Composition for preventing, improving, or treating memory disorder or cognitive dysfunction comprising Piper nigrum extract

Abstract

The present invention relates to a composition for the prevention, improvement, or treatment of memory impairment or cognitive impairment comprising a black pepper (Piper nigrum) extract, and more specifically, to a composition for the prevention, improvement, or treatment of memory impairment or cognitive impairment induced by scopolamine comprising a black pepper 70% (v / v) ethanol extract as an active ingredient. It was confirmed that the black pepper extract of the present invention, particularly the black pepper ethanol extract, contains a high content of piperine and effectively inhibits the inflammatory response induced in LPS-stimulated microglia without cytotoxicity compared to Indian pepper (Piper longum) or black pepper methanol extract. Furthermore, the black pepper extract of the present invention showed memory-enhancing effects in the Y-maze test and passive avoidance test using mice with memory impairment induced by scopolamine, and since the BDNF-CREB pathway activation effect was confirmed, it can be usefully utilized as a composition for the prevention, improvement, or treatment of memory impairment or cognitive impairment induced by scopolamine.

Description

Technology Field

[0001] The present invention is black pepper ( Piper nigrum The present invention relates to a composition for the prevention, improvement, or treatment of memory impairment or cognitive impairment comprising an extract, and more specifically, to a composition for the prevention, improvement, or treatment of memory impairment or cognitive impairment induced by scopolamine comprising a 70% (v / v) ethanol extract of black pepper as an active ingredient. Background Technology

[0003] Microglia, which are phagocytes in the brain, can induce chronic neuroinflammation by generating superoxide and neurotoxins, and the activators and neurotoxic substances produced by microglia are directly related to the pathological progression of diseases induced by neuroinflammation, such as Parkinson's disease.

[0004] In particular, microglia activation is linked to the pathogenesis of multiple neurodegenerative disorders such as Parkinson's disease (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). It has also been demonstrated that it is associated with pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-12 (IL-12), interleukin-6 (IL-6), prostaglandin E2 (PGE2), and cyclooxygenase-2 (COX-2). Other toxins have been established as neurotoxins, such as lipopolysaccharide, 6-OHDA, and hydrogen peroxide (H2O2). These toxins have been reported to stimulate microglia to activate inflammatory pathways such as NF-κB, p38, MAPK, JNK, ERK, and IRF3 (Kim, DC, et al. , Molecules , 22(12):2130, 2017; He, X., et al. , Toxicology letters , 283: 58-68, 2018; Lim, H.-W.; et al. , Food and chemical toxicology , 72: 265-272, 2014; Cho, D.-Y.; et al. , Molecules , 21(12):1718, 2016). Therefore, regulating microglia activation can serve as an important therapeutic strategy to attenuate neuroinflammation.

[0006] Meanwhile, black pepper ( Piper NigrumIt is an evergreen vine of the Piperaceae family, order Piperales, and has been primarily used as an effective treatment for gonorrhea, menstrual cramps, tuberculosis, sleep disorders, respiratory infections, and chronic gastrointestinal pain. The problem to be solved

[0008] Accordingly, as a result of diligent efforts to develop an effective neuroinflammation inhibitor in the present invention, black pepper extract, particularly black pepper ethanol extract, contains not only a high content of piperine, but also Indian pepper ( Piper longum It was confirmed that it effectively suppresses the inflammatory response induced in LPS-stimulated microglia without cytotoxicity compared to ).

[0009] In addition, black pepper extract showed memory-enhancing effects in the Y-maze test and passive avoidance test using mice with scopolamine-induced memory impairment, and confirmed not only acetylcholinesterase inhibitory activity but also the BDNF (Brain-derived neurotrophic factor) - CREB (cAMP responsive element binding protein) pathway activation effect.

[0010] Accordingly, the present invention was completed by confirming that black pepper extract is effective not only in inhibiting neuroinflammation but also in improving memory and preventing dementia, attention deficit hyperactivity disorder (ADHD), and depression.

[0012] The objective of the present invention is to provide a composition for the prevention, improvement, or treatment of memory impairment or cognitive impairment caused by scopolamine, comprising black pepper extract as an active ingredient. means of solving the problem

[0014] In order to achieve the aforementioned purpose,

[0015] The present invention is black pepper ( Piper nigrumA pharmaceutical composition for the prevention or treatment of memory impairment or cognitive impairment induced by scopolamine, comprising a 70% (v / v) ethanol extract as an active ingredient,

[0016] The above 70% (v / v) ethanol extract of black pepper is prepared by adding 70% (v / v) ethanol to black pepper at a 10-fold (v / w) ratio and repeating the extraction process three times for 24 hours under vacuum and room temperature conditions, and then concentrating the recovered extract under reduced pressure and freeze-drying it.

[0017] The above black pepper extract provides a pharmaceutical composition for the prevention or treatment of memory impairment or cognitive impairment induced by scopolamine, characterized by increasing the expression of BDNF (brain-derived neurotrophic factor) in the hippocampus and increasing the phosphorylation ratio of CREB (cAMP response element-binding protein) (p-CREB / CREB).

[0018] In addition, the present invention (black pepper) Piper nigrum A health functional food composition for the prevention or improvement of memory impairment or cognitive impairment induced by scopolamine, comprising a 70% (v / v) ethanol extract as an active ingredient,

[0019] The above 70% (v / v) ethanol extract of black pepper is prepared by adding 70% (v / v) ethanol to black pepper at a 10-fold (v / w) ratio and repeating the extraction process three times for 24 hours under vacuum and room temperature conditions, and then concentrating the recovered extract under reduced pressure and freeze-drying it.

[0020] The above black pepper extract provides a health functional food composition for the prevention or improvement of memory impairment or cognitive impairment induced by scopolamine, characterized by increasing the expression of BDNF (brain-derived neurotrophic factor) in the hippocampus and increasing the phosphorylation ratio (p-CREB / CREB) of CREB (cAMP response element-binding protein).

[0022] According to a preferred embodiment of the present invention, the black pepper extract, the black pepper 70% ethanol extract, can inhibit the expression of iNOS and COX-2 in the hippocampus and cerebral cortex of the brain, and inhibit the phosphorylation and degradation of IκB-α.

[0023] According to another preferred embodiment of the present invention, the black pepper 70% ethanol extract may have lower cytotoxicity to microglia compared to the black pepper methanol extract.

[0024] According to another preferred embodiment of the present invention, the black pepper 70% ethanol extract may be included at a concentration of 50 to 100 μg / ml. Effects of the invention

[0026] The black pepper extract of the present invention, in particular the black pepper ethanol extract, contains a high content of piperine, and Indian pepper ( Piper longum It was confirmed that it effectively inhibits the inflammatory response induced in LPS-stimulated microglia without cytotoxicity compared to black pepper methanol extract.

[0027] Furthermore, the black pepper extract of the present invention showed memory-enhancing effects in the Y-maze test and passive avoidance test using mice with memory impairment induced by scopolamine, and since the BDNF-CREB pathway activation effect was confirmed, it can be usefully utilized as a composition for the prevention, improvement, or treatment of memory impairment or cognitive impairment induced by scopolamine. Brief explanation of the drawing

[0029] Figure 1 shows data representing the results of analyzing the piperine content contained in black pepper extract. Fig. 2 shows black pepper ( Piper nigrum This is data confirming the anti-inflammatory activity and cytotoxicity levels according to the extraction method of ). A is the hot water extract, B is the methanol extract, and C is the 70% ethanol extract. Fig. 3 shows Indian pepper ( Piper longum This is data confirming the anti-inflammatory activity and cytotoxicity levels according to the extraction method of ). A is the hot water extract, and B is the methanol extract. Figure 4 shows black pepper ( Piper nigrum This is data confirming the NO generation inhibition effect (A) and cytotoxicity (B) when treated with extracts according to extraction method at concentrations of 100 µg / ml, and the cytotoxicity (C) and NO generation inhibition effect (D) when treated with 70% ethanol black pepper extract at concentrations. Figure 5 shows the data confirming the expression levels of iNOS mRNA (A and B) and protein (C and D) when LPS-stimulated BV-2 microglia were treated with black pepper extract at different concentrations. A represents the iNOS mRNA expression level, B represents the relative expression level to GAPDH by quantifying the amount of iNOS mRNA amplification product, C represents the iNOS protein expression level, and D represents the relative expression level to β-actin by quantifying the amount of iNOS protein. Figure 6 shows the data confirming the expression levels of COX-2 mRNA (A and B) and protein (C and D) when LPS-stimulated BV-2 microglia were treated with black pepper extract at different concentrations. A represents the level of COX-2 mRNA expression, B represents the relative expression level to GAPDH by quantifying the amount of COX-2 mRNA amplification product, C represents the level of COX-2 protein expression, and D represents the relative expression level to β-actin by quantifying the amount of COX-2 protein. Figure 7 shows the cytoplasmic p65 concentration and the degree of IκB-α phosphorylation when LPS-stimulated BV-2 microglia were treated with black pepper extract at different concentrations. A and B represent the degree of inhibition of IκB-α phosphorylation and degradation, C and D represent the cytoplasmic p65 concentration, and E and F represent the nuclear p65 concentration. B, D, and F represent the relative expression levels relative to β-actin, obtained by quantifying the amount of each protein. Figure 8 shows the phosphorylation levels of IRF3, p38, JNK, and ERK when LPS-stimulated BV-2 microglia were treated with black pepper extract at different concentrations. A and B represent the phosphorylation levels of IRF3, C and D represent the phosphorylation levels of JNK, E and F represent the phosphorylation levels of ERK, and G and H represent the phosphorylation levels of p38. B, D, F, and H represent the phosphorylation levels of proteins relative to the total protein amount. Figure 9 shows the results of a passive avoidance test when black pepper extract was administered to mice whose memory was impaired by scopolamine. Figure 10 shows the results of the Y-maze test when black pepper extract was administered to mice with impaired memory scopolamine. A represents the total number of arm entries, and B represents the spontaneous alteration score (%). Figure 11 shows the concentrations of inhibitory activity on acetylcholinesterase (AChE, B) in the hippocampus (A) and cerebral cortex of the brain when black pepper extract was administered at different concentrations to mice with impaired memory from scopolamine. Figure 12 shows data confirming the levels of iNOS and COX-2 mRNA expression when black pepper extract was administered at different concentrations to mice with memory impaired by scopolamine. A represents the levels of iNOS and COX-2 mRNA expression, while B and C represent the relative expression levels relative to GAPDH by quantifying the amount of each amplification product. Figure 13 shows data confirming the degree of IκB-α phosphorylation when black pepper extract was treated at different concentrations in mice with memory impaired by scopolamine. A represents the degree of inhibition of IκB-α phosphorylation and degradation, and B represents the relative expression level for β-actin by quantifying the amount of amplification product. Figure 14 shows data confirming the protein expression levels of BDNF (Brain-derived neurotrophic factor) and CREB (cAMP-responsive element binding protein) when black pepper extract was administered at different concentrations to mice with memory impaired by scopolamine. A represents the phosphorylation level of CREB and the protein expression level of BDNF, B represents the phosphorylation level of CREB relative to the total protein expression level, and B represents the relative expression level of BDNF relative to β-actin. Specific details for implementing the invention

[0030] The present invention will be described in more detail below.

[0032] Black pepper extract manufacturing

[0033] In order to extract a black pepper extract that has excellent effects of inhibiting neuroinflammation and improving memory impairment and cognitive function impairment, in a specific embodiment of the present invention, water (hot water), methanol, and 70% ethanol were each used as extraction solvents to obtain a black pepper extract.

[0034] In order to determine the content of active substances contained in black pepper extract, the piperine content was checked as an indicator component. As shown in Figure 1 and Table 2, it was confirmed that the piperine content in the ethanol extract was 6 to 8 times higher than that in the hot water extract.

[0035] In addition, the black pepper used in the present invention ( Piper nigrum To determine the difference between ) and other species, Indian pepper ( Piper longum A comparative experiment was performed with the extract.

[0036] As shown in Figure 2, the black pepper extract showed weak anti-inflammatory efficacy in the hot water extract, and the methanol extract showed anti-inflammatory efficacy at a concentration of 25 μg / ml, but exhibited high cytotoxicity at higher concentrations. On the other hand, the 70% ethanol extract was found to effectively inhibit NO production without cytotoxicity at a concentration of 100 μg / ml.

[0037] In the case of Indian pepper, as shown in Figure 3, the hot water extract did not show significant anti-inflammatory efficacy, while the methanol extract showed anti-inflammatory efficacy at a concentration of 200 μg / ml.

[0038] However, it was confirmed that the black pepper extract of the present invention has a superior NO generation inhibitory effect compared to Indian pepper, and among the black pepper extracts, the 70% ethanol extract, which has no cytotoxicity, was found to have the best anti-inflammatory efficacy.

[0040] Composition for the prevention or treatment of neuroinflammatory diseases

[0041] In one aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of neuroinflammatory diseases comprising black pepper extract as an active ingredient.

[0042] In another aspect, the present invention relates to a health functional food composition for the prevention or improvement of neuroinflammatory diseases comprising black pepper extract as an active ingredient.

[0043] In the present invention, the black pepper extract is characterized by being extracted with ethanol, preferably with 70% (v / v) ethanol.

[0044] In the present invention, the black pepper extract is

[0045] i) Inhibition of the production of inflammatory mediators, including NO, iNOS, and COX-2;

[0046] ii) inhibition of the nuclear translocation of NF-κB subunit p65, inhibition of IκB-α phosphorylation and degradation; and

[0047] iii) characterized by regulating neuroinflammation through inhibition of the TLR4 downstream pathway by inhibition of p38, JNK, ERK, and IRF3 phosphorylation.

[0048] In the present invention, the neuroinflammatory disease is characterized by being selected from the group consisting of multiple sclerosis, neuroblastoma, ischemic stroke, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, Creutzfeldt-Jakob disease, post-traumatic stress disorder, depression, attention deficit hyperactivity disorder (ADHD), schizophrenia, or amyotrophic lateral sclerosis.

[0050] In a specific embodiment of the present invention, when BV-2 microglia were pretreated with black pepper extract (100 μg / ml) at various concentrations of extraction solvents and then stimulated with LPS, inhibition of NO production and cytotoxicity were confirmed. As a result, as shown in Fig. 4A, it was confirmed that the inhibition of NO production by the black pepper extract in the 70% ethanol extract (100 μg / ml) was superior compared to other extraction solvents, and there was no cytotoxicity. Accordingly, in the present invention, the degree of NO production and cytotoxicity were confirmed once again by setting the concentration of the black pepper extract to 0.1 μg / ml, 10 μg / ml, and 100 μg / ml. As a result, as shown in Figs. 4C and 4D, it was confirmed that NO production was effectively inhibited in a concentration-dependent manner, and as shown in Fig. 4C, it was confirmed that no cytotoxicity was observed by the black pepper extract using 70% ethanol as the extraction solvent.

[0051] In another specific embodiment of the present invention, we sought to confirm the effects of black pepper extract on the inhibition of the TLR4 downstream pathway through iNOS and COX-2 mRNA and protein expression levels, NF-κB (p65) activity, IκBα phosphorylation / degradation, and inhibition of p38, JNK, ERK, and IRF3 phosphorylation in LPS-stimulated BV-2 microglia.

[0052] As shown in Figures 5 and 6, it was confirmed that the expression levels of iNOS and COX-2 mRNA and protein in LPS-stimulated BV-2 microglia were inhibited in a concentration-dependent manner of black pepper extract.

[0053] Phosphorylation / degradation of IκB-α and nuclear translocation of the p65 subunit to the nucleus are essential for NF-κB activation, and the NF-κB signaling pathway is activated by LPS-induced pro-inflammatory cytokines in BV-2 cells. Furthermore, NF-κB plays a crucial role in the LPS-induced expression of iNOS, COX-2, and pro-inflammatory agents. Therefore, to understand the mechanism of neuroinflammation inhibition by black pepper extract, the degree of p65 translocation and IκB-α phosphorylation / degradation in LPS-stimulated BV-2 cells was investigated.

[0054] As shown in Figures 7A and 7B, it was confirmed that IκB-α phosphorylation and degradation were significantly attenuated by black pepper extract in LPS-stimulated BV-2 cells. Additionally, as shown in Figures 7C to 7F, it was confirmed that cytoplasmic p65 concentrations increased in a concentration-dependent manner, while nuclear p65 concentrations decreased. This implies that the translocation of p65 subunits is blocked by black pepper extract. In other words, the inhibition of NF-κB activation by black pepper extract may cause the inhibition of NO, iNOS, and other pro-inflammatory cytokines in LPS-stimulated BV2 microglia.

[0055] In addition, the present invention investigated the major TLR4 downstream signaling pathway induced by LPS to confirm the mechanism by which black pepper extract inhibits the production of pro-inflammatory mediators and cytokines. Stimulation of BV2 microglia with LPS activates various defense mechanisms, including IRF3, and IRF3 regulates the expression of iNOS and other cytokines.

[0056] Toll-like receptor 4 (TLR4) activates innate immune signaling amplified through MyD88-dependent and MyD88-independent signaling pathways, and TLR4 is activated by LPS. Activated TLR4 induces the initial activation and nuclear translocation of NF-κB through MyD88-dependent signaling, and induces the activation of mitogen-activated protein kinases (MAPKs). The activation of NF-κB and MAPKs leads to the secretion of inflammatory cytokines such as TNF-α, IL-1β, and IL-6.

[0057] As shown in Figure 8, it was confirmed that the phosphorylation of p38, JNK, ERK, and IRF3 was inhibited in a concentration-dependent manner with black pepper extract, which means that the TLR4 downstream pathway is inhibited by black pepper extract, thereby inhibiting the production of pro-inflammatory mediators and cytokines.

[0059] Composition for the prevention or treatment of memory impairment and cognitive dysfunction

[0060] In another aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of memory impairment and cognitive dysfunction comprising black pepper extract as an active ingredient.

[0061] In another aspect, the present invention relates to a health functional food composition for preventing or improving memory impairment and cognitive function impairment, comprising black pepper extract as an active ingredient.

[0062] The present invention is characterized in that acetylcholinesterase (AChE) activity in the hippocampus and cerebral cortex of the brain is inhibited by the black pepper extract.

[0063] The present invention is characterized by the activation of the BDNF (Brain-derived neurotrophic factor)-CREB (cAMP responsive element binding protein) pathway by the black pepper extract.

[0064] In the present invention, the cognitive impairment is characterized by being selected from the group consisting of mild cognitive impairment, attention deficit hyperactivity disorder (ADHD), depression, Alzheimer's disease, and senile dementia.

[0066] In a specific embodiment of the present invention, black pepper extract was administered to an animal model of cognitive impairment induced by scopolamine to confirm the effect of black pepper extract on preventing or treating memory impairment and cognitive dysfunction.

[0067] It is known that the administration of scopolamine leads to the degeneration of cholinergic neuronal function and amnesia, as well as deficits in learning ability, immediate response, and working memory (Smith, 1988; Giacobini, E. and Cuadra, G. (1994)). In patients with Alzheimer's disease, abnormalities occur in almost all neurons, including cholinergic, adrenergic, GABAergic, and glutamatergic neurons, but damage and loss of cholinergic neurons have been identified as particularly severe. Meanwhile, senile dementia, a disease characterized by the progressive loss of cognitive ability, is also associated with the activity of cholinergic neurons in the central nervous system, and this is known to be primarily caused by a significant decrease in the activity of acetylcholine and choline acetyltransferase in the brain.

[0068] In another specific embodiment of the present invention, a passive avoidance test was performed to confirm the effect of black pepper extract on memory enhancement. As a result, as shown in Figure 9, regarding the latency time in the bright room during the passive avoidance behavior test, it was observed that the scopolamine-treated group had a latency time similar to the initial acquisition stage due to memory impairment, while the group administered black pepper extract showed a concentration-dependent increase in latency time. In particular, it was confirmed that the group administered black pepper extract at 50 mg / kg showed a latency time similar to that of the tacrine-treated group (positive control).

[0069] In another specific embodiment of the present invention, a Y-maze test was performed to confirm the effect of black pepper extract on memory enhancement. As a result, as shown in Figure 10, the scopolamine-treated group showed low total arm entry and spontaneous alteration scores (%) due to memory impairment, whereas the group administered black pepper extract showed levels similar to the positive control group.

[0070] In another specific embodiment of the present invention, it was confirmed whether black pepper extract inhibits the activity of acetylcholinesterase increased by scopolamine. Scopolamine is known to overactivate the activity of acetylcholinesterase (AChE), and acetylcholinesterase degrades acetylcholine, which is known as a neurotransmitter for memory, thereby causing it to lose its function.

[0071] As shown in Figure 11, when the production of acetylcholinesterase (AChE) in the hippocampus and cerebral cortex of the brain was measured, it was confirmed that the AChE concentration increased rapidly in the scopolamine-treated group, whereas the AChE concentration decreased in a concentration-dependent manner in the black pepper extract-treated group. In particular, it was confirmed that the group administered 50 mg / kg of black pepper extract had an AChE concentration similar to that of the tacrine-treated group (positive control).

[0072] In another specific embodiment of the present invention, it was confirmed whether black pepper extract inhibits the expression of inflammatory mediators known to affect cognitive decline. As a result, as shown in Fig. 12, it was confirmed that the expression of iNOS and COX-2 in the hippocampus and cerebral cortex of the brain was inhibited by the administration of black pepper extract, and as shown in Fig. 13, it was confirmed that the phosphorylation and degradation of IκB-α were inhibited.

[0073] In another specific embodiment of the present invention, it was confirmed whether the BDNF (Brain-derived neurotrophic factor)-CREB (cAMP responsive element binding protein) pathway is activated by black pepper extract. BDNF (brain-derived neurotrophic factor) is one of the nerve growth factor family and plays an important role in the regulation of neurotransmitters or neuroplasticity, and is known to be associated with dementia, Alzheimer's disease, depression, etc. In addition, CREB (cAMP response element-binding protein) is one of the transcription factors associated with depression and dementia and plays an important role in BDNF gene expression, which is regulated by the upstream signaling molecule ERK (extracellular signal regulated kinase).

[0074] As shown in Figure 14, it was confirmed that BDNF levels increased and the p-CREB to CREB ratio (p-CREB / CREB) was high in the hippocampus of mice administered black pepper extract, and that the group administered black pepper extract at 50 mg / kg showed results similar to the tacrine-treated group (positive control).

[0075] In other words, the black pepper extract of the present invention showed memory-enhancing effects in the Y-maze test and passive avoidance test using mice with memory impairment induced by scopolamine, and confirmed not only acetylcholinesterase inhibitory activity but also BDNF-CREB pathway activation effect. Therefore, it was confirmed that the black pepper extract of the present invention can be usefully utilized in pharmaceutical compositions or health functional foods for the prevention or treatment of memory impairment and cognitive dysfunction.

[0077] The pharmaceutical compositions of the present invention may be formulated into various forms according to conventional methods and used. For example, they may be formulated into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, and may be formulated into external preparations, suppositories, and sterile injectable solutions. Depending on each formulation, they may further include pharmaceutically acceptable carriers, excipients, and diluents. Additionally, they may be formulated into external preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, and sterile injectable solutions according to conventional methods and used.

[0078] The above carriers, excipients, and diluents include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. When the above pharmaceutical composition is formulated or prepared, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0079] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc., with the above composition. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid dosage forms for oral administration include suspensions, oral liquids, emulsions, syrups, etc., and may include various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, etc. As non-aqueous solvents and suspending agents, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As bases for suppositories, Witepsol, Macrogol, Tween 61, cocoa paste, laurin paste, glycerogelatin, etc. may be used.

[0080] The term "administration" as used in the present invention means providing the pharmaceutical composition of the present invention to an individual by any appropriate method. The pharmaceutical composition of the present invention may be administered at a therapeutically effective dose, which is the amount of the active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal, or human as conceived by a researcher, veterinarian, physician, or other clinician, i.e., an amount that induces the alleviation of symptoms of the disease or disorder being treated. It is obvious to those skilled in the art that the therapeutically effective dose and frequency of administration for the pharmaceutical composition of the present invention will vary according to the desired effect. Therefore, the optimal dose to be administered can be easily determined by those skilled in the art and may be adjusted according to various factors including the type of disease, the severity of the disease, the content of the active ingredient and other ingredients contained in the composition, the type of formulation, the patient's age, body weight, general health condition, gender and diet, the time of administration, the route of administration and the secretion rate of the composition, the duration of treatment, and concurrently used drugs. The pharmaceutical composition of the present invention may be administered to an individual by various routes. For example, it may be administered intravenously, intraperitoneally, intramuscularly, intra-arterially, orally, intracardiaclysmically, intramedullaryly, transdermally, intestinally, subcutaneously, sublingually, or topically, but is not limited thereto. The pharmaceutical composition of the present invention may be administered in an amount of 1 to 10,000 mg / kg / day, and may be administered once a day or divided into several doses.

[0082] The health functional food composition of the present invention may be used as a health functional food, a food additive, or a dietary supplement. When the black pepper extract of the present invention is used as a food additive, it may be appropriately used according to conventional methods, such as by adding it as is or by mixing it with other foods or food ingredients.

[0083] In addition, the mixing amount of the above-mentioned health functional food composition may be appropriately changed according to the purpose of use (prevention, health, or therapeutic treatment). As a specific example, when manufacturing food or beverages, the black pepper extract of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less, relative to the raw material. However, when consumed for a long period for the purpose of health and hygiene or health control, it may be added in an amount below the above range, and since there are no issues regarding safety, the active ingredient may also be used in an amount above the above range.

[0084] There are no special restrictions on the types of food mentioned above, but examples of food to which the black pepper extract of the present invention can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, etc., and include all health foods in the conventional sense.

[0085] When the health functional food composition of the present invention is manufactured into a beverage, it may include additional ingredients such as various flavoring agents or natural carbohydrates, as in conventional beverages. The natural carbohydrates may include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; natural sweeteners such as dextrin and cyclodextrin; and synthetic sweeteners such as saccharin and aspartame. The natural carbohydrates are included in an amount of 0.01 to 10% by weight, preferably 0.01 to 0.1% by weight, based on the total weight of the food composition of the present invention.

[0086] The health functional food composition of the present invention may include various nutritional agents, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc., and may include fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages, but is not limited thereto. These ingredients may be used independently or in combination. Although the proportion of the above additives is not significantly limited, it is preferable that they be included in the range of 0.01 to 0.1 weight percent with respect to the total weight of the food composition of the present invention.

[0088] The present invention will be explained in more detail below through examples.

[0089] These examples are solely for illustrating the invention, and it will be obvious to those skilled in the art that the scope of the invention is not to be interpreted as being limited by these examples.

[0091] Confirmation of NO generation and cell viability according to pepper type and extraction solvent

[0092] 1-1: Preparation of Black Pepper Extract

[0093] In the present invention, black pepper ( Piper nigrum To prepare the extract, water (hot water), ethanol (70%), and methanol were used.

[0094] First, to prepare a hot water extract, 10 times the amount of hot water at 50°C was added to dried black pepper, and then vacuum refluxed for 24 hours to obtain the extract. The extraction process was repeated 3 times, and the recovered extract was concentrated to obtain a concentrate, which was then freeze-dried to prepare a black pepper hot water extract.

[0095] For the ethanol extract, 70% ethanol was added to dried black pepper in tenfold amounts, and the extract was stored in a vacuum at room temperature for 24 hours. The extraction process was repeated three times, and the recovered extract was concentrated to obtain a concentrate, which was then freeze-dried to prepare the black pepper ethanol extract.

[0096] The methanol extract was prepared by adding 100% methanol to dried black pepper in a tenfold ratio and storing the mixture under vacuum at room temperature for 24 hours. The extraction process was repeated three times, the recovered extract was concentrated, and the resulting concentrate was freeze-dried to produce the black pepper methanol extract.

[0098] 1-2: Checking Indicator Component Content

[0099] To measure the content of active ingredients contained in the black pepper extract obtained above, the piperine content was measured as an indicator component.

[0100] Piperine content was measured using HPLC, and the analysis was performed under isocratic mobile phase conditions using a C18 (YMC 2.1 mm X 100 mm, 2.7 μm) HPLC column. The specific analysis conditions are as shown in Table 1 below.

[0102] Piperine content analysis conditions Instrument Agilent Technologies 6410 Triple Quad (LC-MS / MS) Column YMC triart-C18 (2.1 mm × 100 mm, 2.7 ㎛) Solvent A; 0.1% Formic acid in Water, B; 0.1% Formic acid in Acetonitrile Flow rate 0.4 mL / min Injection vol. 5 ㎕ Gradient 0 min : 5% B solvent30min : 100% B solvent32min : 100% B solvent

[0104] Piperine content Sample area Concentration (ppm) Piperine content per mg of sample Ethanol extract 530,541,466 100ppm 655.50 ug Hot water extract 377,132,403 500ppm 91.62 ug

[0105] As shown in Figure 1 and Table 2, it was confirmed that the piperine content in the ethanol extract was significantly higher than that in the hot water extract.

[0107] Confirmation of the NO generation inhibitory effect of black pepper extract

[0108] In order to confirm the neuroinflammatory inhibitory efficacy of black pepper extract, the degree of NO generation inhibition and cytotoxicity were determined, and the black pepper used in this invention ( Piper nigrumTo determine the difference between ) and other species, Indian pepper ( Piper longum A comparative experiment was performed with the extract.

[0109] For the black pepper extract, the hot water extract, ethanol extract, and methanol extract of Example 1 above were used, and for the Indian pepper, the hot water extract and methanol extract were prepared in the same way as in Example 1.

[0111] 2-1: Confirmation of Cell Viability According to Black Pepper Extract Concentration

[0112] A known method for measuring the degree of cytotoxicity according to the concentration of black pepper extract (Kim, JJ, et al. , Journal of Ethnopharmacology BV-2 cell viability was measured according to , 140:213-221, 2012).

[0113] BV-2 microglia were obtained by a known method (Kim, BW et al. , PLoS One, 8:e55792. 2013). Briefly, cells were cultured in DMEM (Dulbecco's modified Eagle's medium; Gibco / Invitrogen; USA) supplemented with 5% FBS (Fetal bovine serum; PAA Laboratories Inc.; Canada) and 50 μg / ml penicillin-streptomycin at 37°C and 5% CO2.

[0114] First, 3 × 10 BV-2 microglia per well 5 Cells were dispensed into 6-wells at a concentration of cells / mL and cultured for 6 hours. Then, black pepper extract and Indian pepper extract were added at concentrations of 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL, respectively, and pretreated for 1 hour. Afterward, LPS (200 ng / mL), a neuroinflammatory inducer, was applied, and BV-2 microglia were stimulated for 24 hours.

[0115] After 24 hours, the supernatant was removed, and DMEM medium containing MTT (500 μg / mL) was added to each well and incubated at 37°C for 4 hours. After incubation, the supernatant was removed, and DMSO (dimethyl sulfoxide); Compound Diluent (1%) Vehicle Control, Sigma, USA) was added to each well to dissolve formazan. Optical density was measured at 550 nm using a microplate reader (Tecan Trading AG, Switzerland), and the value was compared with the HTB derivative untreated group (control group).

[0116] In the examples, all data were analyzed using Graph Pad Prism version 5.01 (Graph Pad, Inc., USA). All data were expressed as the mean ± standard error of at least three independent experiments performed in vitro or, in the case of histological and biochemical experiments, in triplicate.

[0118] 2-2: Confirmation of NO generation inhibitory effect according to black pepper extract concentration

[0119] To confirm the neuroinflammation inhibitory activity of black pepper extract, the efficacy of inhibiting nitrite (NO) generation was verified.

[0120] Measurement of cell viability and NO concentration of BV2 microglia was performed at 3.0 × 10⁶ cells per well. 5 Cells were dispensed into a 6-well plate to a concentration of cells / mL, and then black pepper extract and Indian pepper extract were added at concentrations of 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL, respectively, and pretreated for 1 hour. Afterward, LPS (200 ng / mL), a neuroinflammatory inducer, was applied, and BV2 melanocytes were stimulated for 24 hours.

[0121] Nitrite (NO) was measured using a NO assay kit (Abcam) with Griess reagent, and the measurement method was carried out according to the kit's instructions. 100 µl of cell culture medium and Griess reagent were mixed and reacted for 10 minutes, and the absorbance was observed at 540 nm. The concentration was finally confirmed using a standard curve.

[0123] As a result, as shown in Figure 2, the black pepper extract showed weak anti-inflammatory efficacy in the hot water extract, and the methanol extract showed anti-inflammatory efficacy at a concentration of 25 μg / ml, but exhibited high cytotoxicity at higher concentrations. On the other hand, the 70% ethanol extract was found to effectively inhibit NO production without cytotoxicity at a concentration of 100 μg / ml.

[0124] In the case of Indian pepper, as shown in Figure 3, the hot water extract did not show significant anti-inflammatory efficacy, while the methanol extract showed anti-inflammatory efficacy at a concentration of 200 μg / ml.

[0125] However, it was confirmed that the black pepper extract of the present invention has a superior NO generation inhibitory effect compared to Indian pepper, and among the black pepper extracts, the 70% ethanol extract, which has no cytotoxicity, was found to have the best anti-inflammatory efficacy.

[0126] Therefore, in subsequent experiments, the inhibitory effect on neuroinflammation, memory enhancement, and cognitive function improvement of black pepper ethanol extract were confirmed.

[0128] Effects of Black Pepper Extract on Inhibition of NO Generation and Cell Viability in LPS-Stimulated BV-2 Microglia Depending on Extraction Solvent

[0129] 3-1: Confirmation of NO generation inhibitory effect and cell viability depending on black pepper extract solvent

[0130] To confirm the neuroinflammatory inhibitory activity of black pepper extract, an experiment was conducted in the same manner as in Example 2-2 above. Each extraction solvent was prepared by dissolving the extract in hot water (Dw), methanol (Mt), 50% ethanol (50% Et), 70% ethanol (70% Et), and 70% ethanol in water, then centrifuging the supernatant (Sup) and precipitate to treat the black pepper extract to a concentration of 100 μg / ml.

[0131] As a result, as shown in Figure 4A, it was confirmed that the extract of black pepper using 70% ethanol solvent and the supernatant after centrifugation of the 70% ethanol extract effectively inhibited NO generation, and as shown in Figure 4B, cytotoxicity was confirmed in the precipitate after centrifugation of the 70% ethanol extract, but no cytotoxicity was confirmed by the black pepper extract using 70% ethanol as the extraction solvent and the supernatant after centrifugation of the 70% ethanol extract.

[0133] 3-2: Confirmation of NO generation inhibitory effect and cell viability according to black pepper extract concentration

[0134] To confirm the neuroinflammation inhibitory activity of black pepper extract, an experiment was conducted in the same manner as in Example 2-2 above, and black pepper extract was applied at concentrations of 1 μg / ml, 10 μg / ml, and 100 μg / ml, respectively.

[0135] As a result, as shown in Figure 4C, it was confirmed that black pepper extract effectively inhibited NO generation in a concentration-dependent manner, and as shown in Figure 4D, it was confirmed that no cytotoxicity was observed from the black pepper extract.

[0137] Effects of black pepper extract on iNOS and COX-2 mRNA and protein expression levels in LPS-stimulated BV-2 microglia

[0138] 4-1: Confirmation of iNOS and COX-2 mRNA expression levels

[0139] 3 × 10 BV-2 microglia per well 5Cells were seeded into 6-well plates at a rate of cells / well and cultured overnight. Then, black pepper ethanol extract was added at concentrations of 1 µg / ml, 10 µg / ml, and 100 µg / ml, respectively, and the cells were pretreated for 1 hour. Subsequently, BV-2 melanocytes were stimulated for 24 hours with the neuroinflammatory inducer LPS (200 ng / ml), and then total RNA was isolated using TRIzol reagent (Invitrogen Life Technologies, USA). 2.5 µg of total RNA was reverse transcribed using the Superscript™ III kit (Invitrogen), and then cDNA was amplified by PCR using specific primers (Table 3) according to known methods.

[0141] PCR primer sequences and reaction conditions Primer sequence (5'-3') Sequence number Product size (bp) Annealing temperature (°C) cycling iNOS F: 5'- GAG GTA CTC AGC GTG CTC CA-3' 1 444 56 27 R: 5'-AGG GAG GAA AGG GAG AGA GG-3' 2 COX-2 F: 5'-TGA GTG GTA GCC AGC AAA GC-3' 3 319 56 27 R: 5'-CTG CAG TCC AGG TTC ATG G-3' 4 GAPDH F: 5'-ACC ACA GTC CAT GCC ATC AC-3' 5 472 57 25 R: 5'-CCA CCA CCC TGT TGC TGT AG-3' 6

[0142] PCR was performed with denaturation (94°C, 5 min), 20–28 amplification cycles (30 seconds at 94°C, 1 min at 50–57°C, and 1 min at 72°C), and extension (5 min at 72°C), and PCR amplification products were analyzed using a 1% agarose gel. GAPDH mRNA serves as an internal control for sample loading and mRNA integrity. For quantification, the gel was imaged, and the pixel intensity for each band was determined using Image J (NIH). Then, the mRNA expression level for each factor was normalized using the GAPDH mRNA band intensity.

[0144] 4-2: Confirmation of iNOS and COX-2 Protein Expression Levels

[0145] BV-2 microglia were cultured in the same manner as in Example 4-1 above.

[0146] Cells were washed twice with PBS and stored at 4°C. They were then treated with lysis buffer (1 RIPA lysis buffer, protease inhibitor cocktail, and phosphatase inhibitor cocktail) and lysed for 10 minutes. The lysate was centrifuged at 4°C and 14,000 rpm, and the supernatant was collected for analysis. Equal amounts of protein (20 mg or 40 mg) were electrophoresed on a 10% SDS-PAGE gel and transferred to a PVDF membrane (polyvinylidenediuoride membranes; Millipore, Bedford, USA).

[0147] To prevent non-specific binding, the transferred PVDF membrane was reacted for 1 hour using PBS containing 5% skim milk, and then treated with primary antibodies: anti-iNOS antibody (1:1000; Cell Signaling Technology, USA), anti-COX-2 antibody (1:1000; Santa Cruz Biotechnology, USA), and anti-β-actin antibody (1:2000; Sigma-Aldrich, USA), and reacted overnight at 4°C. Next, after washing with PBS at least three times, a secondary antibody (1:4000; Mouse and Goat, Santa Cruz, USA; Rabbit, Cell Signaling Technology, USA) conjugated with horseradish peroxidase (HRP) was applied and reacted at room temperature for 1 hour. The membranes after the reaction were completed were imaged using an ECL detection kit and a luminescent image analyzer (LAS-3000, USA).

[0149] As a result, as shown in Figures 5 and 6, it was confirmed that the expression levels of iNOS and COX-2 mRNA and protein in LPS-stimulated BV-2 microglia were inhibited in a concentration-dependent manner of black pepper extract.

[0151] Effects of Black Pepper Extract on NF-κB (p65) Activity and IκBα Phosphorylation / Degradation in LPS-Stimulated BV-2 Microglia

[0152] To confirm the effects of black pepper extract on NF-κB (p65) activity and IκBα phosphorylation / degradation, cells were cultured in the same manner as in Example 3-2 above, and then proteins were obtained and Western blot was performed.

[0153] Whole cell lysates were used as samples to analyze the degree of IκBα phosphorylation / degradation, while cytoplasmic and nuclear lysates were used as samples to analyze p65 activity, respectively.

[0154] 40 µg of each sample was electrophoresed on a 10% SDS-PAGE gel, and the separated proteins were transferred to a PVDF membrane (Millipore; USA). To block non-specific binding, the PVDF membrane was incubated with PBS containing 5% skim milk for 1 hour. Primary antibodies—anti-NF-κB antibody, anti-IκBα antibody, and anti-p-IκBα antibody (1:1000; Cell Signaling Technology, USA) and anti-β-actin antibody (1:2000; Sigma-Aldrich, USA)—were added and incubated overnight at 4°C. Next, the membrane was washed at least three times with PBS, then treated with a secondary antibody (1:4000; Mouse and Goat, Santa Cruz, USA; Rabbit, Cell Signaling Technology, USA) conjugated with horseradish peroxidase (HRP) and reacted at room temperature for 1 hour. The membrane was then imaged using an ECL detection kit and a luminescent image analyzer (LAS-3000, USA).

[0156] As shown in Figures 7A and 7B, it was confirmed that IκB-α phosphorylation and degradation were significantly attenuated by black pepper extract in LPS-stimulated BV-2 cells. Additionally, as shown in Figures 7C to 7F, it was confirmed that cytoplasmic p65 concentration increased in a concentration-dependent manner, while nuclear p65 concentration decreased. This implies that the translocation of p65 subunits is blocked by black pepper extract.

[0157] In other words, this means that NF-κB activation is inhibited by black pepper extract, which may cause the inhibition of NO, iNOS, and other pro-inflammatory cytokines in LPS-stimulated BV2 microglia.

[0159] Effects of black pepper extract on the TLR4 downstream signaling pathway in LPS-stimulated BV-2 microglia

[0160] In this invention, the major TLR4 downstream signaling pathway induced by LPS was investigated to confirm the mechanism by which black pepper extract inhibits the production of pro-inflammatory mediators and cytokines.

[0161] Cells were cultured in the same manner as in Example 4-2 above, and proteins were obtained and Western blotting was performed. 40 µg of total cell lysate was electrophoresed on a 10% SDS-PAGE gel, and the separated proteins were transferred to a PVDF membrane (Millipore; USA). To block non-specific binding, the PVDF membrane was incubated with PBS containing 5% skim milk for 1 hour. Primary antibodies, including anti-JNK antibody, anti-p-JNK antibody, anti-p38, anti-p-p38 antibody, anti-ERK antibody, anti-p-ERK antibody, anti-IRF3 antibody, anti-p-IRF3 antibody (1:1000; Cell Signaling Technology, USA), and anti-β-actin antibody (1:2000; Sigma-Aldrich, USA), were added and incubated overnight at 4°C. Next, the membrane was washed at least three times with PBS, then treated with a secondary antibody (1:4000; Mouse and Goat, Santa Cruz, USA; Rabbit, Cell Signaling Technology, USA) conjugated with horseradish peroxidase (HRP) and reacted at room temperature for 1 hour. The membrane was then imaged using an ECL detection kit and a luminescent image analyzer (LAS-3000, USA).

[0162] As shown in Figure 8, it was confirmed that the phosphorylation of p38, JNK, ERK, and IRF3 was inhibited in a concentration-dependent manner with black pepper extract, which means that the TLR4 downstream pathway is inhibited by black pepper extract, thereby inhibiting the production of pro-inflammatory mediators and cytokines.

[0164] Passive avoidance test

[0165] In the present invention, to confirm the effect of black pepper extract on preventing or treating memory impairment and cognitive dysfunction, black pepper extract was administered to an animal model of cognitive impairment induced by scopolamine.

[0166] Male C57BL / 6 mice (25 g, 8 weeks old) were purchased from Daehan Biolink. They were divided into five groups and acclimatized under standard conditions for one week before being used in experiments (temperature 22±2 ℃, humidity 55±5 %, 12 h-light / dark cycle, free food / water). All experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee (NIH publication No. 85-23, revised 1985) and the Konkuk University IACUC. Mice were divided into five groups of six, and the black pepper extract and scopolamine administered to the mice were dissolved in physiological saline.

[0167] 1) vehicle (untreated group, negative control group),

[0168] 2) Scopolamine 2 mg / kg treatment group

[0169] 3) Scopolamine 2 mg / kg + Black Pepper Extract 25 mg / kg Treatment Group

[0170] 4) Scopolamine 2 mg / kg + Black Pepper Extract 50 mg / kg Treatment Group

[0171] 5) Scopolamine 2 mg / kg + tacrine treatment group (positive control)

[0173] Black pepper extract was orally administered to mice at a dose of 25 mg / kg or 50 mg / kg daily for 7 days of scopolamine treatment, and scopolamine 2 mg / kg was administered via intraperitoneal injection.

[0174] Passive avoidance experiments were conducted to evaluate memory. For the training and testing of passive avoidance behavior, an active and passive avoidance system from GEMINI (San Diego Instruments, San Diego, CA, USA), consisting of two identical light and dark square boxes, was used. This device features a light chamber and a dark chamber, separated by a computer-controlled door. After placing the mouse in the light chamber, light was applied to guide it to the dark chamber, and an electric current was passed through it to allow the mouse to remember this sequence. Specifically, when the mouse entered the dark chamber, the door automatically closed, and an electric foot shock of 0.5 mA was administered for 5 seconds. During this time, the step-through latency time of the animal in the light compartment was measured for up to 300 seconds. The latency was recorded and stored via a computer, and the electric shock and the opening and closing of the door were controlled by an attached device system.

[0175] As a result, as shown in Figure 9, in the passive avoidance behavior experiment, the scopolamine-treated group showed a latency time in a bright room similar to the initial acquisition stage due to memory impairment, while the group administered black pepper extract showed a concentration-dependent increase in latency time. In particular, the group administered 50 mg / kg of black pepper extract showed a latency time similar to that of the tacrine-treated group (positive control).

[0177] Y-maze test

[0178] In the present invention, a Y-maze test was performed to confirm the effect of black pepper extract on memory enhancement. Black pepper extract was administered to an animal model of cognitive impairment induced by scopolamine in the same manner as in Example 8, and the experiment was performed 30 minutes after the administration of scopolamine.

[0179] The Y-maze experiment apparatus consists of three arms (designated as A, B, and C). The passage length of each arm is 40 cm, the width is 12 cm, and the height is 30 cm. The angle at which the three arms meet adjacent arms is 120 degrees, and the entire experimental apparatus is made of polyvinyl plastic. A mouse was carefully placed in the center of the maze and allowed to move freely for 7 minutes. The arm the mouse entered was then recorded, but only if the mouse had entered completely up to its tail; repeated entry into an arm was also recorded. The number of times and the order of entry into each arm were measured to evaluate the mouse's total arm entry and spontaneous alteration score (%). Spontaneous alteration score was defined as entering the three arms sequentially, i.e., ABC, BCA, CAB, etc., and was calculated using the following Equation 1.

[0180] [Mathematical Formula 1]

[0181] % alteration = [Total number of alterations] / [Total number of times entered arm - 2] X 100

[0183] As a result, as shown in Figure 10, the scopolamine-treated group showed low total arm entry and spontaneous alteration scores, while the group administered black pepper extract showed improved memory and a level similar to the positive control group.

[0185] Efficacy of black pepper extract on acetylcholinesterase activity in an animal model of cognitive impairment induced by scopolamine

[0186] In the present invention, it was confirmed whether black pepper extract inhibits the activity of acetylcholinesterase increased by scopolamine. Black pepper extract was administered to an animal model of cognitive impairment induced by scopolamine using the same method as in Example 8 above. Then, 30 minutes after scopolamine administration, the animals were sacrificed, their brains were extracted, and the hippocampus and cerebral cortex were separated. Proteins were isolated from the hippocampus and cerebral cortex of the animal brains and then quantified using the BCA protein quantification method.

[0187] Acetylcholinesterase activity was measured at an absorbance of 410 nm using a UV microplate reader according to the manufacturer's test method using the Biochain Kit (Biochain, CA, USA).

[0189] As shown in Figure 11, when the production of acetylcholinesterase (AChE) in the hippocampus and cerebral cortex of the brain was measured, it was confirmed that the AChE concentration increased rapidly in the scopolamine-treated group, whereas the AChE concentration decreased in a concentration-dependent manner in the black pepper extract-treated group. In particular, it was confirmed that the group administered 50 mg / kg of black pepper extract had an AChE concentration similar to that of the tacrine-treated group (positive control).

[0191] Efficacy of black pepper extract on inflammatory proteins in an animal model of cognitive impairment induced by scopolamine

[0192] In this invention, it was confirmed whether black pepper extract inhibits the expression of inflammatory mediators known to affect cognitive decline.

[0193] Black pepper extract was administered to an animal model of cognitive impairment induced by scopolamine in the same manner as in Example 8 above, and then the animal was sacrificed 30 minutes after scopolamine administration, the brain was extracted, and the hippocampus and cerebral cortex were separated. Proteins were isolated from the hippocampus and cerebral cortex of the animal brain and then quantified using the BCA protein quantification method.

[0194] Equal amounts of protein (20 mg or 40 mg) were electrophoresed on a 10% SDS-PAGE gel and then transferred to a PVDF membrane (polyvinylidenediuoride membranes; Millipore, Bedford, USA). To prevent non-specific binding, the transferred PVDF membrane was incubated for 1 hour using PBS containing 5% skim milk, and then treated with primary antibodies: anti-iNOS antibody (1:1000; Cell Signaling Technology, USA), anti-COX-2 antibody (1:1000; Santa Cruz Biotechnology, USA), anti-IκBα antibody and anti-p-IκBα antibody (1:1000; Cell Signaling Technology, USA), and anti-β-actin antibody (1:2000; Sigma-Aldrich, USA), and incubated overnight at 4°C. Next, after washing with PBS at least three times, a secondary antibody (1:4000; Mouse and Goat, Santa Cruz, USA; Rabbit, Cell Signaling Technology, USA) conjugated with horseradish peroxidase (HRP) was applied and reacted at room temperature for 1 hour. The membranes after the reaction were completed were imaged using an ECL detection kit and a luminescent image analyzer (LAS-3000, USA).

[0196] As a result, as shown in Figure 12, it was confirmed that the expression of iNOS and COX-2 in the hippocampus and cerebral cortex of the brain was inhibited by the administration of black pepper extract, and as shown in Figure 13, it was confirmed that the phosphorylation and degradation of IκB-α were inhibited.

[0198] Efficacy of black pepper extract on cognitive function mechanisms in an animal model of scopolamine-induced cognitive impairment

[0199] In the present invention, it was confirmed whether the BDNF (Brain-derived neurotrophic factor)-CREB (cAMP responsive element binding protein) pathway is activated when black pepper extract is administered to an animal model of cognitive impairment induced by scopolamine.

[0200] Black pepper extract was administered to an animal model of cognitive impairment induced by scopolamine in the same manner as in Example 8 above, and then the animal was sacrificed 30 minutes after scopolamine administration, the brain was extracted, and the hippocampus and cerebral cortex were separated. Proteins were isolated from the hippocampus and cerebral cortex of the animal brain and then quantified using the BCA protein quantification method.

[0201] Equal amounts of protein (20 mg or 40 mg) were electrophoresed on a 10% SDS-PAGE gel and then transferred to a PVDF membrane (polyvinylidenediuoride membranes; Millipore, Bedford, USA). To prevent non-specific binding, the transferred PVDF membrane was incubated for 1 hour using PBS containing 5% skim milk, and then treated with primary antibodies: anti-BDNF antibody (1:1000; abcam), anti-CREB antibody (1:1000; abcam), anti-p-CREB antibody (1:1000; abcam), and anti-β-actin antibody (1:2000; Sigma-Aldrich, USA), and incubated overnight at 4°C. Next, after washing with PBS at least three times, a secondary antibody (1:4000; Mouse and Goat, Santa Cruz, USA; Rabbit, Cell Signaling Technology, USA) conjugated with horseradish peroxidase (HRP) was applied and reacted at room temperature for 1 hour. The membranes after the reaction were completed were imaged using an ECL detection kit and a luminescent image analyzer (LAS-3000, USA).

[0202] As a result, as shown in Figure 14, it was confirmed that BDNF levels increased in the hippocampus of mice administered black pepper extract and the p-CREB ratio to CREB (p-CREB / CREB) was high, and that the group administered black pepper extract at 50 mg / kg showed results similar to the tacrine-treated group (positive control).

[0203] In other words, the black pepper extract of the present invention showed memory-enhancing effects in the Y-maze test and passive avoidance test using mice with memory impairment induced by scopolamine, and confirmed not only acetylcholinesterase inhibitory activity but also BDNF-CREB pathway activation effect. Therefore, it was confirmed that the black pepper extract of the present invention can be usefully utilized in pharmaceutical compositions or health functional foods for the prevention or treatment of memory impairment and cognitive dysfunction.

Claims

Claim 1 Black pepper ( Piper nigrum A pharmaceutical composition for the prevention or treatment of memory impairment or cognitive impairment induced by scopolamine, comprising a 70% (v / v) ethanol extract as an active ingredient, wherein the black pepper 70% (v / v) ethanol extract is prepared by adding 70% (v / v) ethanol to black pepper at a 10-fold (v / w) ratio and repeating the process of extraction under vacuum and room temperature conditions for 24 hours three times, followed by vacuum concentration and freeze-drying the recovered extract, and wherein the black pepper extract is characterized by increasing the expression of BDNF (brain-derived neurotrophic factor) in the hippocampus and increasing the phosphorylation ratio (p-CREB / CREB) of CREB (cAMP response element-binding protein). Claim 2 A pharmaceutical composition for the prevention or treatment of memory impairment or cognitive impairment induced by scopolamine, wherein the black pepper 70% ethanol extract inhibits the expression of iNOS and COX-2 in the hippocampus and cerebral cortex of the brain, and inhibits the phosphorylation and degradation of IκB-α. Claim 3 A pharmaceutical composition for the prevention or treatment of memory impairment or cognitive impairment induced by scopolamine, wherein, in claim 1, the black pepper 70% ethanol extract has lower cytotoxicity to microglia compared to the black pepper methanol extract. Claim 4 A pharmaceutical composition for the prevention or treatment of memory impairment or cognitive impairment induced by scopolamine, characterized in that, in claim 1, the black pepper 70% ethanol extract is included at a concentration of 50 to 100 μg / ml. Claim 5 Black pepper ( Piper nigrum A health functional food composition for the prevention or improvement of memory impairment or cognitive impairment induced by scopolamine, comprising a 70% (v / v) ethanol extract as an active ingredient, wherein the black pepper 70% (v / v) ethanol extract is prepared by adding 70% (v / v) ethanol to black pepper at a 10-fold (v / w) ratio and repeating the process of extraction under vacuum and room temperature conditions for 24 hours three times, followed by vacuum concentration and freeze-drying the recovered extract, and wherein the black pepper extract is characterized by increasing the expression of BDNF (brain-derived neurotrophic factor) in the hippocampus and increasing the phosphorylation ratio (p-CREB / CREB) of CREB (cAMP response element-binding protein). Claim 6 A health functional food composition for the prevention or improvement of memory impairment or cognitive impairment induced by scopolamine, wherein, in claim 5, the black pepper 70% ethanol extract inhibits the expression of iNOS and COX-2 in the hippocampus and cerebral cortex of the brain, and inhibits the phosphorylation and degradation of IκB-α. Claim 7 A health functional food composition for the prevention or improvement of memory impairment or cognitive impairment induced by scopolamine, wherein, in claim 5, the black pepper 70% ethanol extract has lower cytotoxicity to microglia compared to the black pepper methanol extract. Claim 8 A health functional food composition for the prevention or improvement of memory impairment or cognitive impairment induced by scopolamine, characterized in that, in claim 5, the black pepper 70% ethanol extract is included at a concentration of 50 to 100 μg / ml.

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