of 2022, the elderly population aged 65 and older in South Korea is expected to reach approximately 9 million, and the country is aging at the fastest rate in the world due to factors such as a declining birth rate and increasing life expectancy. Due to this rapid population aging, the prevalence of dementia is also rapidly increasing. According to statistics from the National Dementia Center, approximately 10.3% of the elderly population aged 65 and older and approximately 35% of the elderly population aged 85 and older have been diagnosed with dementia. This increase is expected to continue, with the number of dementia patients projected to reach approximately 1 million by 2025 and 3 million by 2050. As the number of dementia patients increases, the social burden caused by dementia also increases. In 2021, the total annual cost of dementia management was approximately 18.7 trillion won, accounting for approximately 11% of the gross domestic product (GDP). Annual management costs also increase as the severity of dementia increases. Furthermore, because dementia is characterized by psycho-behavioral symptoms, the burden of caregiving and informal family care is increased in addition to medication and medical expenses, so the economic and social costs of dementia can be said to have a significance that goes beyond statistics.

[0004] The exact causes of neurodegenerative diseases such as dementia and the resulting cognitive decline are not yet clearly understood. However, various studies have highlighted the formation of plaques due to beta-amyloid aggregates, deposition due to mutations in tau protein, and tissue damage caused by oxidative stress or neuroinflammation as major causes. While neuroinflammation generally serves as a protective mechanism against direct and indirect damage caused by infections, toxic substances, and other factors, if an imbalance in inflammatory signals within the body arises due to various additional factors, it can lead to a chronic inflammatory response, negatively affecting nerve cells and tissues and directly inducing neurodegeneration. Furthermore, this inflammatory response further promotes the formation of beta-amyloid plaques and tau protein aggregates, which in turn trigger an inflammatory response, creating a vicious cycle of disease. Therefore, research targeting neuroinflammation is actively underway.

[0005] Meanwhile, current dementia treatments aim to improve cognitive abilities in dementia patients. For patients with mild to moderate dementia, the focus is on maintaining normal acetylcholine levels by inhibiting the activity of the cholinesterase enzyme, which hydrolyzes acetylcholine, a neurotransmitter that plays a crucial role in memory and cognitive function in the brain. Representative drugs used include donepezil, rivastigmine, and galantamine. However, these drugs have been reported to have side effects such as decreased appetite, nausea, vomiting, diarrhea, headache, and weight loss, which can negatively impact the health of the elderly. Furthermore, these side effects can lead to significant deterioration in cognitive function when the drug is suddenly discontinued. Therefore, there is a growing demand for alternative therapies based on combinations of natural ingredients that minimize side effects and harm to the human body while also providing clear preventive and improving effects.

[0006] Numerous references and citations are provided throughout this specification. The disclosures of these references are incorporated herein by reference in their entirety to further clarify the state of the art and the scope of the present invention.

[0007]

[0008] The purpose of the present invention is to provide a health functional food and pharmaceutical composition capable of preventing and improving cognitive decline and exhibiting a dementia treatment effect by simultaneously providing an anti-inflammatory effect, an acetylcholinesterase inhibitory effect, and an improvement effect on cognitive decline caused by beta-amyloid through a natural product-derived ingredient or a complex thereof, and a method for producing the same.

[0009]

[0010] In order to solve the above-mentioned problem, the present invention provides a health functional food and pharmaceutical composition for preventing, improving, or treating dementia and cognitive decline, comprising a complex of pine needle extract and galangal extract as an active ingredient, and a method for preparing the same. The complex in the present invention can inhibit acetylcholinesterase activity and nitric oxide production, but is not limited thereto, and exhibits excellent cognitive function improvement efficacy in a beta-amyloid-induced animal model, and thus can be used as a composition for preventing, improving, or treating dementia and cognitive decline. More specifically, it has been confirmed that the complex has anti-inflammatory activity by inhibiting the production of nitric oxide, which is produced by an inflammatory response and causes damage to cells and tissues, while activating neurotransmission through inhibition of acetylcholinesterase enzyme, a representative cause of cognitive decline, and improving cognitive decline induced by beta-amyloid administration in an animal model. Therefore, the complex of pine needle and galangal extracts of the present invention can be used as an active ingredient in a composition for preventing, improving, or treating dementia and cognitive decline.

[0011] The pine needles used in the present invention are the leaves of pine (Pinus densiflora). Pine is a type of evergreen gymnosperm belonging to the Pinaceae family of the Pinales order. It grows natively throughout Korea and is also distributed in most of Northeast Asia, including eastern Russia, northeastern China, and Japan. It grows wild on ridges of mountainous areas with abundant sunlight and is dry, and is about 35 m tall and about 1.8 m in diameter. The leaves grow in pairs, are needle-shaped and have a pointed tip, and are about 8 to 9 cm long and 1.5 mm wide. The leaves, pollen, and bark are used for food or medicinal purposes. Among them, the leaves are dried and made into powder and used in foods such as tea and rice cakes, and are included as medicinal materials in ancient books such as Donguibogam and Bencao Gangmok, and have been used for a long time. Pine needles are known to contain essential oil components of the terpene series such as alpha-pinene and beta-pinene, polyphenols such as chlorophyll, lutein, and beta-carotene, as well as vitamin C, vitamin K, flavonoids, and organic acids. Various physiological activities such as serum cholesterol lowering effect, anti-diabetic effect, lipid lowering and antioxidant effect, and anticancer and antibacterial effect due to these physiologically active components have been reported.

[0012] In the present invention, galangal is the rhizome of Alpinia officinarum. It was formerly known as Gaoliang Jiang in China and began to be called galangal after being introduced by the Italian botanist Prospero Alpini in the 17th century. In Korea, it is also known as Gaoliang Jiang, Asian ginger, new ginger, yang ginger, etc. As a plant belonging to the Zingiberaceae family of the Zingiberales order, it originated in China and is mainly cultivated in Southeast Asia. It grows to a height of about 1.5 to 2 m, with long leaves and inconspicuous white flowers. The rhizome, galangal, has been used as a raw material for curry and perfume throughout Asia due to its sweet and spicy taste and aroma, and has also been widely used in Europe. Galangal is known to contain essential oil components such as 1,8-cineole, methyl cinnamate, and alpha-cadinene, flavonoids such as galangin, campheride, and alpinin, and galangol. Various physiological activities such as inhibition of pancreatic lipase activity, inhibition of fatty acid synthesis, antioxidant effect, defense effect against cell and DNA damage by reactive oxygen species, inhibition of cyclooxygenase-2, and inhibition of prostaglandin biosynthesis, i.e., anti-inflammatory effects, have been reported.

[0013] Therefore, the inventors of the present invention have discovered that a complex of pine leaf extract and galangal extract simultaneously exhibits excellent nitric oxide production inhibitory and acetylcholinesterase inhibitory effects, thereby conferring both anti-inflammatory effects and inhibitory effects on the decomposition of neurotransmitters, and can exhibit preventive, ameliorative, and therapeutic effects on dementia and cognitive function decline, and thus completed the present invention.

[0014] In one embodiment of the present invention, the pine leaf extract is characterized by being extracted from pine leaves with water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof and concentrated.

[0015] In another embodiment of the present invention, the galangal extract is characterized by being obtained by extracting galangal with water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof and then concentrating the extracted galangal.

[0016] In another embodiment of the present invention, the pine needle extract or galangal extract is characterized in that it is a concentrate or a dry powder.

[0017] In another embodiment of the present invention, the composite of the pine needle extract and the galangal extract is characterized by mixing the galangal extract with the pine needle extract in a weight ratio of 1:1 to 1:8 (pine needle extract: galangal extract). More specifically, the weight ratio of the pine needle extract to the galangal extract may be 1:2 to 1:8, or 1:4 to 1:8. In another embodiment, the weight ratio of the pine needle extract to the galangal extract may be 1:8±1, 1:8±0.5, or 1:8±0.1. The composite of the present invention can exhibit a synergistic effect due to the composite compared to each extract alone by including the pine needle extract and the galangal extract in the above-mentioned weight ratio, and can further exhibit a more remarkable synergistic effect compared to including the two extracts in a different weight ratio.

[0018] In another embodiment of the present invention, the complex may be composed of a pine needle extract and a galangal extract, and the weight ratio of the extracts in the complex is as described above.

[0019] In another embodiment of the present invention, the composition of the present invention may not contain any other natural extract components other than the pine needle extract and galangal extract as active ingredients.

[0020] In another embodiment of the present invention, the complex of the pine needle extract and galangal extract is characterized by acetylcholinesterase inhibitory activity, improvement of cognitive function in cognitive decline due to beta-amyloid, and inhibition of nitric oxide production.

[0021] In another embodiment of the present invention, the composition is characterized in that it is a health functional food composition or a pharmaceutical composition.

[0022] The present invention also provides health functional foods and pharmaceuticals comprising the composition.

[0023] All ingredients described in the present invention preferably do not exceed the maximum usage levels stipulated in relevant laws and regulations of Korea, China, the United States, Europe, Japan, etc. (e.g., Food Code (Korea), Food Additives Code (Korea), Health Functional Food Code (Korea)). That is, preferably, the food, health functional food, or pharmaceutical composition according to the present invention contains the ingredients according to the present invention within the content limits permitted by relevant laws and regulations of each country.

[0024]

[0025] The complex provided by the present invention has anti-inflammatory activity that inhibits the production of nitric oxide, which is produced by an inflammatory reaction and causes damage to cells and tissues, while activating neurotransmission through inhibition of acetylcholinesterase enzyme, which is a representative cause of dementia and cognitive decline, and has been confirmed to have an improvement activity on cognitive decline in an animal model in which dementia was induced by beta-amyloid administration, and because its effect is excellent, it can be utilized as a health functional food and pharmaceutical composition for preventing, improving, or treating dementia and cognitive decline.

[0026]

[0027] Figure 1 is a diagram showing the concentration-dependent anti-inflammatory effect of Example 7.

[0028] Figure 2 is a diagram showing the cognitive function improvement effect due to oral administration of Examples 4, 6, and 7 in mice in which an Alzheimer's disease-like condition is induced due to beta-amyloid.

[0029] The experimental groups and graphs in Fig. 2 are described below.

[0030] NC: Control group

[0031] AB: Beta-amyloid administration group (induction group)

[0032] Example 4: Induction group + Example 4 (pine needle extract: moringa extract = 1:1) 100 mg / kg administration group

[0033] Example 6: Induction group + Example 6 (pine needle extract: moringa extract = 1:4) 100 mg / kg administration group

[0034] Example 7: Induction group + Example 7 (pine needle extract: moringa extract = 1:8) 100 mg / kg administration group

[0035] Morin: Induction group + Morin 10 mg / kg administration group

[0036] **: p<0.01 vs NC by unpaired t-test

[0037] ## : p<0.01 vs AB by unpaired t-test

[0038]

[0039] The present invention provides a composition for preventing, improving or treating dementia and / or cognitive decline, comprising a complex of pine needle extract and galangal extract (HL-BCRS) as an active ingredient, and more specifically, relates to a health functional food and pharmaceutical composition for preventing, improving or treating dementia and cognitive decline.

[0040]

[0041] [extract]

[0042] In the present invention, the term 'extract' includes the extract itself and all formulations that can be formed using the extract, such as an extract obtained by an extraction process using water or other solvents, a diluted or concentrated extract, a dried product obtained by drying the extract, a controlled or purified product of the extract, or a mixture thereof.

[0043] Within the scope that does not impede the purpose of the present invention, the extract according to the present invention may include, in addition to the specific parts of the materials described above, leaves, stems, roots, bark, flowers, fruits, seeds, sap, the whole plant, etc.

[0044] In order to manufacture the extract according to the present invention, any suitable method known in the industry can be used in the pre- and post-extraction processes, such as pre-treatment of raw materials, extraction of raw materials, and processing of the extract. For example, the whole or any part of the raw material to be used can be pulverized, and then an extraction solvent can be treated to obtain a liquid solvent extract. The extraction efficiency can be increased through heating during the extraction process, and an extraction method that minimizes solvent loss can be used by simultaneously performing a reflux cooling process to minimize solvent loss caused by heating during this process. In addition, the raw material can be dried and pulverized during the entire extraction process, and the solvent extract obtained after extraction can be manufactured into a filtrate, concentrate, or powder through an additional process, such as filtration, distillation under reduced pressure, freeze-drying, or spray drying.

[0045] The type of extraction solvent used in the above extraction process is not particularly limited, and any solvent commonly used in the industry can be used. For example, water; C1 to C4 lower alcohols such as methanol, ethanol, propyl alcohol, butyl alcohol, etc.; hydrocarbon solvents such as ethyl acetate, acetone, hexane, dichloromethane, etc.; or mixtures thereof can be used, and preferably, water and lower alcohols can be used alone or in combination of two or more. The solvent can be used to perform extraction once or more repeatedly to prepare a solvent extract, and the solvent extract can be dried to prepare a dry extract.

[0046] In the above extraction process, the amount of extraction solvent may vary depending on the type of solvent used, but for example, it may be used in an amount of 5 to 30 times, more preferably 10 to 20 times, the dry weight of the target raw material. Conditions such as extraction temperature, time, and pressure are not limited, but may be performed at 60 to 120°C, 1 to 10 hours, and 0.1 to 0.3 MPa.

[0047] In addition, various extraction processes commonly used in the industry, such as leaching, microwave-assisted extraction, ultrasonic extraction, and supercritical fluid extraction, can be selected and used, and these methods can be performed simultaneously or sequentially in parallel.

[0048] The pine needle extract according to the present invention may contain 0.05 to 0.35 mg / g of protocatechuic acid, and it is presumed that the reason why such concentration of protocatechuic acid can be obtained is due to differences in the origin of raw materials, types of parts used, differences in the manufacturing process and process variables described above, and thus the present invention is not limited to these matters.

[0049] The galangal extract according to the present invention may contain 0.04 to 0.20 mg / g of 4-hydroxybenzaldehyde, and it is presumed that the reason why 4-hydroxybenzaldehyde in such a concentration can be obtained is due to differences in the origin of the raw material, types of parts used, differences in the manufacturing process and process variables described above, and thus the present invention is not limited to these matters.

[0050]

[0051] [Composition]

[0052] The present invention provides a composition that can be used for the prevention, improvement, or treatment of dementia and cognitive decline. More specifically, the present invention provides a composition that can be used for the prevention, improvement, or treatment of dementia and cognitive decline, comprising a complex of pine needle extract and galangal extract as an active ingredient.

[0053] In the present invention, the meaning of 'included as an active ingredient' means that the component is added to the composition according to the present invention to an extent that the component can exhibit the effect of preventing, improving, or treating dementia and cognitive decline, and in this process, it includes the meaning that it can be formulated in various forms through the addition of auxiliary ingredients other than the active ingredient for delivery to target tissues or stabilization of the composition.

[0054] In the composition according to the present invention, the pine needle extract and galangal extract may have a weight ratio of 1:1 to 1:8. Depending on the intended effect of the composition or the form of the product into which the composition is incorporated, the extract, concentrate, and complex may be included in the final form in a range of about 0.0001% to 99% or more, which may be calculated based on the weight relative to the total weight of the composition or the volume relative to the total volume of the composition.

[0055] The composition according to the present invention can be manufactured by mixing the pine needle extract and galangal extract prepared by the above-described method, and in addition, various processes and additives can be added for the purpose of improving the storability, distribution, and stability of the composition, within the limits that do not impede the purpose of the present invention.

[0056] The composition according to the present invention can be commercialized as a health functional food composition or a pharmaceutical composition.

[0057] The present invention provides a pharmaceutical composition comprising one of the above compositions. The present invention also provides a method for preventing, improving, or treating dementia and cognitive decline by administering one of the above compositions.

[0058] The pharmaceutical composition according to the present invention may contain a pharmaceutically effective amount of pine needle extract and galangal extract alone, or may additionally contain a carrier, excipient or diluent commonly used in the art.

[0059] The above 'pharmaceutically effective amount' refers to an amount that shows a greater response than the negative control group, and preferably refers to an amount sufficient to show a preventive and therapeutic effect on dementia and cognitive decline.

[0060] The above 'pharmaceutically acceptable' refers to a physiologically acceptable, non-toxic composition that does not inhibit the action of the active ingredient when administered to the human body and does not typically cause allergic reactions such as gastrointestinal disorders or dizziness or similar side effects.

[0061] The above carrier, excipient, and diluent may be raw materials commonly used in the industry as long as they do not impede the purpose of the present invention, and may additionally include various auxiliary raw materials such as fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives.

[0062] Additionally, the composition may be formulated using methods commonly known in the industry to achieve the desired effect using the active ingredient. This may include powders, granules, tablets, syrups, emulsions, aerosols, sterile injections, soft or hard capsules, etc.

[0063] The pharmaceutical composition of the present invention may be administered orally or parenterally, and the present invention is not limited thereto. When oral administration is included, the composition may be prepared by conventional means with pharmaceutically acceptable excipients, and may be formulated in a form suitable for sustained release, controlled release, sustained release, etc. to smoothly achieve the intended purpose. In the case of parenteral administration, various routes may be included, such as transdermal, nasal, intraperitoneal, subcutaneous, intramuscular, and intravenous, and these may also be prepared by conventional means with pharmaceutically acceptable excipients.

[0064] The preferred dosage of the pharmaceutical composition of the present invention may vary depending on the patient's condition, the degree of the disease, the form of the drug, the route of administration, and the period of administration, and therefore the concentration and dosage of the composition may be appropriately selected by a person skilled in the art.

[0065] The pharmaceutical composition of the present invention can be used alone or in combination with methods for preventing and treating dementia and cognitive decline commonly used in the industry.

[0066] The present invention provides a health functional food composition comprising one of the above compositions. The present invention provides a method for preventing, improving, or treating dementia and cognitive decline by administering one of the above compositions as a diet.

[0067] The health functional food composition according to the present invention may contain only a pine needle extract and galangal extract in a food-scientifically effective amount, or may additionally contain a carrier, excipient, or diluent acceptable in the art.

[0068] The health functional food composition according to the present invention includes all processed forms, including foods, functional foods, nutritional supplements, health foods, and food additives. The food composition can be manufactured in various forms according to conventional methods known in the art, and the form, type, etc. of the finished product are not limited.

[0069] The food composition according to the present invention can be added directly to food or used in combination with other foods or food ingredients, and can be used appropriately according to conventional methods. The amount of active ingredient mixed can be appropriately determined depending on the intended use (prevention, improvement, or treatment).

[0070] The food composition according to the present invention has no particular limitations on other ingredients other than the above-mentioned mixing ratio of the essential ingredients, pine needle extract and galangal extract, and may contain, like conventional products, various flavoring agents or natural carbohydrates, nutrients, vitamins, minerals, flavoring agents, coloring agents and enhancers, and other food additives for various purposes commonly used in the industry, as well as fruit pulp for the production of natural fruit and vegetable beverages.

[0071]

[0072] [Uses and treatment methods for preventing, improving, or treating dementia and cognitive decline]

[0073] The composition according to the present invention is effective in preventing, improving or treating dementia and / or cognitive decline.

[0074] Dementia is an irreversible intellectual disability caused by an underlying brain disorder. It refers to the loss of a person's mental (intellectual) abilities and social functioning. It occurs when previously developed intelligence is progressively deteriorated due to a brain disease such as a degenerative or cerebrovascular disease.

[0075] Cognitive decline, also known as cognitive disorder, refers to a state in which memory, attention, language ability, visuospatial ability, judgment, etc. decline. Cognitive decline is an intermediate stage between normal aging and dementia, and can progress to dementia. It is a comprehensive concept that includes everything from memory decline that accompanies the normal aging process to cognitive impairment that is a pre-dementia stage. 'Benign senescent forgetfulness', 'age-associated memory impairment', 'age-associated cognitive decline', and 'cognitive impairment-no dementia (CIND)' may also be included in the scope of cognitive decline of the present invention.

[0076] In a further aspect, the present invention provides food and medical uses of the complex and / or compositions comprising the complex. The complex and / or compositions comprising the complex are provided for use as foods, health functional foods, pharmaceuticals, and the like.

[0077] In some embodiments, a method of treating or preventing dementia and / or cognitive decline is provided, comprising administering to a subject in need thereof a complex of the invention, or a composition comprising the complex. In certain embodiments, such uses provide an effect of treating or ameliorating symptoms of dementia and / or cognitive decline in at least 5%, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70% or more, of subjects administered the complex and / or composition. For example, the improved effect may include an improvement in individual abilities such as memory, attention, language, visuospatial skills, judgment, as well as an improvement in social skills. In certain embodiments, the use of the present invention is for adults aged 65 years or older, but is not particularly limited to age, and adults suffering from early-onset dementia, vascular dementia, etc., or adults suffering from cognitive impairment at a young age may also be included within the scope of the present invention.

[0078] Administration of the above complex and / or composition may be oral, sublingual, intravenous, intramuscular, intradermal, transdermal or subcutaneous, and preferably oral.

[0079] In one specific example, depending on the degree of disease of the subject, the complex and / or composition of the present invention may be provided in an adhesive formulation such as a microneedle patch or a transdermal patch.

[0080] In one specific example, a combination of a pine needle extract and a galangal extract and / or a composition comprising the combination is provided for use in a food or pharmaceutical for preventing, improving, or treating dementia and / or cognitive decline. Preferably, the combination of the pine needle extract and the galangal extract is provided by mixing the galangal extract with the pine needle extract in a weight ratio of 1:1 to 1:8 (pine needle extract: galangal extract).

[0081]

[0082] Preventing, ameliorating, or treating dementia and / or cognitive decline involves, for example, preventing the full development of dementia and / or cognitive decline in individuals at risk for such conditions. "Treatment" refers to a therapeutic intervention that improves the signs or symptoms of a disease or pathological condition after they have already developed. The term "amelioration" refers to a reduction in the severity of some or all clinical symptoms of the disease, a delay in the onset of clinical symptoms, or a slow progression of the disease. "Prevention" can be understood as reducing the risk of developing pathological symptoms in individuals who do not show signs of the disease or who show only early signs.

[0083] The selection of a specific effective dosage can be determined by those skilled in the art in consideration of several factors, including the disease to be treated or prevented, related symptoms, the subject's medical history, the subject's physical condition such as age, weight, and / or immune status, the administered composition, and other factors known to those skilled in the art. The dosage to be used in a preparation comprising the complex of the extracts of the present invention and / or a composition containing the complex may vary depending on the subject's age, weight, sex, dosage form, health condition, and degree of disease, and may be administered once or several times a day at regular intervals at the discretion of a physician or pharmacist. For example, the daily dosage may be 0.001 to 2 g / kg, preferably 0.5 to 500 mg / kg, based on the active ingredient content. The above dosage is an example of an average case, and the dosage may be higher or lower depending on individual differences.

[0084]

[0085] In one embodiment, the subject requiring this is a mammalian subject, for example a human subject.

[0086]

[0087] More specifically, it was confirmed that the complex and / or composition according to the present invention has anti-inflammatory activity by inhibiting the activity of acetylcholinesterase, an enzyme that hydrolyzes the neurotransmitter acetylcholine, and at the same time inhibiting the production of nitric oxide, an inflammatory mediator, in raw 264.7 cells, a mouse-derived macrophage cell line, in which an inflammatory response was induced with lipopolysaccharide (LPS).

[0088]

[0089] Hereinafter, the present invention will be described in detail with examples and the like to aid understanding. However, the examples according to the present invention can be modified in various other ways, and the scope of the present invention is not limited to the examples below. The examples of the present invention are provided to more fully explain the present invention through the level of technology generally known in the art. Unless otherwise specified, the mixing ratio of each component is understood to be a weight ratio.

[0090]

[0091] Example

[0092] <Manufacturing Example 1> Manufacturing of pine needle and galangal extracts

[0093] 1. Preparation of pine needle extract

[0094] For the production of pine needle extract, dried raw materials were purchased through a domestic distributor and used. The dried pine needles were quantified, washed, and placed in an extractor at a weight ratio of raw material:solvent = 1:10. The extraction process was repeated at 60℃ ± 10% for 6 hours, for a total of two extractions. 50% ethanol was used as the solvent. The pine needle extract was filtered and concentrated under reduced pressure to obtain a concentrate of 30 brix (± 5 brix), which was then dried. The dried powder was homogenized to produce the pine needle extract to be used in the present invention.

[0095]

[0096] 2. Preparation of galangal extract

[0097] For the production of galangal extract, dried raw material was purchased through a domestic distributor and used. The dried galangal was quantified, washed, and placed in an extractor at a weight ratio of raw material:solvent = 1:10. The extraction process was repeated twice in total at 100°C ± 10% for 3 hours. Water was used as the solvent. The galangal extract was filtered and concentrated under reduced pressure to obtain a concentrate of 20 brix (± 5 brix), which was then dried. The dried powder was homogenized to produce the galangal extract to be used in the present invention.

[0098]

[0099] 3. Composite manufacturing

[0100] The pine needle extract and galangal extract manufactured in the above process were mixed as follows and used as test substances (Table 1: Preparation of composite).

[0101] Pine needle extract (weight %) Galanga extract (weight %) Sodium phosphate buffer (weight %) Complex ratio (pine needle : galangal) Example 188.9 11.1-8 : 1 Example 280.0 20.0-4 : 1 Example 366.7 33.3-2 : 1 Example 450.0 50.0-1 : 1 Example 533.3 66.7-1 : 2 Example 620.0 80.0-1 : 4 Example 711.1 88.9-1 : 8 Control Example 111.1-88.9- Control Example 2-88.9 11.1-

[0102] <Test Example 1> Confirmation of the content of test substances

[0103] 1. Check the ingredient content of pine needle extract

[0104] The pine needle extract prepared from the manufacturing example was analyzed using high-performance liquid chromatography to determine the content of major components of the pine needle extract.

[0105] More specifically, to prepare a standard solution, an appropriate amount of standard material was dissolved in methanol to make 0.5 mg / mL to prepare a standard stock solution. The standard stock solution was dissolved by shaking, then diluted with methanol to prepare a standard solution. To prepare a test solution, each extract was precisely weighed, methanol was added, and ultrasonic extraction was performed. The solution was filtered through a 0.22 to 0.45 um PTFE or PVDF membrane filter to prepare a test solution.

[0106] Under the high-performance liquid chromatography analysis conditions, the detector used was an ultraviolet-visible spectrophotometer (measuring wavelength 254 nm), and the column was a stainless steel tube with an inner diameter of 4 to 6 mm and a length of 15 to 25 cm, filled with 5 to 10 μm octadecylsilyl silica gel for liquid chromatography. The column temperature was a constant temperature around 30°C, and the mobile phases were controlled stepwise or in a concentration gradient manner using mobile phase A and mobile phase B. Mobile phase A was set to 0.1% phosphoric acid aqueous solution, and mobile phase B was set to methanol.

[0107] As a result, protocatechuic acid was identified as the main component in the pine needle extract, and the protocatechuic acid content in the pine needle extract manufactured according to the above manufacturing example was confirmed to be in the range of 0.05 to 0.35 mg / g (Table 2: Protocatechuic acid content in pine needle extract).

[0108] Content of protocatechuic acid (mg / g) measured 10.05 measured 20.35 measured 30.26

[0109] 2. Check the ingredient content of galangal extract

[0110] To determine the content of major components of the galangal extract prepared from the manufacturing example, the galangal extract was analyzed using high-performance liquid chromatography.

[0111] More specifically, to prepare a standard solution, the appropriate amount of standard substance was dissolved in 50% methanol to make 0.5 mg / mL to prepare a standard stock solution. The standard stock solution was dissolved by shaking, then diluted with 50% methanol to prepare a standard solution. To prepare a test solution, each extract was precisely weighed, methanol was added, and ultrasonic extraction was performed. The solution was filtered through a 0.22 to 0.45 um PTFE or PVDF membrane filter to prepare a test solution.

[0112] Under the high-performance liquid chromatography analysis conditions, the detector used was an ultraviolet-visible spectrophotometer (measuring wavelength 254 nm), and the column was a stainless steel tube with an inner diameter of 4 to 6 mm and a length of 15 to 25 cm, filled with 5 to 10 μm octadecylsilyl silica gel for liquid chromatography. The column temperature was a constant temperature around 40°C, and the mobile phases were controlled stepwise or in a concentration gradient manner using mobile phase A and mobile phase B. Mobile phase A was set to be a 2% phosphoric acid aqueous solution, and mobile phase B was set to be methanol.

[0113] As a result, 4-hydroxybenzaldehyde was confirmed as the main component in the pine needle extract, and the 4-hydroxybenzaldehyde content in the pine needle extract manufactured according to the above manufacturing example was confirmed to be in the range of 0.04 - 0.20 mg / g (Table 3: 4-hydroxybenzaldehyde content in galangal extract).

[0114] Category 4 - Hydroxybenzaldehyde content (mg / g) Measurement 10.048 Measurement 20.152 Measurement 30.193

[0115] <Test Example 2> Evaluation of the acetylcholinesterase inhibitory activity of test substances

[0116] In order to evaluate the efficacy of the test substance manufactured from the examples on cognitive decline, the inhibitory ability of acetylcholinesterase, an enzyme that hydrolyzes acetylcholine, a neurotransmitter, was evaluated using the modified Ellman method.

[0117] More specifically, each test substance manufactured from the examples, tacrine as a positive control, and sodium phosphate buffer (pH 8.0) as a control were dispensed 10 ㎕ into each well of a 96-well microplate, and then 120 ㎕ of sodium phosphate buffer, 10 ㎕ of 3 mM ATCI, and 30 ㎕ of 10 mM DTNB reagent were dispensed into the wells in sequence and reacted at room temperature for 15 minutes. After that, 20 ㎕ of AChE enzyme was dispensed into the experimental group and 20 ㎕ of sodium phosphate buffer into the blank group, reacted for 30 minutes, and then the absorbance was measured at 412 nm using a microplate reader. The result was expressed as the inhibition rate compared to the control group, as shown in Equation 3 below, and the suspension was corrected through the absorbance measured at 880 nm (Equation 1), and the color was corrected using the blank group (Equation 2).

[0118]

[0119] [Formula 1]

[0120] Suspension correction = A 412 -A 880

[0121] A 412 : absorbance at 412 nm after incubation

[0122] A 880 : absorbance at 880 nm after incubation

[0123] [Formula 2]

[0124] Color Correction = A test -A blank

[0125] A test : turbidity-adjusted absorbance of test well

[0126] A blank : turbidity-adjusted absorbance of blank well

[0127] [Formula 3]

[0128] Inhibition rate (%) = {1 - (abs / abc)} x 100

[0129] abs = turbidity and color-adjusted absorbance of test well after incubation

[0130] abc = turbidity and color-adjusted absorbance of control well after incubation

[0131]

[0132] The expected efficacy of the complex was calculated using Colby's formula (Formula 4), and the synergistic effect was compared with the measured value, and is shown in Table 4 (Table 4: Acetylcholinesterase inhibitory activity of test substances). In order to calculate the predicted acetylcholinesterase inhibitory activity of the complex using Colby's formula, the acetylcholinesterase inhibitory activity of pine needle extract and galangal extract alone according to the mixing ratio of each example was measured and applied to the calculation.

[0133]

[0134] [Formula 4]

[0135] E = X + Y - (X x Y) / 100

[0136] X: Inhibition rate when compound A is used at a concentration of a

[0137] Y: Inhibition rate when compound B is used at concentration b

[0138]

[0139] Classification Predicted value (%) Actual value (%) Synergy effect compared to predicted value (%) Example 191.2280.60- Example 275.2778.023.65 Example 351.0866.4930.17 Example 432.0335.4110.55 Example 532.8643.0130.89 Example 638.4256.4546.93 Example 747.0372.0553.20 Control example 1-31.75- Control example 2-22.39-

[0140] As a result of the experiment, it was confirmed that the actual measured value was higher than the value predicted by the Colby equation in all examples except Example 1, and that there was a synergistic effect. Among them, a remarkable synergistic effect was confirmed in Examples 3, 5, 6, and 7.

[0141] <Test Example 3> Cytotoxicity Evaluation of Test Substances

[0142] To evaluate the cytotoxicity of the test substance prepared from the examples, cell viability was evaluated in the human-derived neural cell line SH-SY5Y.

[0143] More specifically, SH-SY5Y cells cultured in a separate culture plate were harvested and 2 x 10 6 After inoculating the cells / well into a 96-well plate, the cells were cultured for 24 hours, treated with the test substance, and reacted for an additional 24 hours. Afterwards, the WST reagent was treated, cultured for 3 hours, and cell viability was evaluated by measuring the absorbance at 450 nm using a microplate reader, which is shown in Table 5 (Table 5: Cytotoxicity Evaluation of Test Substances).

[0144] Cell viability (%) Pine needle extract 12 ug / ml 93.17 ± 9.5 125 ug / ml 84.97 ± 9.6 450 ug / ml 77.90 ± 7.92 Galangal extract 25 ug / ml 102.81 ± 5.2 450 ug / ml 98.10 ± 1.05 100 ug / ml 95.29 ± 3.81

[0145] As a result of the experiment, the galangal extract showed almost no cytotoxicity even at a concentration of 100 μg / ml, but the pine needle extract showed concentration-dependent cytotoxicity. In addition, in the complex of the present invention, when the galangal extract was more than half of the weight of the complex, it was confirmed that there was almost no cytotoxicity even at a concentration of 100 μg / ml. Therefore, test substances having the compositions of Examples 4 to 7 in Table 1 were used in the cell test.

[0146]

[0147] <Test Example 4> Evaluation of the anti-inflammatory effect of the test substance

[0148] To evaluate the anti-inflammatory effect of the test substance prepared from the examples, the inhibition response of the production of nitric oxide, an inflammatory mediator, was evaluated in raw 264.7 cells, a mouse-derived macrophage cell line, in which an inflammatory response was induced with lipopolysaccharide (LPS).

[0149] More specifically, raw 264.7 cells cultured in a separate culture plate were harvested and 5 x 10 4 After inoculating the cells / well into a 96-well plate and culturing for 24 hours, the test substance was treated at a concentration of 100 μg / ml and reacted for an additional 24 hours. Afterwards, the cells were stimulated for 24 hours by treating with lipopolysaccharide at a concentration of 10 ng / ml, and the supernatant was treated with Griess reagent and reacted for 30 minutes. The nitric oxide production in the cell supernatant was evaluated by measuring the absorbance at 540 nm using a microplate reader. The results are expressed as the inhibition rate compared to the stress group induced by inflammatory response through LPS (Table 6: Inhibitory ability of test substances to inhibit nitric oxide production).

[0150]

[0151] [Formula 5]

[0152] Inhibition rate (%) = {1 - (abt / abs)} x 100

[0153] abt = absorbance of test well after incubation

[0154] abs = absorbance of stress well after incubation

[0155] NO generation inhibition ability (%) Example 44.58 ± 0.90 Example 55.01 ± 0.58 Example 613.71 ± 0.91 Example 720.03 ± 2.45

[0156] As a result of the experiment, inhibition of nitric oxide production was confirmed in all examples, and excellent NO production inhibition activity was confirmed in examples 6 and 7.

[0157]

[0158] <Test Example 4-1> Evaluation of concentration-dependent anti-inflammatory effect of test substance

[0159] In order to evaluate the concentration-dependent anti-inflammatory effect of Example 7, which was confirmed to have the highest synergistic effect in Test Example 2, the inhibition response of the production of nitric oxide, an inflammatory mediator, was evaluated in raw 264.7 cells, a mouse-derived macrophage cell line, in which an inflammatory response was induced with lipopolysaccharide (LPS).

[0160] More specifically, raw 264.7 cells cultured in a separate culture plate were harvested and 5 x 10 4 After seeding the cells / well in a 96-well plate, the cells were cultured for 24 hours, and then treated with test substances at various concentrations for an additional 24 hours. Afterwards, the cells were stimulated for 24 hours by treating with lipopolysaccharide at a concentration of 10 ng / ml, and the supernatant was treated with Griess reagent and reacted for 30 minutes. After that, the nitric oxide production in the cell supernatant was evaluated by measuring the absorbance at 540 nm in a microplate reader. The results were expressed as the inhibition rate compared to the stress group that induced an inflammatory response through LPS (Fig. 1).

[0161]

[0162] [Formula 6]

[0163] Inhibition rate (%) = {1 - (abt / abs)} x 100

[0164] abt = absorbance of test well after incubation

[0165] abs = absorbance of stress well after incubation

[0166]

[0167] As shown in Fig. 1, when the complex of pine needle extract and galangal extract of Example 7 was treated, nitric oxide production was suppressed in a concentration-dependent manner, confirming that the test substance has excellent anti-inflammatory activity.

[0168]

[0169] <Experimental Example 5> Evaluation of the Cognitive Function Improvement Effect of Test Substances - Y Maze Experiment

[0170] To evaluate the cognitive function improvement effect of the test substance manufactured from the examples, cognitive function was evaluated through a Y maze experiment using mice in which an Alzheimer's disease-like state was induced by intracerebroventricular beta-amyloid injection.

[0171]

[0172] [Laboratory animals]

[0173] Forty-eight 8-week-old male mice (C57BL / 6N) weighing 20 ± 2.2 g were divided into 6 groups of 8 mice each, and were designated as the normal group, the induced group, the pine needle extract:galangal extract 1:1 administration group (Example 4), the pine needle extract:galangal extract 1:4 administration group (Example 6), the pine needle extract:galangal extract 1:8 administration group (Example 7), and the positive control group. Beta-amyloid was administered to the remaining 5 groups, excluding the normal group, to induce an Alzheimer's disease-like state.

[0174]

[0175] [Beta-amyloid administration]

[0176] Beta-amyloid was dissolved in sterile 0.1 M phosphate-buffered saline (pH 7.4) at a concentration of 1 μg / μl and stored at 37°C for 1 week before administration to induce aggregation, and then administered into the third ventricle of the brain. The experimental animals were anesthetized with avertin, and 5 μl of the aggregated beta-amyloid solution was administered intracerebral at a rate of 2 μl / min using a stereotaxic apparatus.

[0177]

[0178] [Test substance administration]

[0179] The test substance was administered orally via direct gastrointestinal administration using a syringe equipped with a sonde for three weeks starting from the day of beta-amyloid injection. To facilitate administration, 0.5% carboxymethylcellulose was used as an excipient. The normal and induced groups were administered only the excipient without the test substance (Table 7: Test substance and dosage by test group).

[0180] Test groupAdministration substanceAdministration amount(mg / kg / day)Administration amount(ml / kg / day)Normal group (NC)Vessel-10Induction group (AB)Vessel-10AB-induced+ 1:1 administration groupPine needles:galangal 1:1(Example 4)10010AB-induced+ 1:4 administration groupPine needles:galangal 1:4(Example 6)10010AB-induced+ 1:8 administration groupPine needles:galangal 1:8(Example 7)10010AB-induced+positive control groupMorin1010

[0181] [Y-maze experiment] A Y-maze test was conducted to examine the spatial cognitive ability of laboratory animals. The Y-maze consists of three identical passages (A, B, C: 6 cm wide, 30 cm long, and 20 cm high, respectively) with the ends of the Y shaped block. The laboratory animals were carefully placed at one end of one passage and allowed to acclimate for 2 minutes, after which they were allowed to freely wander the Y passage for 8 minutes. During this time, if their body entered the space beyond the first 10 cm, it was recognized as entering the passage. The number of times and the order in which they entered each passage were measured by a camera installed on the ceiling, and a video tracking device was used to evaluate spontaneous alternation. Spontaneous alternation was defined as entering the three passages sequentially, i.e., ABC, BCA, CAB, etc. Therefore, the % spontaneous alternation was calculated using the following formula.

[0182]

[0183] Formula 7.

[0184] % spontaneous alternation

[0185] = Actual number of changes / (Total number of entries - 2) × 100

[0186]

[0187] As a result of the above experiment, it was confirmed that there was no significant difference in the number of times of entering and exiting the maze when comparing the normal group, induced group, test substance administration group, and positive control group. However, it was confirmed that the spontaneous alternation (spontaneous alternation, %) was significantly reduced in the induced group compared to the normal group, and the group administered pine needles: galangal 1:8 complex (Example 7) showed a result that exceeded the normal group when compared to the induced group, confirming a significant cognitive function improvement effect (39.21% improvement compared to the induced group). The pine needles: galangal 1:1 complex administration group (Example 4) and the pine needles: galangal 1:4 complex administration group (Example 6) showed an excellent cognitive function improvement effect with an improvement of 16.73% and 11.59% in spontaneous alternation, respectively, compared to the induced group (Table 8: spontaneous alternation, Fig. 2).

[0188] Test group Spontaneous cross-over behavior (%) Effect (compared to induction group) Normal group (NC) 65.55 ± 4.81 - Induction group (AB) 50.39 ± 12.66 - Induction + 1:1 administration group (Example 4) 58.82 ± 11.13 16.73% Improvement Induction + 1:4 administration group (Example 6) 56.23 ± 5.48 11.59% Improvement Induction + 1:8 administration group (Example 7) 70.15 ± 11.72 39.21% Improvement Induction + Morin 70.68 ± 9.32 -

[0189]

[0190] The complex and / or composition of the present invention has a treatment, prevention, and improvement effect on dementia, and provides a prevention, improvement, or treatment effect on cognitive decline, and can be provided as a health functional food or pharmaceutical.

Claims

1. A composition for preventing, improving or treating dementia and / or cognitive decline, characterized in that it comprises a complex of pine needle extract and galangal extract as an active ingredient.

2. A composition for preventing, improving or treating dementia and / or cognitive decline, characterized in that in paragraph 1, the pine needle extract is obtained by extracting pine needles with water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof and concentrating the extracted pine needles.

3. A composition for preventing, improving or treating dementia and / or cognitive decline, characterized in that in paragraph 1, the galangal extract is obtained by extracting galangal with water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof and concentrating the extract.

4. A composition for preventing, improving or treating dementia and / or cognitive decline, characterized in that the pine needle extract or galangal extract in the first paragraph is a concentrate or dry powder.

5. A composition for preventing, improving or treating dementia and / or cognitive decline, characterized in that the complex of the pine needle extract and galangal extract in the first paragraph is a mixture of the pine needle extract and the galangal extract in a weight ratio of 1:1 to 1:8 (pine needle extract: galangal extract).

6. In the first paragraph, the complex of pine needle extract and galangal extract is a composition for preventing, improving or treating dementia and / or cognitive decline characterized by anti-inflammatory action, inhibition of acetylcholinesterase and improvement of cognitive decline caused by beta-amyloid.

7. A composition for preventing, improving or treating dementia and / or cognitive decline, characterized in that the pine needle extract in paragraph 1 contains 0.05 to 0.35 mg / g of protocatechuic acid.

8. A composition for preventing, improving or treating dementia and / or cognitive decline, characterized in that the galangal extract in paragraph 1 contains 0.04 to 0.20 mg / g of 4-hydroxybenzaldehyde.

9. A composition according to any one of claims 1 to 8, characterized in that the composition is a health functional food composition for preventing or improving dementia and / or cognitive decline.

10. Health functional food containing the composition of Article 9.

11. A composition according to any one of claims 1 to 8, characterized in that the composition is a pharmaceutical composition.

12. A pharmaceutical product containing the composition of Article 11.

13. A method for improving or treating dementia and / or cognitive decline in an individual by administering an effective amount of a complex of pine needle extract and galangal extract to the individual in need thereof.

14. A method according to claim 13, wherein the complex of pine needle extract and galangal extract is characterized in that the galangal extract is mixed with the pine needle extract in a weight ratio of 1:1 to 1:8 (pine needle extract: galangal extract).

15. Use of a combination of pine needle extract and galangal extract in food or medicine for preventing, improving or treating dementia and / or cognitive decline.

16. In the 15th paragraph, the use is characterized in that the complex of the pine needle extract and the galangal extract is mixed with the pine needle extract in a weight ratio of 1:1 to 1:8 (pine needle extract: galangal extract).

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