KR102610305B1 - Composition for the prevention or treatment of neurodegenerative disease and cognitive dysfunction comprising bamboo leaf extract or bioactive compound, tricin - Google Patents

Abstract

The present invention relates to a health functional food composition for improving cognitive function or memory ability containing bamboo leaf extract or tricin as an active ingredient, and a composition for preventing or treating neurodegenerative diseases. More specifically, bamboo leaf or tricin is a glial cell. It effectively reduces neuroinflammation caused by and inhibits neuroinflammation in a mouse model of dopamine neuron death, effectively reducing neuron death. Bamboo leaf extract or tricine inhibits the activity of acetylcholinesterase, and scopolamine is administered. In memory disorder models, bamboo leaf extract improves antioxidant enzymes such as SOD, Cat, and GPx and suppresses lipid peroxides (malondealdehyde, MDA) and neuroinflammatory factors accumulated by oxidative stress in the hippocampus and cerebral cortex of experimental animals. Provides a composition that can improve memory and cognitive function or prevent or treat neurodegenerative diseases by confirming it in the (Cerebral cortex) tissue and analyzing behavioral experiments. Diseases to be treated by the present invention include Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, multiple sclerosis, mild cognitive impairment, and amnesia.

Description

Composition for preventing and treating neurodegenerative diseases and cognitive dysfunction containing bamboo leaf extract or tricin as an active ingredient {COMPOSITION FOR THE PREVENTION OR TREATMENT OF NEURODEGENERATIVE DISEASE AND COGNITIVE DYSFUNCTION COMPRISING BAMBOO LEAF EXTRACT OR BIOACTIVE COMPOUND, TRICIN}

The present invention relates to a health functional food composition for improving cognitive function or memory, containing bamboo leaf extract or tricin as an active ingredient, and a composition for preventing or treating neurodegenerative diseases. More specifically, it relates to the bamboo leaf extract or tree of the present invention. Sour effectively reduces neuroinflammation caused by glial cells and effectively reduces neuron death by suppressing neuroinflammation in experimental animal (mouse) models of dopamine neuron death. Bamboo leaf extract or tricine inhibits the activity of acetylcholinesterase. In a memory impairment model administered with scopolamine, bamboo leaf extract improves antioxidant enzymes such as SOD, Cat, and GPx and inhibits lipid peroxide (malondealdehyde, MDA) and neuroinflammatory factors accumulated by oxidative stress in experimental animals. Provides a composition that can improve memory and cognitive function or prevent or treat neurodegenerative diseases by confirming it in the tissues of the hippocampus and cerebral cortex and analyzing behavioral experiments.

It has been established that neuroinflammatory response is one of the main factors causing neurodegeneration. In other words, glial cells, which are immune cells present in the central nervous system, can be activated by various exogenous and endogenous substances, and activated glial cells produce inflammatory cytokines such as TNF-α and IL-1β, nitric oxide, prostaglandins, and superoxide. Produce and release substances (Gao et al., J Neurochem, 81, 1285-97, 2002; Nelson, PT. et al., Ann Med, 34, 491-500, 2002; Griffin, W.S. et al., J Neuroinflammation, 3, 5, 2006). The production of these substances triggers an immune response in the short term, but excessive or continuous production induces the death of nearby nerve cells, ultimately causing neurodegeneration.

Considering the importance of the neuroinflammation response in degenerative cranial nerve diseases, neuroinflammation and the degenerative cranial nerve diseases that may arise from it can be treated by reducing the expression level of pro-inflammatory mediators in glial cells. something to do.

Activated glial cells sequentially produce nitric oxide (NO), reactive oxygen species (ROS), inflammatory enzymes Inducible nitric oxidesynthase (iNOS), Cyclooxygenase-2 (COX-2), and pro-inflammatory mediators IL-1β and IL. It is reported to cause various central nervous system diseases such as multiple sclerosis, Parkinson's disease, Alzheimer's disease, and Huntington's disease by inducing the activity of -6 and TNF-α (Gao et al., 2002; Nelson et al., 2002; Eikelenboom) and Van Gool, 2004).

At this time, if pro-inflammatory mediators are reduced within glial cells, the expression of these diseases can be suppressed (Matsumoto et al., 1992; Liu and Hong, 2003). It is widely known through mechanistic studies that various pro-inflammatory factors and neurotoxins in glial cells induced by LPS in cell-based models contribute to nerve damage during the inflammatory process (Kim et al., 2004a,b). In addition, it has been reported that glial cells activated by LPS increase neurotoxicity due to pro-inflammatory mediators and cytotoxins (Dawson et al., 1994). Considering the importance of inflammatory responses in neuroinflammation, inhibiting the production of these inflammation-related factors in activated glial cells would be a goal for treating various diseases caused by neuroinflammation.

Cognitive dysfunction encompasses memory impairment, language impairment, visual-spatial ability impairment, perceptual impairment, and performance impairment. Cognitive impairment is a mental and behavioral impairment caused by brain disease, brain injury, addiction, aging, etc. and endocrine system abnormalities, metabolic and nutritional abnormalities, and mental diseases caused by drugs, etc., including amnesia, which is determined to be a temporary short-term memory disorder depending on the severity of the symptom, as well as loss of reasoning ability, forgetting, and learning. It is a type of brain disease that also occurs in neurodegenerative diseases such as cognitive dysfunction and dementia that cause disability, decreased concentration, decreased intelligence, and impaired judgment. Mild cognitive impairment refers to a condition in which cognitive ability is reduced compared to people of the same age. Cognitive areas such as memory, language ability, and judgment are deteriorated. As we become an aging society, the number of elderly people has increased rapidly. In this social environment, senile dementia and Parkinson's disease are recognized as degenerative central nervous system diseases, and as the number of patients increases day by day, social issues increase and prevention or improvement efforts are needed accordingly. and the importance of treatment is emerging day by day.

Meanwhile, senile dementia, a disease in which cognitive ability is gradually lost, is also related to the activity of cholinergic neurons in the central nervous system, and is mainly caused by a significant decrease in the activity of acetylcholine and choline acetyltransferase in the brain. It is known as Accordingly, the development of therapeutic agents is being actively conducted in the direction of recovery and improvement to complement the ideal cholinergic nerve. It is known that administration of scopolamine causes degeneration of cholinergic nerve function, memory loss, and defects in learning ability, immediate response, and working memory. (Smith, 1988; Giacobini, E. and Cuadra, G. (1994)).

Meanwhile, 12 to 13 species of bamboo grow naturally in Korea, including Wangdae, cotton bamboo, and bamboo bamboo. In oriental medicine, they include Phyllostachys Bambusoids S. et Z , Phyllostachys nigra var . Henesis Stapf, and Sasa borealis (Hackel). Makino), Sasa Kurilensis (Rupr.) Makino et al, Shibata), etc. are used together. Bamboo leaves (Bambusac Folium) belong to the Graminae family and refer to the leaves of the bamboo and bamboo genera. Bamboo leaves are composed of substances that are beneficial to the human body and have been used as herbal medicine since ancient times. Bamboo leaves have a bitter taste and are cold in nature, so they are effective in relieving cancer, tumors, fever, and conflict. They are also effective in relieving diarrhea, hematemesis, expectoration, stroke, diabetes, headaches, high blood pressure, dizziness, nervous breakdown, anemia of pregnancy, epilepsy, insomnia, excessive drinking, and fatigue. It is recorded to be effective in recovery, etc. Bamboo leaves have the effect of promoting biochemical metabolism in the body, so they are good for treating high blood pressure or stroke, and are known to have an anti-obesity effect because they contain a lot of essential unsaturated fatty acids, linoleic acid and linolenic acid.

Korean Patent Publication No. 2016-0024243 discloses a method for producing fermented bamboo leaf extract and a fermented bamboo leaf extract using the same, and Korean Patent Publication No. 1402936 discloses a composition containing bamboo leaf extract and golden extract showing obesity-suppressing activity, and Although its use has been disclosed, there has been no disclosure regarding a composition for improving, relieving, treating neuroinflammation and improving cognitive function containing the bamboo leaf extract of the present invention as an active ingredient.

Accordingly, the present inventors found that the bamboo leaf extract or the active ingredient tricin not only does not show toxicity to cells, but also significantly suppresses the increase in inflammatory cytokines in glial cells, effectively reduces neuroinflammation caused by glial cells, and increases dopaminergic nerve activity. In a mouse model of cell death, it effectively reduces neuronal death by suppressing neuroinflammation. Bamboo leaf extract or tricin inhibits the activity of acetylcholinesterase, and in a memory impairment model administered with scopolamine, bamboo leaf extract is used to treat SOD, Cat, Enhancing antioxidant enzymes such as GPx and suppressing lipid peroxides (malondealdehyde (MDA)) and neuroinflammatory factors accumulated by oxidative stress were confirmed in the hippocampus and cerebral cortex tissues of experimental animals and analyzed through behavioral experiments. Through this, the present invention, which is a composition that can improve memory and cognitive function or prevent or treat neurodegenerative diseases, was completed.

The present invention was made to solve the above problems, and the first object of the present invention is to provide a pharmaceutical composition containing bamboo leaf extract and tricin for the prevention or treatment of neurodegenerative diseases.

The second object of the present invention is to provide a food composition for improving memory and cognitive function containing bamboo leaf extract and tricin as active ingredients.

In order to solve the above-mentioned problems of the present invention, the present invention provides a pharmaceutical composition for preventing or treating neuroinflammatory diseases or improving memory and cognitive function containing bamboo leaf extract or tricin as an active ingredient.

According to a preferred embodiment of the present invention, the tricin may be extracted from bamboo leaves.

In addition, the bamboo leaf extract can inhibit the production of nitric oxide (NO) in nerve cells, and the bamboo leaf ethanol extract can be characterized by inhibiting the activity of acetylcholinesterase.

According to another preferred embodiment of the present invention, it is possible to induce improvement in cognitive function by suppressing scopolamine-induced neuroinflammation. The bamboo leaf extract can increase antioxidant enzymes, inhibit lipid peroxidation accumulation, and suppress the expression of neuroinflammatory factors in the hippocampus and cerebral cortex of mice injected orally with scopolamine, thereby improving cognitive function. The improvement effect can be confirmed through behavioral experiments.

According to another preferred embodiment of the present invention, the bamboo leaf extract may contain tricin at a concentration of 1 to 50 μg/ml in the total composition. According to one embodiment of the present invention, the tricin can reduce NF-κB activation by reducing phosphorylation of IκBα or p65 in nerve cells. In addition, the tricin can reduce mitogen-activated protein kinase (MAPK) activation by reducing phosphorylation of extracellular signal regulated kinase (ERK) or p38 MAP kinase (p38) in nerve cells.

According to another embodiment of the present invention, the tricin can inhibit neuroinflammation caused by MPTP, a neurotoxin.

According to a preferred embodiment of the present invention, the neuroinflammatory disease may be at least one selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, and multiple sclerosis.

According to another embodiment of the present invention, a food composition for improving neuroinflammatory disease or memory and cognitive function containing bamboo leaf extract or tricin as an active ingredient is provided.

According to a preferred embodiment of the present invention, the bamboo leaf extract may be extracted from bamboo leaves.

In addition, the bamboo leaf extract may contain tricin at a concentration of 1 to 50 μg/ml in the total composition, and the neuroinflammatory diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, and multiple sclerosis. It may be at least one selected disease.

The present invention also provides a food composition for improving cognitive dysfunction, comprising bamboo leaf extract or tricin as an active ingredient.

According to one embodiment of the present invention, the cognitive dysfunction may be at least one selected from the group consisting of amnesia, loss of reasoning ability, forgetting, learning disability, decreased concentration, and decreased intelligence, and may be a patient with memory loss. It can be targeted.

Additionally, the memory decline may be due to damage or loss of the cholinergic system.

The bamboo leaf extract of the present invention not only does not exhibit toxicity to cells, but also induces tumor necrosis factor-α (TNF-α; tumor necrosis factor-alpha), nitric oxide (NO), and nitric oxide in microglial cells activated by LPS. It significantly suppresses the increase in inflammatory cytokines such as interleukin-6 (IL-6) and reduces the phosphorylation of MAPK (mitogen-activated protein kinases) and the activity of NF-κB (nuclear factor-kappaB). It effectively suppressed the activity of microglial cells related to neuroinflammation. In addition, bamboo leaf extract inhibited the activity of acetylcholinesterase, suppressed antioxidant enzymes induced by scopolamine, accumulated lipid peroxidation, and increased neuroinflammation, and improved memory and spatial cognition. Tricine, the active ingredient, inhibits the activity of acetylcholinesterase and neuroinflammation induced by MPTP, protecting dopaminergic nerve cell death, thereby improving memory and treating Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, and multiple diseases. It can be useful as a composition for preventing or improving neurodegenerative diseases such as sclerosis.

Figure 1A shows the inhibitory effect on the production of nitric oxide (NO) induced by LPS (100 ng/ml) by the ethanol extract of bamboo leaves obtained from Ojuk in BV-2 glial cells. Figure 1B shows the effect of suppressing the production of nitric oxide (NO) in BV-2 glial cells. This is a graph showing the cytotoxicity test of bamboo leaf ethanol extract through MTT assay.
Figure 2A shows the inhibitory effect of NO production induced by LPS (200 ng/ml) of bamboo leaf hot water extract in BV-2 glial cells, and Figure 2B shows cytotoxicity test of bamboo leaf hot water extract through MTT assay. This is a graph showing .
Figure 3 is a graph measuring the activity of acetylcholinesterase after treating tacrine, bamboo leaf fraction, and bamboo leaf extract, respectively.
Figure 4 shows the results of a passive avoidance behavior experiment analyzing the transfer latency time (TLT), the time it takes to avoid moving from a bright room (light chamber) to a dark room (dark chamber) through a passive avoidance behavior experiment after bamboo leaf extract treatment.
Figure 5 shows the results of analyzing spatial cognitive ability (Y-maze experiment), which measures the relative frequency of sequentially entering the maze when processing bamboo leaf extract in the scopolamine model.
Figure 6 is a graph showing the increase in the expression of antioxidant enzymes (SOD, CAT, GPX) necessary for bamboo leaf extract to reduce oxidative damage caused by scopolamine.
Figure 7 is a graph analyzing the inhibitory effect of lipid peroxide (Malondealdehyde, MDA) generated by active oxygen during treatment of bamboo leaf extract in the scopolamine model.
Figure 8 shows neuroinflammatory factors (TNF-α, IL-1β, IL-6) and anti-neuroinflammatory factors (IL-10) in mouse brain tissue (hippocampus, cerebral cortex) upon treatment of bamboo leaf extract in the scopolamine model. This is the result of analyzing the expression of .
Figure 9 shows the NO production inhibition efficacy of fractions and six components of bamboo leaf extract.
Figure 10 is a graph showing the activity of acetylcholinesterase after treating each mouse model with components isolated from bamboo leaf extract.
Figure 11 shows the observation of BV-2 glial cells treated with LPS after treatment with tricin, which has the highest efficacy among the active ingredients of bamboo leaves. Figure 11 A is a graph showing the amount of NO produced, and Figure 11 B is a graph. Figure 11C is a graph showing the expression level of iNOS compared to GAPDH, and iNOS expression level compared to β-actin.
Figure 12A is a graph showing the expression level of PGE ₂ after pretreatment with tricin for 1 hour and LPS treatment, Figure 12B is a graph showing the expression level of COX-2 compared to GAPDH, and Figure 12C is a graph showing the expression level of COX-2 compared to GAPDH. This is a graph showing the expression level of COX-2 compared to β-actin.
Figure 13A shows the expression levels of inflammatory cytokines such as TNF-α, IL-1β, and IL-6 that change when pretreated with tricine for 1 hour and then treated with LPS, and Figure 13B shows the expression level of inflammatory cytokines such as TNF-α, IL-1β, and IL-6 compared to GAPDH. A graph showing the expression level of -α, Figure 13C shows the expression level of IL-1β compared to GAPDH, and Figure 13D shows the expression level of IL-6 compared to GAPDH.
Figure 14A is an image observed with a fluorescence microscope showing the movement of p65 induced by LPS in BV-2 glial cells, and Figure 14B is an image observed by LPS after treating BV-2 glial cells with tricine. I An image showing the degree of phosphorylation of Bα and p65, C in Figure 14 is an image showing the degree of phosphorylation of ERK, and D in Figure 14 is an image showing the degree of phosphorylation of p38.
Figure 15 is an image showing the protective effect of tricine on dopaminergic neurons in the midbrain in a mouse model of dopaminergic neuron death administered with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine).
Figure 16 is an image showing the protective effect of tricine on dopamine fibers in the striatum in a mouse model of dopaminergic neuron death administered with MPTP.
Figures 17A and B are graphs evaluating the behavioral (exercise) ability of mice through the Rota-Rod test of the MPTP-administered dopaminergic neuron death mouse model, and Figure 17C is a graph of the MPTP-treated mouse model. This is a graph evaluating the mouse's motor ability through the Pole test.
Figures 18A and B measure the expression level of iNOS according to tricine treatment in an MPTP-treated mouse model of dopaminergic neuron death, and Figures 18C and D show COX- 2 This is the result of measuring the expression level.

Hereinafter, the present invention will be described in detail with reference to the drawings. The advantages and features of the present invention and the embodiments for achieving them will become clear with reference to the embodiments described below. However, the present invention is not limited to the embodiments disclosed below and may be implemented in various different forms. The present embodiments are merely intended to ensure that the disclosure of the present invention is complete and to provide common knowledge in the technical field to which the present invention pertains. It is provided to fully inform those who have the scope of the invention, and the present invention is only defined by the scope of the claims. Like reference numerals refer to like elements throughout the specification.

Unless otherwise defined, all terms (including technical and scientific terms) used in this specification may be used with meanings that can be commonly understood by those skilled in the art to which the present invention pertains. Additionally, terms defined in commonly used dictionaries are not interpreted ideally or excessively unless clearly specifically defined. The terminology used herein is for describing embodiments and is not intended to limit the invention. As used herein, singular forms also include plural forms, unless specifically stated otherwise in the context.

As described above, activated glial cells produce and release substances such as inflammatory cytokines TNF-α and IL-1β, nitric oxide, prostaglandins, and superoxide (Gao et al., J Neurochem, 81, 1285-97, 2002; Nelson, PT. et al., Ann Med, 34, 491-500, 2002; Griffin, W.S. et al., J Neuroinflammation, 3, 5, 2006). The production of these substances triggers an immune response in the short term, but excessive or continuous production can lead to the death of nearby nerve cells, ultimately leading to neurodegeneration. Research is needed on natural products that can alleviate and improve these neurodegenerative and neuroinflammatory diseases.

Accordingly, the present inventors confirmed that bamboo leaf extract can suppress nitric oxide (NO) production when treated with glial cells, suppress the expression of iNOS, and suppress the production of inflammatory cytokines, etc., and proposed the present invention. Completed.

The present invention provides a pharmaceutical composition for the prevention or treatment of neuroinflammatory diseases or a food composition for the prevention or improvement of neuroinflammatory diseases, comprising the bamboo extract, especially the bamboo leaf extract or tricin, as an active ingredient. The bamboo leaf extract or tricin of the present invention significantly inhibits the production of inflammatory mediators occurring in activated glial cells, thereby preventing or treating neuroinflammatory diseases more effectively.

As used herein, the term “treatment” refers to an approach to obtain beneficial or desirable clinical results. For the purposes of this invention, beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, reduction of disease extent, stabilization of the disease state (i.e., not worsening), delay or reduction in the rate of disease progression, disease state, etc. improvement or temporary relief and relief (partial or total) of, whether detectable or undetectable. It may mean increasing the survival rate compared to the expected survival rate without treatment. “Treatment” refers to both therapeutic treatment and prophylactic or preventive measures. The treatments include treatment required for disorders that have already occurred as well as disorders that are being prevented. “Alleviating” a disease means reducing the extent and/or undesirable clinical signs of the condition and/or slowing or prolonging the time course of its progression compared to no treatment. It means losing.

In the present invention, the bamboo extract may be extracted from bamboo leaves. All parts of bamboo can be used, including bamboo leaves, stems, roots, and seeds. However, parts other than bamboo leaves may have insufficient efficacy in suppressing nerve inflammation, so it is most desirable to use bamboo leaves.

The bamboo leaf extract can be extracted with a single or mixed solvent such as water, alcohol, ethyl acetate, chloroform, butanol, or hexane, and more preferably methanol or ethanol. It can be extracted by applying a general solvent extraction method using the above solvent, and can also be a fraction purified using column chromatography. The method for producing the extract according to the present invention can apply all plant extraction methods known to those skilled in the art, such as extraction with water, alcohol or mixed solvent, extraction with double boiling or room temperature, etc. Including, but not limited to.

In one embodiment of the present invention, the bamboo leaf extract can be extracted using methanol, ethanol, butanol, hexan, chloroform, and ethyl acetate as solvents, and methanol or ethanol can be used. When extracted, it can inhibit the neuroinflammatory activity of activated glial cells more effectively than other solvents such as butanol, chloroform, or hexane.

The pharmaceutical composition for preventing or treating neuroinflammatory diseases of the present invention may include the bamboo leaf extract at a concentration of 1 to 100 μg/ml. The solvent of the composition is not particularly limited as long as it does not interfere with the neuroinflammation inhibitory effect of the bamboo leaf extract, but more preferably, it may be water, ethanol, propyl alcohol, DMSO, etc. If the concentration of the bamboo extract composition is less than 1 ㎍/㎖, the effect of inhibiting the production of inflammatory mediators in microglial cells may be insufficient, and the effect of suppressing neuroinflammation may be reduced. In addition, although bamboo extract may be included in a higher amount than 100 ㎍/㎖, sufficient neuroinflammation inhibitory activity can be confirmed even within the above range, and if it exceeds 100 ㎍/㎖, the improvement in efficacy due to increase in concentration is small. There may be problems with reduced efficiency. The concentration of the bamboo extract composition, which exhibits excellent neuroinflammation inhibition effect without cytotoxic effect, may be more preferably 10 to 100 μg/ml.

In one embodiment of the present invention, in order to determine the effect of each extract on suppressing neuroinflammation, BV2 cells, which are rat-derived glial cells, were pretreated with bamboo extract, and then the BV2 cells were stimulated with LPS to mediate inflammation caused by LPS stimulation. The efficacy of inhibiting the production of sex substances and their cytotoxicity were observed.

LPS (lipopolysaccharide), well known as an endotoxin, is the most well-known external factor involved in the activation of macrophages, especially tumor necrosis factor-alpha (TNF-α) and IL-6 in macrophages such as RAW 264.7 cells and monocytes. It is known that pro-inflammatory cytokines such as (interleukin-6) and IL-1β (interleukin-1 beta) are secreted and increased in inflamed areas. Additionally, when LPS stimulates macrophages, nitric oxide (NO) is generated in the process of converting L-arginine into L-citrulline by an enzyme called iNOS (inducible nitric oxide synthase), thereby producing NO from the macrophages. In mammals, NO is synthesized by three types of NO synthase (NOS, nitric oxide synthase): neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). Among these, NO produced by nNOS and eNOS is produced for normal biological functions, and its concentration in tissues is maintained at a low level. However, NO produced by iNOS is excessively produced and exhibits harmful effects on the living body, such as pathological vasodilation, cytotoxicity, and tissue damage.

According to another embodiment of the present invention, bamboo leaf extract or tricin has an effect of improving memory or cognitive function.

Hereinafter, the effects of bamboo leaf extract or tricin in preventing or treating neuroinflammatory diseases or improving memory or cognitive function will be explained through experimental examples.

According to one embodiment of the present invention, in order to study the effect of ethanol extract of Phyllostachys nigra MUNRO leaves (PNL) on NO production stimulated by LPS, accumulation of nitric oxide (NO) in BV-2 glial cells and cell Toxicity was confirmed.

Referring to Figure 1A, it can be seen that pretreatment of BV-2 glial cells with a certain concentration of bamboo leaf extract inhibits the increase in NO stimulated by LPS. In this case, it can be seen from Figure 1B that there was no change in the survival rate of the overall cells.

In another example of the present invention, LPS-stimulated BV-2 glial cells were treated with different concentrations of bamboo leaf extract to confirm the level of nitric oxide (NO) production. In this case, referring to Figure 2B, it can be seen that there is no significant change in the survival rate of the overall cells even if the concentration of the bamboo leaf extract changes. However, referring to Figure 2A, it can be seen that as the treatment concentration of bamboo leaf extract increases, nitric oxide (NO) production significantly decreases.

Additionally, in one example of the present invention, the acetylcholinesterase inhibitory ability of the bamboo leaf fraction was confirmed. Acetylcholine, known as one of the neurotransmitters, is known to be involved in neurotransmission between synapses, and acetylcholinesterase (AChE), an enzyme that hydrolyzes acetylcholine into acetate and choline, plays a role in cognitive functions. It is used as a target substance for drugs used to improve and treat dementia diseases. Due to these properties, it is known that the levels of this enzyme are reduced in the brains of patients with Alzheimer's disease. In the present invention, it was confirmed that the bamboo leaf fraction and bamboo leaf ethane extract reduce the activity of acetylcholinesterase similar to tacrine (see Figure 3). According to these results, it can be seen that bamboo leaf fraction and bamboo leaf ethanol extract can be used as a treatment for cognitive function improvement and dementia disease.

It is known that the decline in memory and cognitive function is closely related to the cholinergic nervous system, and scopolamine, an antagonist of muscarinic receptors in the post-synapse, affects the pre-synapse. ), it impairs learning and memory by temporarily blocking information transmission by inhibiting the binding of acetylcholine, a neurotransmitter released from muscarinic receptors, and is commonly used in animal models of memory loss to verify learning and memory enhancement effects. is being used. In one embodiment of the present invention, the improving effect of bamboo leaf extract on scopolamine-induced neuroinflammation and cognitive dysfunction was investigated in a mouse model. Impairment of cognitive function induced by scopolamine was observed by conducting a passive avoidance test and impairment of spatial cognitive ability by conducting a Y-maze test. Injection of bamboo leaf extract was able to improve cognitive dysfunction (see Figure 4) and spatial cognitive impairment (see Figure 5) induced by scopolamine. These experimental results show that bamboo leaf extract improves scopolamine-induced cognitive dysfunction.

In the present invention, the damage-inhibiting effect of bamboo leaf extract on scopolamine-induced neuroinflammation was observed in a mouse model of neuroinflammation and cognitive dysfunction. It was confirmed that when bamboo leaf extract was injected orally, the antioxidant enzymes SOD (Superoxide Dismutase), CAT (Catalase) activity, and GPx (Glutathione peroxidase) activity were increased in the hippocampus and cerebral cortex of mice (see Figure 6). In addition, it suppressed the accumulation of lipid peroxides increased by scopolamine (see Figure 7), decreased the expression of the neuroinflammatory factors TNF-alpha, IL-1, and IL-6, and increased the expression of the anti-neuroinflammatory factor IL-10. It was confirmed that expression increased (see Figure 8). Accordingly, it can be seen that bamboo leaf extract suppresses scopolamine-induced neuroinflammation and cognitive dysfunction.

The results of component analysis of the ethanol extract of Phyllostachys nigra MUNRO leaves (PNL) according to an embodiment of the present invention were as shown in Figure 9 below. Referring to Figure 9, the bamboo leaf extract of the present invention contains Isoorientin, Orientin, Vitexin, Luteolin, 6-C-(6“-O-trans-caffeolyglucoside ), Vittariflavone, and Tricin may be included as some of the ingredients. According to one embodiment of the present invention, the bamboo leaf extract of the present invention may contain tricin at a concentration of 1 to 50 μg/ml in the total composition. In the present invention, when tricin is included at a concentration of less than 1㎍/ml, there is no sufficient cognitive function improvement, treatment or alleviation effect of neuroinflammatory disease, and when the concentration of tricin exceeds 50㎍/ml, cytotoxicity occurs. Increasing problems may occur.

In one example of the present invention, it was confirmed that each ingredient had the ability to inhibit acetylcholinesterase (see Figure 10).

Accordingly, the bamboo leaf extract of the present invention may be a bamboo leaf extract extracted from bamboo leaves, and may contain tricin as one component to prevent, treat, or improve cognitive function of neuroinflammatory diseases.

In another example of the present invention, the expression of nitric oxide (NO) and iNOS was confirmed in BV-2 glial cells to study the effect of tricin on nitric oxide (NO) production stimulated by LPS.

Referring to Figure 11A, it can be seen that pretreatment of BV-2 glial cells with a specific concentration of tricine inhibits the production of nitric oxide (NO) stimulated by LPS. Additionally, referring to Figures 11B and C, as the concentration of tricine treatment increased, there was no change in GAPDH and β-actin, but the expression level of iNOS decreased. In other words, it can be seen that treatment with tricine can inhibit neurodegeneration mediated by inflammation.

In addition, prostaglandins, TNF-α, etc. produced by COX-2 are toxic to nerve cells, which ultimately worsens damage to nerve cells. Therefore, in the present invention, activated glial cells are treated with tricine to reduce PGE2, COX -2 Gene expression was confirmed. As a result, it was found that as the treatment concentration of tricin increased, the expression of PGE2 and COX-2 decreased (see A, B, and C of Figure 12), but there was no change in GAPDH and β-actin. Accordingly, it was confirmed that tricine exerts an anti-inflammatory effect on BV-2 glial cells induced by LPS-induced neuroinflammation.

In another example of the present invention, LPS-stimulated BV-2 glial cells were treated with tricine to determine whether inflammatory cytokines were produced. It is known that increased production of inflammatory cytokines is involved in the development of inflammation in the CNS through secretion of chemokines that promote the migration of white blood cells to the brain, induction of adhesion molecules, and destruction of the BBB (Blood Brain Barrier). The bamboo extract of the present invention can inhibit the production of inflammatory cytokines, including tricin, and thus can exert a protective effect against damage caused by activated glial cells to nerve cells. According to one embodiment of the present invention, as the treatment concentration of tricin increases, tumor necrosis factor-α (TNF-α; tumor necrosis factor-alpha), interleukin-1β (IL-1β), or interleukin-6 (IL-6) -6) suppressed the production of inflammatory cytokines (see Figure 13).

The bamboo leaf extract of the present invention also contains at least one MAPK (mitogen-ac

tivated protein kinases) or the activity of one or more NF-κB (nuclear factor kappaB) selected from the group consisting of IκBα and p65.

In one embodiment of the present invention, the inhibitory effect of tricine on MAPK activity of BV2 cells stimulated with LPS was observed. MAPK (mitogen-activated protein kinase) is a key protein as a signal transduction mediator within cells. It is activated in response to various extracellular signals such as inflammatory response, death, cell differentiation, and growth in the human body, and activates transcription factors to control the production of necessary genes. Regulating transcription, Erk (extracellular signal regulated kinase), p38 MAP kinase, etc. are known. Erk is closely related to cell growth and is regulated by many cell growth factors. p38 MAP kinase is closely related to most inflammatory responses, cell stress signals, and cell death signals.

In addition, NF-κB is an important transcription factor in inflammation, and NF-κB is known to regulate the expression of proteins including iNOS, TNF-α, COX-2, and MARK signaling mechanisms in activated microglial cells.

First, to determine whether changes in MAPK (mitogen-activated protein kinases) activity occur in BV2 cells by LPS stimulation, the degree of phosphorylation of p38 (p38 MAP kinase) and ERK (extracellular signal regulated kinase) was measured over time after LPS treatment. Measured. As shown in Figure 14, in BV2 cells, phosphorylation of IκBα (Figure 14B), p65 (Figure 14B), ERK (Figure 14C), and p38 (Figure 14D) were processed by LPS stimulation. It was confirmed that the concentration of tricin decreased as the concentration increased. Additionally, referring to Figure 14A, it can be seen that tricin delays the movement of p65, a NF-κB subunit, from the cytoplasm to the nucleus induced by LPS. In other words, it can be seen that the inhibition of NO production and inflammatory cytokine production by tricin in BV-2 glial cells is linked through blocking the NF-κB and MAPK signaling systems.

Therefore, the bamboo extract (preferably bamboo leaf extract or tricin) of the present invention not only does not show toxicity to cells, but also significantly reduces inflammatory mediators generated in activated glial cells, phosphorylation of MAPK and NF- It was confirmed that it has the effect of significantly reducing the activity of κB, which means that the bamboo extract of the present invention can be seen as effectively suppressing neuroinflammation caused by microglial cells. Therefore, the bamboo extract of the present invention effectively inhibits the activity of glial cells related to neuroinflammation, and is therefore used for the prevention or treatment of neuroinflammatory diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, multiple sclerosis, etc., or neuroinflammation. It can be useful as a health functional food for preventing or improving diseases.

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) can cause activation of microglia in the central nervous system (CNS: Central Nervous System), leading to neuroinflammation and dopaminergic neuron death. It is widely known as a neurotoxin. In an example of the present invention, the neuroprotective effect of tricine against neuroinflammation induced after MPTP administration was investigated in a mouse model of dopaminergic neuron death. The loss of dopaminergic neurons induced by MPTP in SNpc (Substantia Nigra pars compacta) and STR (Striatum) was observed through TH (Tyrosine Hydroxylase) immunostaining. It was confirmed that administration of tricine reduced the loss of dopaminergic neurons induced by MPTP (see Figures 15 and 16). These experimental results show that tricin protects nerve cells by suppressing MPTP-induced neuroinflammation.

The present invention also confirmed the neuroinflammation inhibitory effect and cognitive function improvement effect of tricine by orally injecting tricine into a mouse model of dopaminergic neuron death treated with MPTP. In one embodiment of the present invention, the behavioral improvement effect of tricine on neuroinflammation was observed in a mouse model of dopaminergic neuron death. It was confirmed that injection of MPTP suppressed mouse behavioral ability, but after tricine administration, inhibition of behavioral ability induced by MPTP was reduced (see FIG. 17).

In the present invention, the neuroinflammation inhibitory effect of tricine on MPTP-induced neuroinflammation was observed in a mouse model of dopaminergic neuron death. When intraorally injected with tricin, the expression of iNOS (A and B in Fig. 18) and COX-2 (C and D in Fig. 18) decreased, confirming that tricin suppresses neuroinflammation induced by MPTP.

In particular, in the present invention, it may be composed of a pharmaceutical composition containing the bamboo leaf extract or tricin, and a pharmaceutical composition for preventing, treating, or improving cognitive function of neuroinflammatory diseases containing the bamboo leaf extract or tricin according to the present invention as an active ingredient. It may further contain other natural substances or compounds that inhibit or reduce neuroinflammation, and the pharmaceutical composition of the present invention can be administered orally, transdermally, subcutaneously, intravenously, or intramuscularly to mammals such as rats, mice, livestock, and humans. It can be administered through several routes, including:

In addition, the pharmaceutical composition for preventing, treating, or improving cognitive function of neuroinflammatory diseases containing bamboo leaf extract or tricin as an active ingredient according to the present invention can be formulated into various dosage forms. When formulating, it can be formulated using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules. These solid preparations contain bamboo extract and at least one or more excipients (for example, starch, sucrose, lactose, and gelatin). It can be mixed and prepared. Additionally, lubricants other than simple excipients may also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives are included. may be included. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. Glycerol and gelatin can be used as a base for suppositories.

The dosage of the pharmaceutical composition may be applied differently depending on the subject's age, gender, condition, absorption, inactivation rate and excretion rate of the active ingredient in the body, and concomitant drugs.

When the pharmaceutical composition for preventing, treating, or improving cognitive function of neuroinflammatory diseases containing the bamboo leaf extract or tricin of the present invention as an active ingredient is administered to the human body, there is less risk of side effects compared to other synthetic drugs because it is a natural extract. You can use it with confidence.

According to another embodiment of the present invention, the bamboo leaf extract of the present invention not only does not exhibit toxicity to cells, but also significantly reduces inflammatory mediators generated in activated microglial cells, phosphorylation of MAPK and NF-κB. It was confirmed that it has the effect of significantly reducing activity, which means that the bamboo extract of the present invention can be seen as effectively suppressing neuroinflammation caused by microglial cells. Therefore, since the bamboo extract of the present invention effectively inhibits the activity of microglial cells related to neuroinflammation, it can be used as a preventative or therapeutic agent for neuroinflammatory diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, multiple sclerosis, etc. It can be useful as a health functional food to prevent or improve inflammatory diseases.

In addition, the present invention provides a food composition for preventing neuroinflammatory diseases or improving memory or cognitive function containing the bamboo leaf extract or tricin as an active ingredient.

The type of health functional food containing the food composition for preventing neuroinflammatory disease or improving cognitive function containing the bamboo extract or tricin of the present invention as an active ingredient is not particularly limited, and includes, for example, meat, sausage, bread, chocolate, These may include candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes.

The health food can be used with other foods or food additives in addition to the bamboo leaf extract, and can be used appropriately according to conventional methods. For example, the beverage for preventing neuroinflammatory diseases containing the bamboo extract as an active ingredient is filled with a drink bottle by mixing calcium, acanthus extract, high fructose corn syrup, purified water, etc. in addition to the bamboo extract as an active ingredient. After sterilization, the beverage can be prepared by cooling to room temperature. In addition, the health supplement for preventing neuroinflammatory diseases containing the bamboo extract as an active ingredient contains bamboo extract, nutritional supplements (vitamins B1, B2, B5, B6, E and acetic acid ester, nicotinic acid amide), oligosaccharides, 50% ethanol, Add purified water and mix to form granules, dry in a vacuum dryer, pass through 12~14 mesh to produce granules uniformly, extrude in appropriate amounts to form tablets or powder, or fill into hard capsules. It can be manufactured into a product.

The effective dose of the bamboo leaf extract or tricin contained in the health food can be used in accordance with the effective dose of the pharmaceutical composition, and the mixing amount of the active ingredients can be appropriately determined depending on the purpose of use, such as preventive or therapeutic treatment. In the case of long-term intake for health and hygiene purposes or health control, it may be below the above range.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be obvious to those skilled in the art that the scope of the present invention should not be construed as limited by these examples.

Preparation of bamboo leaf extract

First, a certain amount of bamboo leaves was taken, six times the amount of water was added, and hot water extraction was performed at 100°C for 1 hour. After filtering this solution, it was centrifuged at 6,000 rpm for 30 minutes (Dupont Sorvall RC-5C, SORVALL, USA) and the supernatant was filtered again. The extract obtained by concentrating the filtrate again was filled into a diaion column and left overnight. After repeated extraction with 60% ethanol twice, the 60% ethanol extract was collected and concentrated under reduced pressure (Rotary vacuum evaporator R-114, Swiss) to obtain a bamboo leaf extract.

Bamboo leaves (100g) obtained from Ojuk and Joriti were placed in 1L distilled water and subjected to heat extraction in an extractor (DW-290, Daewoong Electronics) at a temperature of 100°C for 150 minutes. After filtering the extract, it was freeze-dried using a freeze dryer (Clean Vac 8, Hanil Science Co., LTD) to obtain a bamboo leaf hot water extract (6.56 g).

Bamboo leaves (200g) were added to 1L ethanol 95.0% (GR Grade) and extracted repeatedly 30 times (40 KHz, 1500W for 15 minutes per time, sonication for 120 minutes) at room temperature. The ultrasonic extractor used here was SDN-900H (SD-Ultrasonic Co., LTD), and the extract was filtered and dried under reduced pressure to obtain an ethanol extract of bamboo leaves (7.7 g).

Effect of bamboo leaf ethanol extract on the production of nitric oxide (NO) in LPS-stimulated BV-2 glial cells

After seeding BV-2 glial cells on a microplate, treatment with LPS and bamboo leaf ethanol extract, and reacting in a culture medium for 24 hours, the supernatant was dispensed into 96 wells, and 100 ㎕ of Griess reagent (1% sulfanyl) was added to each well. After reacting with amide/0.1% N-(1-naphthyl)=ethylenediamine dihydrochloride/2.5% H3PO4), the value was measured using a Vmax 96-well microplate spectrophotometer with a wavelength of 550 nm. Specifically, BV-2 glial cells were treated with bamboo leaf ethanol extract at a concentration of 100 ng/ml for 24 hours, and then treated with LPS for 18 hours. The culture fluid was collected and analyzed for NO production and cell survival. The results are shown as mean values ± SEM in Figure 1 (n=3).

Referring to Figure 1A, when LPS was treated with BV-2 glial cells, it was observed that NO production was significantly increased compared to the control group (in this case, there was no change in the overall cell survival rate (Figure 1 (see B). However, pretreatment of BV-2 glial cells with a certain concentration of bamboo leaf ethanol extract inhibited the increase in NO stimulated by LPS.

Effect of bamboo leaf hydrothermal extract on NO production in LPS-stimulated BV-2 glial cells.

BV-2 glial cells were treated with bamboo leaf water extract at a concentration of 100 ng/ml for 24 hours, and then treated with LPS for 18 hours. The culture fluid was collected and analyzed for NO production and cell survival. The results are shown as mean values ± SEM in Figure 2 (n=3).

Referring to Figure 2B, when LPS was treated with BV-2 glial cells, it was observed that NO production was significantly increased compared to the control group (in this case, there was no change in the overall cell survival rate (Figure 2B) However, pretreatment of BV-2 glial cells with a certain concentration of bamboo leaf hot water extract inhibited the increase in NO stimulated by LPS.

Acetylcholinesterase inhibitory activity of bamboo leaf fractions

Acetylcholine, known as one of the neurotransmitters, is known to be involved in neurotransmission between synapses, and acetylcholinesterase (AChE), an enzyme that hydrolyzes acetylcholine into acetate and choline, plays a role in cognitive functions. It is used as a target substance for drugs used to improve and treat dementia diseases. Due to these properties, it is known that the levels of this enzyme are reduced in the brains of patients with Alzheimer's disease.

Specific concentrations of bamboo leaf fraction (0.1 μg/ml) and bamboo leaf ethanol extract (0.1 μg/ml) reduced the activity of acetylcholinesterase similarly to 0.1 mM tacrine (Figure 3).

Analysis of the efficacy of bamboo leaf hot water extract and ethanol extract on passive avoidance ability in a scopolamine-induced cognitive dysfunction mouse model

Male C57BL/6 mice (25 g, 8 weeks old) were purchased from Daehan Biolink. They were divided into four groups, acclimatized for one week under standard conditions, and used in subsequent experiments (temperature 22±2°C, humidity 55±5%, 12 h-light/dark cycle, food/water free diet). All experiments were performed in accordance with the guidelines of the National Institute of Laboratory Animal Care (NIH publication No. 85-23, revised 1985) and the Institutional Animal Care and Use Committee (IACUC) of Konkuk University. Mice were divided into 4 groups of 6 each (1. Vehicle (control group), 2. Scopolamine 2 mg/kg, 3. Tacrine (TAC) 10 mg/kg, 4. Bamboo leaf hot water extract 100 mg/kg, Bamboo leaf ethanol extract 100 mg/kg It was divided into kg and then put into the experiment. All treatment groups were used by dissolving in physiological saline. Bamboo leaf hot water extract, bamboo leaf ethanol extract, and Tacrine (TAC) were administered orally at 100 mg/kg daily for 7 days before Scopolamine treatment. It was administered to mice, and 2 mg/kg of Scopolamine was administered via intraperitoneal injection. A passive avoidance experiment was conducted to evaluate memory, and for training and testing of passive avoidance behavior, two identical light/dark square boxes were used. An active and passive avoidance system from GEMINI (San Diego Instruments, San Diego, CA, USA) was used. This device has a light chamber (bright room) and a dark chamber (dark room). , It is distinguished by a door controlled by a computer. After putting the mouse in a bright room, light is given, it is led to a dark room, and then an electric current is passed through it so that the mouse remembers this. In other words, when the mouse enters the dark room, the door automatically opens. After closing, an electric foot shock of 0.5 mA is given for 5 seconds. At this time, the transfer latency time (TLT), the time it takes to avoid a bright room to a dark room, was recorded and stored through a computer, and the electric shock and opening and closing of the door were recorded and stored. It was controlled by an attached device system. When the mouse stayed in the bright room for 180 seconds, the results of the passive avoidance experiment were analyzed by assuming that the mouse remembered the passive avoidance training after one training attempt.

Figure 4 is a graph showing the results of a passive avoidance experiment to confirm the effect of bamboo leaf extract on improving cognitive dysfunction caused by scopolamine. Figure 4A shows the results immediately after the passive avoidance experiment, and Figure 4B shows the results on the 7th day of the experiment after the manual avoidance experiment. Referring to Figure 4, when the experimental mice were treated with only scopolamine, the TLT time was shorter than that of the control group, and the difference between immediately after the experiment (A) and on the 7th day (B) was found to be minimal. However, when TAC and bamboo leaf hot water extract were treated with scopolamine, the TLT time was significantly increased on the 7th day (B) compared to immediately after the experiment (A).

That is, referring to Figure 4, in the passive avoidance behavior experiment, the transfer latency time (TLT), the time taken to avoid moving from a bright room (light chamber) to a dark room (dark chamber), significantly increased in the final result in the control group. In the final results, TLT was significantly increased in the group administered Tacrine for 7 days and the group pretreated with hot water extract of bamboo leaves and ethanol extract. By confirming the efficacy of the extract similar to that of Tacrine, it was confirmed that the bamboo leaf hot water extract and ethanol extract showed improvement effects on cognitive function in a passive avoidance experiment using scopolamine.

Effect of bamboo leaf ethanol extract on spatial cognitive ability in a mouse model of scopolamine-induced cognitive dysfunction.

Mice were divided into four groups, acclimatized for one week under standard conditions, and used in subsequent experiments. Mice were divided into 4 groups of 6 each (1. vehicle (control group), 2. Scopolamine 2 mg/kg (comparison group 1), 3. Tacrine (TAC) 10 mg/kg (experimental group 1) 4. Bamboo leaf ethanol extract It was divided into 100 mg/kg (experimental group 2) and then administered to the experiment. All treatment groups were used by dissolving them in physiological saline solution. Bamboo leaf ethanol extract and tacrine were administered to mice at 100 mg/kg orally daily for 7 days before Scopolamine treatment, and 2 mg/kg of Scopolamine was administered via intraperitoneal injection. A spatial cognitive test (Y-maze) was conducted to evaluate memory. The Y-maze test is a method of testing a mouse's will to explore a new environment. It is a method of measuring the relative frequency of experimental animals identifying clues around them and sequentially entering the maze. Mice have an instinctive tendency to explore new environments, but the ability of memory loss mice treated with scopolamine to explore new environments (Spontaneous alteration, %) was significantly reduced. However, it was confirmed that mice with memory loss that were orally administered ethanol extract of bamboo leaves recovered their exploration instinct (see Figure 5).

Figure 5 shows the results of analyzing spatial cognitive ability (Y-maze), which measures the relative frequency with which the scopolamine model sequentially enters the maze during bamboo leaf extract treatment. A in Figure 5 is a graph showing the results immediately after the experiment, and B in Figure 5 is a graph showing the results 7 days after the start of the experiment. Referring to Figure 5, the relative frequency of entering the maze decreased in the comparison group compared to the control group, but in experimental groups 1 and 2. It can be seen that there is an increase. In other words, it can be seen that memory decline induced by scopolamine is improved when treated with bamboo leaf extract.

Effect of bamboo leaf ethanol extract on oxidative stress in a scopolamine-induced cognitive dysfunction mouse model

Brains were extracted from mice in the control group administered scopolamine for 30 minutes and the group treated with tacrine and bamboo leaf ethanol extract. Afterwards, the hippocampus and cerebral cortex were carefully separated from the mouse brain, and SOD (Superoxide Dismutase), CAT (Catalase) activity, and GPx (Glutathione peroxidase) activity were measured.

To confirm the activation of antioxidant enzymes SOD, CAT, and GPx, brain tissue homogenate extract was prepared using 10% w/v 0.1 M Phosphate buffer. Superoxide dismutase and catalase assay kits (Cell Biolabs, Dan Diego, CA) were used, and glutathione peroxidase activity was measured using the GPx colorimetric assay kit (BioVision, USA, CA). SOD was measured at a wavelength of 490 nm, CAT at 520 nm, and GPx at 340 nm.

In an experiment in which normal and treated groups were treated with scopolamine to check the levels of SOD, CAT, and GPx to determine the activity of antioxidant enzymes, the bamboo leaf ethanol extract treated group showed levels of enzymes reduced by scopolamine similar to the normal group. It was improved to the point where it was confirmed that it showed the same increase in the cerebral cortex and hippocampus of the brain (see Figure 6).

Figure 6 shows the effect of bamboo leaf extract on oxidative stress in the scopolamine-induced cognitive dysfunction mouse model confirmed in the brain tissue hippocampus and brain tissue cerebral cortex. Figure 6A shows SOD and Figure 6B shows the effect of bamboo leaf extract on oxidative stress. CAT and C in FIG. 6 are graphs confirming the expression level of GPx. Referring to Figure 6, when treated with scopolamine alone, the degree of activation of enzymes SOD, CAT, and GPx, which are antioxidant enzymes, was significantly lowered, but when treated together with bamboo leaf extract, the degree of activation of SOD, CAT, and GPx was recovered. Able to know. In other words, it can be seen that the bamboo leaf extract according to the present invention is effective in treating cognitive dysfunction induced by scopolamine by alleviating oxidative stress.

Effect of bamboo leaf ethanol extract on lipid peroxidation in a scopolamine-induced cognitive dysfunction mouse model

The brains of mice in the control group administered scopolamine for 30 minutes and the group treated with tacrine and bamboo leaf ethanol extract were extracted, and the hippocampus and cerebral cortex were carefully separated to measure MDA (Malondialdehyde) activity.

To check lipid peroxidation (MDA), a brain tissue homogenate extract was prepared using 10% w/v 0.1 M Phosphate buffer. For MDA, a colorimetric/fluorometric assay kit (BioVision, USA, CA) was used. MDA was measured at a wavelength of 410 nm.

Lipid peroxidation is caused by oxygen free radicals generated by external as well as internal factors. The brain has a high content of polyunsaturated fatty acids that are prone to oxidative reactions, and oxygen consumption in the body is higher than other organs, resulting in oxidative damage. It is reported that it is susceptible to , and the resulting accumulation of lipid peroxides is known to cause cell dysfunction and deteriorate the function of the central nervous system. The level of lipid peroxidation was decreased in the bamboo leaf ethanol extract treatment group, and the same decrease was observed in the cerebral cortex and hippocampus of the brain (see Figure 7).

Referring to Figure 7, it can be seen that the degree of lipid peroxide accumulation (MDA Eq) in the brain tissue hippocampus and brain tissue cerebral cortex is lower when bamboo leaf extract is treated together with scopolamine (scop) than when treated alone. In other words, it can be seen that bamboo leaf extract prevents deterioration of the function of the central nervous system by reducing the accumulation of lipid peroxides in the brain tissue hippocampus and brain tissue cerebral cortex.

Effect of bamboo leaf extract on neuroinflammatory factors in a scopolamine-induced cognitive dysfunction mouse model

The hippocampus and cerebral cortex were carefully separated from the brains of mice in the control group administered scopolamine and the group treated with tacrine and bamboo leaf ethanol extract, and the expression levels of TNF-α, IL-1, IL-6, and IL-10 were measured. .

To identify the above neuroinflammatory factors, a brain tissue homogenate extract was prepared using 10% w/v 0.1 M Phosphate buffer. TNF-α, IL-1, IL-6, and IL-10 Duoset ELISA kit (R&D systems, MN) was used and each was measured at a wavelength of 450 nm. This is shown in Figure 8.

Referring to A to C of Figure 8, in an experiment in which the normal group and the treated group were treated with scopolamine to check the levels of TNF-alpha, IL-1, and IL-6 in order to see the anti-inflammatory effect, the cells were killed. The ethanol extract treatment group reduced the levels of inflammatory cytokines increased by scopolamine, and this was equally reduced in the cerebral cortex and hippocampus of the brain.

On the other hand, referring to Figure 8D, the level of anti-inflammatory cytokine (IL-10) showed a tendency to recover compared to scopolamine, which increased equally in the cerebral cortex and hippocampus of the brain. showed results.

Analysis of NO production inhibition ability of active ingredients of bamboo leaf extract

BV-2 glial cells were treated with 6 active ingredients (isoorientin, orientin, luteolin, vitariflavone, tricin, and vitexin) identified through component analysis of bamboo leaf extract for 1 hour, and then treated for 18 hours. During treatment with LPS. The culture fluid was collected and analyzed for NO production and cell survival. The results are shown in Figure 9 (n=4).

Referring to Figure 9, when LPS is treated with BV-2 glial cells, NO production is significantly increased compared to the control group (in this case, the survival rate of the overall cells was not affected). Six types of bamboo leaf extracts at specific concentrations were observed. The increase in NO stimulated by LPS was suppressed by pretreatment with the active ingredient, and among them, Tricin showed the highest inhibitory effect.

Acetylcholinesterase inhibition activity analysis of active ingredients of bamboo leaf extract

Six active ingredients isolated from bamboo leaf extract: Isoorientin, Orientin, Vitexin, Luteolin, and Vittariflavon.

avone) and tricine acetylcholinesterase activity levels were confirmed. Referring to Figure 10, it was confirmed that each active ingredient reduces the activity of acetylcholinesterase, and these results show that among the six active ingredients isolated from the extract, tricine can be used as a treatment for cognitive function improvement and dementia disease. It is assumed that there is (Figure 10).

Effect of the active ingredient Tricine on NO production and iNOS expression in LPS-stimulated BV-2 glial cells.

To analyze the anti-inflammatory effect of tricin, the inhibitory effect of NO was evaluated in BV-2 glial cells stimulated with LPS, the same neuroinflammatory factor. The production of NO was quantitatively measured. In order to investigate changes in the gene and protein expression patterns of iNOS, an enzyme involved in inflammation, BV-2 glial cells were treated with different concentrations of LPS and tricine and cultured. Specifically, after treatment with LPS for 6 hours, the cell supernatant was removed, RNA was identified from the cells, and the iNOS gene was analyzed using RT-PCR. Additionally, after treatment with LPS for 18 hours, the cell supernatant was removed, proteins were identified in the cells, and iNOS protein was analyzed using Western blot.

When LPS was treated with BV-2 glial cells, it was observed that NO production and iNOS expression were significantly increased compared to the control group (in this case, there was no change in the overall cell survival rate. However, in BV-2 glial cells, specific Pretreatment with a high concentration of tricine suppressed the increase in nitric oxide (NO) stimulated by LPS in a concentration-dependent manner (Figure 11A) and also suppressed the expression of iNOS at the mRNA and protein levels (Figure 11B, C).

Effect of the active ingredient Tricin on PGE2 and COX-2 expression in LPS-stimulated BV-2 glial cells

To confirm the expression of PGE2, which is synthesized from arachidonic acid and induces inflammation, a prostaglandin E2 parameter assay kit (R&D System, MN, USA) was used and analyzed using the manufacturer's recommended method. Quantification was performed using the BCA protein quantitative method. An equal amount of protein was electrophoresed on a 10% SDS-PAGE gel and then transferred from the gel to the membrane using 0.45 μm polyvinylidene fluoride (PVDF, Milipore). When using antibodies, dilute anti-COX-2 (1:1000, Santa Cruz) as the primary antibody and react overnight at 4°C, and react with an antibody containing horseradish peroxidase (HRP) as the secondary antibody at room temperature for 1 hour. After doing so, the reaction was performed using an ECL kit (Pierce, CA), and the results were confirmed using a DAVINCH CAS-400SM (Davinch-K, Korea). When LPS was treated with BV-2 glial cells, it was observed that the production of PGE2 and COX-2 expression were significantly increased compared to the control group. However, pretreatment of BV-2 glial cells with a certain concentration of Tricin inhibited the increase in PGE2 stimulated by LPS in a concentration-dependent manner (see A in Figure 12), and increased the level of COX-2 at the mRNA and protein levels. Expression was also suppressed (see B and C of Figure 12).

Therefore, it can be seen that Tricin exhibits an anti-inflammatory effect on BV-2 glial cells induced by LPS-induced neuroinflammation.

Effect of Tricine on the production of inflammatory cytokines in LPS-stimulated BV-2 glial cells.

It is known that increased production of inflammatory cytokines is involved in the development of inflammation in the CNS through secretion of chemokines that promote the migration of white blood cells to the brain, induction of adhesion molecules, and destruction of the BBB (Blood Brain Barrier). Therefore, BV-2 glial cells were treated with 2.5, 5, and 10 μM tricine for 1 hour, and then treated with LPS for 6 hours. The cell supernatant was removed, RNA was identified from the cells, and mRNA expression levels of TNF-α, IL-1β, and IL-6 were analyzed.

Stimulation of BV-2 glial cells with LPS increases the levels of inflammatory cytokines such as TNF-α, IL-1β, and IL-6. However, when treated with tricin, the gene expression of these cytokines was lowered and the production of cytokines was also significantly suppressed in a concentration-dependent manner (see FIG. 13). Through this, it was confirmed that tricin can suppress the production of inflammatory cytokines stimulated by LPS, and it can also be seen that this suppression occurs at the transcription stage.

Effect of Tricin on NF-κB and MPAK activation in LPS-stimulated BV-2 glial cells.

LPS is known to regulate iNOS gene expression through NF-κB activation. Therefore, in order to determine whether Tricin inhibits the activation of the NF-κB signaling system, what effect Tricin has on the phosphorylation and nuclear movement of IκBα and p65 in the cytoplasm in BV-2 glial cells induced by LPS. It was investigated whether it was effective. In addition, to confirm whether the MPAK signaling system, another neuroinflammatory signaling mechanism, is regulated, the effect of tricine on the phosphorylation of the MAPK substances ERK and p38 was examined.

Tricine delayed the movement of p65, a NF-κB subunit, from the cytoplasm to the nucleus induced by LPS (Figure 14A), and reduced the phosphorylation of IκBα and p65 induced by LPS in the cytoplasm in a concentration-dependent manner. This was confirmed (B in Figure 14).

In addition, tricine attenuated the phosphorylation of ERK and p38 induced by LPS in a concentration-dependent manner (Figure 14, C and D).

Therefore, it can be seen that the inhibition of NO production and inflammatory cytokine production by tricin in BV-2 glial cells may be linked through blocking the NF-κB and MAPK signaling systems.

Inhibitory effect of the active ingredient Tricine on dopaminergic neuron death in a mouse model of dopaminergic neuron death induced by MPTP

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is widely known as a neurotoxin that can induce neuroinflammation and dopaminergic neurodegeneration, which can cause activation of microglial cells in the CNS. . The neuroprotective effect of tricine against neuroinflammation induced by MPTP was analyzed in a mouse model of dopaminergic neuron death. The loss of dopaminergic neurons induced by MPTP in the SNpc (Substantia Nigra pars compacta, substantia nigra) and STR (striatum, striatum) of the medial brain was analyzed by measuring neurons stained for TH (Tyrosine Hydroxylase). did. It was confirmed that injection of MPTP significantly reduced the number of dopaminergic neurons and nerve fibers in SNpc and STR, and also reduced the expression of TH. However, orally administering tricine was able to reduce the loss of dopaminergic neurons induced by MPTP and also reduce the loss of TH (see Figures 15 and 16). Therefore, it was confirmed that tricin protects nerve cells by suppressing MPTP-induced neuroinflammation.

Efficacy of the active ingredient Tricin in improving behavioral disorders in a mouse model of dopaminergic neuron death induced by MPTP

MPTP (1-methyl-4-phenyl-1,2,3,6-tetr) in a mouse model of dopaminergic neuron death.

The behavioral improvement effect of tricine on ahydropyridine-induced neuroinflammation was confirmed. Behavioral inhibition induced by MPTP was observed using the Rota-Rod and Pole tests. Behavioral impairment was judged by dividing the time taken for the mouse to fall (latency to fall), distance traveled (distance traveled), and latency time. Injection of MPTP inhibited mouse behavioral ability, but intraoral injection of tricine reduced behavioral inhibition induced by MPTP (see Figure 17). Through these experimental results, it was found that tricine suppresses behavioral impairment induced by MPTP administration.

Inhibitory effect of the active ingredient Tricine on neuroinflammation in a mouse model of dopaminergic neuron death induced by MPTP.

MPTP (1-methyl-4-phenyl-1,2,3,6-tetr) in a mouse model of dopaminergic neuron death.

The neuroinflammation inhibitory effect of tricine on neuroinflammation induced by ahydropyridine was analyzed. Neuroinflammation induced by MPTP in STR was observed by measuring iNOS (Figure 18, A and B) and COX-2 (Figure 18, C and D). Referring to Figures 18A and B, the production of iNOS was reduced in the Parkinson's disease mouse model when treated with tricine, and referring to Figures 18C and D, the production of COX-2 was decreased in the Parkinson's disease mouse model when treated with tricine. suppressed. Injection of MPTP promoted neuroinflammation, but intraoral injection of tricine reduced neuroinflammation induced by MPTP. Accordingly, it can be seen that tricin inhibits neuroinflammation induced by MPTP.

As above, the specific parts of the present invention have been described in detail, and those skilled in the art will understand that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (17)

A pharmaceutical composition for preventing or treating neuroinflammatory diseases,
Contains bamboo leaf ethanol extract as an active ingredient,
A pharmaceutical composition for preventing or treating a neuroinflammatory disease, wherein the neuroinflammatory disease is multiple sclerosis or Parkinson's disease. According to claim 1,
The bamboo leaf ethanol extract is a pharmaceutical composition for preventing or treating neuroinflammatory diseases, containing tricin as an active ingredient. According to claim 1,
The bamboo leaf ethanol extract is a pharmaceutical composition for preventing or treating neuroinflammatory diseases, characterized in that it inhibits the production of nitric oxide (NO) in nerve cells. According to claim 1,
The ethanol extract of bamboo leaves is a pharmaceutical composition for preventing or treating neuroinflammatory diseases, characterized in that it inhibits the activity of acetylcholinesterase. According to claim 1,
A pharmaceutical composition for preventing or treating neuroinflammatory diseases, wherein the ethanol extract of bamboo leaves contains tricin at a concentration of 1 to 50 μg/ml in the total composition. According to clause 5,
A pharmaceutical composition for preventing or treating neuroinflammatory diseases, wherein the tricin reduces NF-κB activation by reducing phosphorylation of IκBα or p65 in nerve cells. According to clause 6,
The tricin is used for the prevention, treatment or cognitive function of neuroinflammation, characterized in that it reduces mitogen-activated protein kinase (MAPK) activation by reducing phosphorylation of ERK (extracellular signal regulated kinase) or p38 (p38 MAP kinase) in nerve cells. Pharmaceutical composition for improvement. delete A food composition for improving symptoms of neuroinflammatory diseases,
Contains bamboo leaf ethanol extract as an active ingredient,
A food composition for improving symptoms of a neuroinflammatory disease, wherein the neuroinflammatory disease is multiple sclerosis or Parkinson's disease. According to clause 9,
The bamboo leaf ethanol extract is a food composition for improving symptoms of neuroinflammatory disease, comprising tricin as an active ingredient. According to clause 9,
A food composition for improving symptoms of neuroinflammatory disease, wherein the bamboo leaf ethanol extract contains tricin at a concentration of 1 to 50 μg/ml in the total composition. delete delete delete delete delete delete