KR20220126841A - The Pharmaceutical composition for the improvements and prevention of the symptoms in the dementia comprising the extracts from Lespedeza bicolor Turcz. - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing Lespedeza bicolor as an active component. The pharmaceutical composition for preventing or treating dementia according to an embodiment of the present invention can include a compound represented by chemical formula selected from chemical formulas 1 to 8, or a pharmaceutically formulated salt thereof as an active component.

Description

The pharmaceutical composition for the prevention or treatment of dementia {The Pharmaceutical composition for the improvements and prevention of the symptoms in the dementia comprising the extracts from Lespedeza bicolor Turcz.}

The present invention relates to a pharmaceutical composition for preventing or treating dementia comprising a compound derived from an extract of Lespedeza bicolor Turcz. It relates to a pharmaceutical composition for preventing or treating dementia.

Dementia is common enough to occur in about 8.2 to 10.8% of the elderly population over 65 in Korea. It is a representative old-age disease that is emerging as a serious social problem along with the rapid aging of the population. It is a degenerative disease with clinical symptoms called devastation. In particular, Alzheimer's disease (AD) is the most common type of dementia, with brain atrophy and senile plaques, neurofibrillary tangles, and granulocytes in nerve cells occurring in the cortex or hippocampus. It is characterized by histological findings such as granulovacuolar degeneration and Hirano body, and beta-amyloid (Aβ) is a major component of senile plaque and is presumed to be an important cause of AD. It is known to induce neuronal cell death due to oxidative stress by generating reactive oxygen species (ROS).

The main symptom is cognitive function and memory loss due to brain cell destruction, which leads to death if it progresses slowly. Onset between the ages of 40 and 65 is classified as early-onset or familial AD, and onset between the ages of 65 and older is classified as late-onset or sporadic AD. The family type is heavily influenced by genetic factors. A case in which the progression is slow due to neurodegenerative disease due to aging is called senile dementia, and AD is progressive and the rate of progression (exacerbation) is faster than that of senile dementia.

Anti-dementia drugs clinically used for dementia prevent and restore cognitive decline in dementia patients, and cholinesterase blockers tacrine and donapezil ), revastigmine, galantamine, MAO-B blocker selegilin, vasodilators, and nootropics. .

Since the treatment of dementia has been mainly developed around Alzheimer's disease, drug treatment for Alzheimer's disease is essential. For the treatment of non-cognitive disorders and abnormal behavior, haloperidol, an antipsychotic drug, is used for the treatment of thinking disorders and aggressive behavior. However, small doses of clozapine, risperidone, and olanzapine, which are currently known as atypical tranquilizers, are recommended, and for the treatment of affective disorders, currently, prozac, which blocks serotonin reuptake, is recommended. Antidepressants such as zoloft and seroxat are relatively widely used.

Laid-open Patent Publication No. 10-2008-0103321

The problem to be solved by the present invention relates to the neuroprotective effect of 1-methoxylespeflorin G11 (MLG) against glutamate-induced neurotoxicity in neuronal HT22 hippocampal cells.

The protective effect of MLG was assessed using MTT assay and microscopic analysis. The extent of apoptosis was studied by flow cytometry performed on damaged cells probed with annexin V/propidium iodide.

In addition, mitochondrial reactive oxygen species (ROS) was evaluated through flow cytometry through MitoSOXTM Red staining. To determine the mitochondrial membrane potential, flow cytometry was performed after staining with tetramethylrhodamine and JC-1.

The results showed that MLG attenuated glutamate-induced apoptosis in HT22 cells by inhibiting intracellular ROS generation and mitochondrial dysfunction. In addition, MLG prevented the glutamate-induced apoptosis pathway in HT22 cells through upregulation of Bcl-2 and downregulation of the cleaved PARP-1, AIF and phosphorylated MAPK cascade.

In addition, MLG treatment induced HO-1 expression in HT22 cells. These results suggest that MLG has a neuroprotective effect against glutamate-induced neurotoxicity in HT22 cells of neurons by inhibiting pre-oxidative stress and apoptosis.

Meanwhile, Alzheimer's disease (AD), the most common form of dementia, is a progressive age-related neurodegenerative disease. Oxidative stress and excitotoxic disorders are strongly associated with neuronal loss and synaptic dysfunction, which are key hallmarks of AD.

Glutamate, a major excitatory neurotransmitter, is responsible for cell survival, migration and differentiation during brain development. However, excessive amounts of glutamate cause neuronal cell death through oxidative stress or excitotoxicity.

Neurons are particularly vulnerable to the production of reactive oxygen species (ROS) and are very sensitive to redox stress. Thus, AD is known to be associated with the generation of ROS, which results in neuronal cell death.

Furthermore, oxidative stress causes mitochondrial dysfunction; This causes the translocation of apoptosis-inducing factor (AIF) to the nucleus, resulting in the activation of a caspase-independent apoptosis pathway. Excessive production of ROS promotes phosphorylation of mitogen-activated protein kinase (MAPK).

Therefore, inhibition of glutamate-induced oxidative stress using antioxidants may be a good strategy for the prevention and treatment of AD.

Flavonoids, naturally occurring polyphenolic compounds, are normal constituents of the human diet and are known for a variety of biological activities. Pterocarpans constitute the second largest group of isoflavonoids. These include a tetracyclic ring system of benzofuran-benzopyran with two chiral centers at positions 6a and 11a derived from the flavonoid backbone.

Pterocarpans (pterocarpans) exhibit various biological activities, such as anti-neuroinflammatory, anti-cancer, antioxidant, anti-malarial, and antibacterial agents.

In the present invention, in the process of examining the multifunctional agent of medicinal plants having both antioxidant and antiapoptotic properties, the methanol extract of Lespedeza bicolor Turcz. It has been found to have protective activity.

Pterocarpans derived from L. bicolor are known to have beneficial effects such as anticancer, antioxidant, and neuraminidase inhibitory activity.

Previously, four novel pterocarpans were isolated from L. bicolor showing antiproliferative activity in Jurkat cells. However, to the best of our knowledge, the neuroprotective activity of pterocarpans against glutamate-induced toxicity in HT22 hippocampal cells has not yet been investigated. The present inventors, in order to study the neuronal cell death caused by glutamate-induced oxidative stress, the physiologically active protein in L. bicolor showing a neuroprotective effect in HT22 cells, a hippocampal neuronal cell line of murine pterocarpans were further identified.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

A pharmaceutical composition for preventing or treating dementia according to an embodiment of the present invention for achieving the above object is a compound represented by a formula selected from the following Chemical Formulas 1 to 8 or a pharmaceutically composition salt thereof as an active ingredient. may include

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

The compound is derived from the extract of Lespedeza bicolor Turcz.

The extract is a crude extract, polar solvent-soluble extract, or non-polar solvent-soluble extract of serrata.

The crude product includes an extract soluble in a solvent selected from water including purified water, lower alcohols having 1 to 4 carbon atoms, or a mixed solvent thereof.

The non-polar solvent-soluble extract includes an extract soluble in methylene chloride, hexane, chloroform, dichloromethane or ethyl acetate.

The compound can inhibit the neurotoxicity of glutamate, which causes oxidative stress or cell death in neurons.

The compound can alleviate the accumulation of reactive oxygen species (ROS) due to oxidative stress in neurons.

The compound can modulate mitochondrial membrane potential (MMP, ΔΨm) disruption.

The compound may inhibit phosphorylation of mitogen-activated protein kinases (MAPK).

The compound may increase the expression of heme oxygenase 1 (HO-1) in neurons.

A pharmaceutical composition for preventing or treating dementia according to another embodiment of the present invention for achieving the above object is a compound represented by a formula selected from the following Chemical Formulas 1 to 8, or a pharmaceutically composition thereof, as an active ingredient. may include

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

The compound is derived from the extract of Lespedeza bicolor Turcz.

The extract is a crude extract, polar solvent-soluble extract, or non-polar solvent-soluble extract of serrata.

The crude product includes an extract soluble in a solvent selected from water including purified water, lower alcohols having 1 to 4 carbon atoms, or a mixed solvent thereof.

The non-polar solvent-soluble extract includes an extract soluble in methylene chloride, hexane, chloroform, dichloromethane or ethyl acetate.

The compound can inhibit the neurotoxicity of glutamate, which causes oxidative stress or cell death in neurons.

The compound can alleviate the accumulation of reactive oxygen species (ROS) due to oxidative stress in neurons.

The compound can modulate mitochondrial membrane potential (MMP, ΔΨm) disruption.

The compound may inhibit phosphorylation of mitogen-activated protein kinases (MAPK).

The compound may increase the expression of heme oxygenase 1 (HO-1) in neurons.

The present invention relates to a pharmaceutical composition for preventing or treating dementia comprising a compound derived from an extract of Lespedeza bicolor Turcz. A pharmaceutical composition for preventing or treating dementia comprising a salt as an active ingredient is provided.

1 is an isoflavonoid compound having an efficacy of preventing or treating dementia obtained from Psoriasis extract.
2 is a result showing the neuroprotective effect (*p<0.05, **p<0.01 vs. glutamate group) of compound 1-8 in (A), (B) glutamate-treated HT22 cells, (C) form The microscopic (400 × magnification) results for the change are shown.
Figure 3 shows the inhibitory effect of MLG on glutamate-induced apoptosis, (A) annexin V / PI (annexin V / PI) using double staining to evaluate the level of apoptosis, apoptosis is flow cytometry analysis (B) Immunoblot test results of whole cell lysates of HT22 cells with antibodies specific for Bcl-2, cleaved PARP, AIF, IF-1 and GAPDH (*p<0.05 vs. glutamate) group).
4 shows that MLG attenuated glutamate-induced cell death by preventing reactive oxygen species (ROS) generation and mitochondrial dysfunction. (A) the result of ROS detection through fluorescence intensity measurement using a fluorescence microscope, (B) the result of MMP analysis using flow cytometry, and (C) the result of staining with JC-1 staining (*p<0.05) versus glutamate group).
Figure 5 shows the effect of MLG on the MAPK cascade of HT22 cells undergoing apoptosis induced by glutamate. That is, the expression levels of p-JNK, JNK, p-ERK, and ERK are shown (*p<0.05 vs. glutamate group).
6 shows the effect of MLG on HO-1 expression in HT22 cells. (A) The mRNA levels of HO-1 and GST are the results measured by RT-qPCR (*p<0.05 vs. glutamate group), and (B) the expression of HO-1 evaluated in the presence of MLG at various concentrations. results (*p<0.05 vs. glutamate group), (C) HO-1 inhibitor SnPP inhibited the cytoprotective effect of MLG, HT22 cells were treated with 4 mM glutamate and treated with 0.2 μM SnPP or 10 μM MLG HO-1 expression was measured after incubation (#p <0.05 vs glutamate group; *p <0.05 vs glutamate and MLG treated group).

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described below in detail in conjunction with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in a variety of different forms, only these embodiments allow the disclosure of the present invention to be complete, and common knowledge in the technical field to which the present invention belongs It is provided to fully inform the possessor of the scope of the invention, and the present invention is only defined by the scope of the claims. Detailed contents for carrying out the present invention will be described in detail with reference to the accompanying drawings below. Irrespective of the drawings, like reference numbers refer to like elements, and "and/or" includes each and every combination of one or more of the recited items.

The terminology used herein is for the purpose of describing the embodiments and is not intended to limit the present invention. In this specification, the singular also includes the plural, unless specifically stated otherwise in the phrase. As used herein, “comprises” and/or “comprising” does not exclude the presence or addition of one or more other components in addition to the stated components.

Unless otherwise defined, all terms (including technical and scientific terms) used herein may be used with the meaning commonly understood by those of ordinary skill in the art to which the present invention belongs. In addition, terms defined in a commonly used dictionary are not to be interpreted ideally or excessively unless clearly defined in particular.

Hereinafter, the present invention will be described in more detail through examples. However, these are only for describing the present invention in more detail, and the scope of the present invention is not limited thereto.

[Example 1] Extraction and separation of compounds

For the separation of Compound 1 represented by Equation 1, the ethyl acetate fraction (EA) was subjected to silica gel column chromatography (CC) to obtain four fractions (E1-E4) eluted with CH2Cl2-MeOH. E1 (16.4 g) was further separated using silica gel CC (hexane-ethyl acetate) to give 5 subfractions (H1-H6). Seven subfractions (H18-H24) were obtained from subfraction H1 by RPC18-MLPC. Separation of subfraction M22 (1.5 g) gave compound 1 (44.2 mg) by semi-preparative HPLC using 75% CH ₃ CN in H ₂ O. 2-Geranylbicolosin A (1): brown-yellow solid; [α] ²⁰ _D -163 (c 0.21, MeOH); ECD (Δε+5.85) at 287 nm (MeOH); UV(MeOH)λmax(logε) 284 (3.98), 230 (4.27) nm; IR(MeOH) vmax 3421, 2969, 2923, 1615, 1462, 1348, 1169, 1128, 1072 cm-1; 1H (DMSO-d ₆ , 400 MHz); ¹³ C NMR (DMSO-d ₆ , 100 MHz) data, see Supplementary Table 1; HRTOFMS m/z 504.2877 [M+] (calcd for C ₃₂ H ₄₀ O ₅ , 504.2870).

[Example 2] Cell viability (cell viability)

Various effects of compounds 1 to 8 represented by Formulas 1 to 8 on the viability of HT22 cells were determined by MTT assay. Cells were plated in 96-well plates at 105 cells per well containing 100 μL of growth medium, and cultured for 24 hours. Various concentrations of each compound were added to each well. In addition, MTT solution (5 mg/mL) was added to each well.

The formed formazan precipitate was dissolved in 100 μL of dimethyl sulfoxide, and the cells were incubated for 2 hours. After incubation, absorbance was measured at 562 nm using an automated microplate reader (Bio-Tek, San Diego, CA, USA). Relative cell viability (%) was calculated as the ratio between the absorbance of treated and control (untreated) cells and expressed as a percentage. Experiments were performed three or more times, and each condition was incubated in triplicate.

[Example 3] Microscopy

To observe morphological changes, glutamic acid (4 mM)-treated HT22 cells with or without MLG treatment were examined with a phase-contrast microscope (Olympus, Tokyo, Japan).

[Example 4] Analysis of apoptosis

Apoptotic cell populations were assessed using the Annexin V APC/PI Apoptosis Detection Kit (BioLegend, San Diego, CA, USA). Briefly, HT22 cells were seeded in 6-well plates and treated with various concentrations of MLG. They were then exposed to 4 mM glutamate for 12 and 24 hours. Cells were harvested, washed with PBS, and then stained with annexin V/PI at 37° C. and 5% CO ₂ for 30 minutes. Cells were then analyzed using a flow cytometer (BD FACSVerse, BD Biosciences, San Jose, CA, USA). Data were analyzed using Flow Jo software.

[Example 5] Mitochondrial membrane potential (Δψm)

MMP is tetramethylrhodamine methyl ester perchlorate (TMRM) and MitoProbe JC-1 (5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzyl) Midazolylcarbocyanine (tetraethylbenzimidazolylcarbocyanine) iodide; Thermo Fisher Scientific, Waltham, MA, USA) staining was used. Cells were seeded in 6-well plates and treated with MLG, followed by exposure to 4 mM glutamate for 12 and 24 hours.

After harvesting, cells were incubated with 100 nM cell permeability fluorescent indicator TMRM for 20 minutes. To detect Δψm with MitoProbe JC-1, cells were incubated with 2 μM JC-1 at 37° C. and 5% CO 2 for 30 min.

Samples stained with TMRM or JC-1 were washed with PBS and resuspended in PBS with 1% FBS. Samples were further analyzed on a flow cytometer (BD FACSverse, BD Biosciences, San Jose, CA, USA). Results were analyzed with FlowJo software (Becton, Dickinson and Company, Frankiln Lakes, NJ, USA).

[Example 6] Intracellular ROS analysis

To measure intracellular mitochondrial ROS levels, MitoSOX™ Red mitochondrial superoxide indicator kit (Thermo Fisher Scientific, Waltham, MA, USA) was used. Briefly, MitoSOX™ Red reagent work (5 μM) was added to cells pretreated with MLG and glutamate. In addition, the cells were incubated at 37°C in the dark for 10 minutes. Cells were washed and resuspended in 500 μl PBS with 1% FBS. ROS levels were analyzed by measuring intracellular fluorescence using a flow cytometer (BD FACSVerse, BD Biosciences, San Jose, CA, USA).

[Example 7] Western blot

For Western blot, cells were lysed with RIPA buffer containing 0.1 mg/mL phenylmethylsufony fluoride. The cell lysate was centrifuged at 13000 rpm and 4° C. for 10 minutes.

Protein levels were quantified using a Bradford assay. The same amount of protein was administered by 10% SDS-PAGE, transferred to polyvinylidene difluoride membrane, and incubated with primary antibody in TBST in 1% non-fat dry milk at 4°C.

Specific primary antibodies against Bcl-2, pp38, p38, pJNK, JNK, IF-1, GAPDH and -tubulin were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against cleaved PARP, AIF, HO-1, pERK and ERK were purchased from Cell Signaling Technologies (Danvers, MA, USA). After washing with 1×TBST, incubated with an anti-mouse or anti-rabbit horseradish-peroxidase-conjugated secondary antibody (Jackson Laboratory) for 1 hour for 1 hour. Signals were detected with an Image Quant LAS mini (Fujifilm, Tokyo, Japan).

[Example 8] Reverse transcription polymerase chain reaction (RT-qPCR)

HT22 cells were plated in 6-well plates and incubated with MLG in the presence of 4 mM glutamate. Total RNA was extracted with the RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol.

cDNA was synthesized from 1 μg total RNA using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) and amplified by PCR using specific primers. The PCR reaction mixture consisted of 10 µL of iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA), 1 µL of PCR forward primer (10 µM), 1 µL of PCR reverse primer (10 µM), 1 µL of cDNA template and ddHO. contains 7 µl of qRT-PCR conditions were as follows: 95 °C for 10 min (denaturation), followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s.

This procedure was performed using a StepOnePlus Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA, USA).

All values are expressed as mean ± standard error. Statistical significance was determined by Student's t-test. p<0.05 is considered statistically significant.

[Result analysis]

One) Structural description of novel pterocarpans, 2-geranylbicolosin A (1)

Novel pterocarpans, 2-geranylbicolosin A (1) and 7 isoflavonoids previously reported in L. bicolor after bioassay-induced fractionation and chemical investigation has been separated

When the ¹ H and ¹³ C NMR data of compound 1 represented by Formula 1 and the data of Compound 2 (bicolosin A) represented by Formula 2 are compared in detail, Compound 1 is a geranyl derivative of bicolosin A was shown (Table 1).

The positions of the geranyl side chains range from H-1'[δ _H 3.19 (2H, m)] to C-1 (δ _C 160.0), C-2 (δ _C 115.4) and C-3 (δ _C 157.9) and H From -2′[δ _H 5.16 (1H, m)] to C-2 was identified in C-2 through HMBC correlation. The cis configuration of protons H-6a and H-11a was observed based on the ¹ H- ¹ H binding constant (J = 6.4 Hz) and the ROESY experiment (Fig. 1).

ε+5.85)와 음의 광학 회전([α]²⁰ _D -163, c 0.21, MeOH)에 따라, 화합물 1의 절대 배열은 R형태로 표시되었으며, 앞서 보고된 바와 같이 분리된 프테로카르판(pterocarpans)의 그것과 비교되었다. 따라서, 화합물 1은 게라닐기의 치환에 대해서만 비콜로신 A(bicolosin A)와 다르기 때문에 2-게라닐비콜로신 A(2-geranylbicolosin A)로 명명되었다 (도 2A).The positive Cotton effect at 287 nm (

According to ε + 5.85) and negative optical rotation ([α] ²⁰ _D -163, c 0.21, MeOH), the absolute configuration of compound 1 was represented by the R form, and as previously reported, the isolated pterocarpane ( pterocarpans) compared to that of Therefore, compound 1 was named 2-geranylbicolosin A (2-geranylbicolosin A) because it differs from bicolosin A only with respect to the substitution of the geranyl group ( FIG. 2A ).

2) fight ( L. bicolor ) neuroprotective effect of the isolated compound on glutamate-treated HT22 cells

To evaluate the neuroprotective effect of the isolated compound, HT22 cells treated with glutamate were incubated with the isolated compound. Glutamate is a toxic substance that induces oxidative stress in HT22 cells, causing various damage and neuroexcitotoxicity. HT22 cells were exposed to glutamate (4 mM) for 24 h at concentrations ranging from 0.1 to 10 μM in the presence of isolated compounds.

Cell viability is 3-(4,5-dimethylthizaol-2-yl)-2,5-diphenyltetrazolium bromide (3-(4,5-dimethylthizaol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay and expressed as a percentage of untreated controls.

After incubation with 4mM glutamate for 24 hours, the viability of HT22 cells decreased to 20.5%±0.1%, indicating that 4mM glutamate induced significant toxicity ( FIG. 2B ).

However, its toxic effect was reduced by some compounds obtained from L. bicolor . Structural requirements for protective measures were further analyzed.

Arylbenzofuran (arylbenzofuran) type compound 8 (Formula 8) did not protect HT22 cells from glutamate-induced apoptosis. Similarly, coumestan type compound 7 (Formula 7) did not show a protective effect at a concentration of up to 10 μM.

On the other hand, pterocarpans type compounds (1-6) showed significant neuroprotective effects against glutamate-neurotoxicity.

Of these, 1-methoxylespeflorin G11 (MLG) (5) having a geranyl group at C-2, a prenyl group at C-10, and a methyl group at C-8 is It showed a strong protective effect (Fig. 2B).

In addition, microscopic analysis showed that MLG strongly inhibited glutamate-induced apoptosis in HT22 cells (Fig. 2C). In addition, no significant cytotoxicity was observed when the MLG concentration was 10 μM or less, and a slight cytotoxicity was observed at 25 μM MLG (Supplementary Fig. 1).

Therefore, to further investigate the neuroprotective mechanism, 5 and 10 μM of MLG were used.

3) Attenuation of glutamate-induced apoptosis by MLG in HT22 cells

To investigate the protective activity of MLG against glutamate-induced apoptosis, flow cytometry was performed on damaged cells probed with annexin V/propidium iodide ( FIG. 3A ). The proportion of early apoptotic cells increased after exposure to glutamate for 24 h.

However, when cells were treated with 10 μM of MLG before exposure to glutamate, the proportion of early apoptotic cells was reduced as shown in FIG. 3A. These data demonstrated that MLG attenuated glutamate-mediated premature cell death in HT22 cells as revealed by PI staining.

In western blot analysis, the level of antiapoptotic protein Bcl-2 decreased after treatment with 4 mM glutamate was recovered after treatment with 5 and 10 μM MLG ( FIG. 3B ).

On the other hand, elevation of AIF and cleavage of poly(ADP-ribose) polymerase (PARP), which promote cell death by preventing DNA repair, were dose-dependently inhibited by MLG. IF-1 levels were not significantly different in cells treated with or without MLG.

4) Prevention of glutamate-induced intracellular ROS generation and mitochondrial dysfunction by MLG

Since oxidative stress is a major factor in neuronal cell death, preventing ROS may be a strategy for AD treatment. ROS of mitochondria were investigated using flow cytometry with MitoSOX™ Red staining.

FIG. 4A shows that mitochondrial ROS levels were significantly improved in HT22 cells treated with glutamate compared to controls not treated with glutamate. The results showed that MLG significantly inhibited the accumulation of intracellular ROS increased after glutamate treatment ( FIG. 4A ).

Apoptosis-induced mitochondrial dysfunction was demonstrated by decreased mitochondrial membrane potential (MMP; ΔΨm). To determine MMP, cells were treated with MLG, stained with tetramethylrhodamine methyl ester perchlorate (TMRM), and analyzed using flow cytometry. Glutamate treated cells showed significant membrane potential depolarization after 24 h (Fig. 4B).

The results showed that MLG prevented destruction of MMP by glutamate in a time- and dose-dependent manner. In addition, under glutamate treatment, cells treated with or without MLG were stained with JC-1 to examine whether MMP was disrupted by flow cytometry (FIG. 4C).

Cells treated with glutamate had a more rapid decrease in MMP than the control group.

JC-1 forms aggregates with red fluorescence in the mitochondria of normal cells and residues in the form of green fluorescent monomers in the cytoplasm of apoptotic cells, the latter showing a decrease in MMP. 4C, MLG significantly improved the mitochondrial red/green fluorescence intensity ratio, indicating that MLG prevented glutamate-induced mitochondrial dysfunction.

5) Inhibition of glutamate-induced apoptosis-related proteins and MAPK activation by MLG

MAPK activation plays an important role in the regulation of cell survival and death. Inhibition of MAPK phosphorylation may be a protective mechanism against glutamate-induced neuronal cell death. Additionally, preventing intracellular ROS generation has been reported to inhibit MAPK phosphorylation and apoptosis, suggesting the involvement of ROS-mediated phosphorylation of MAPK in AD. Therefore, the effect of MLG on MAPK activation was further evaluated by examining the phosphorylated ERK, JNK and p38 cascades.

Treatment of HT22 cells with glutamate significantly induced the phosphorylated MAPK cascade. In contrast, MLG inhibited glutamate-induced phosphorylation of MAPK ( FIG. 5 ). These results suggested that inhibition of MAPK phosphorylation is the underlying molecular mechanism of MLG-mediated neuroprotective effects in glutamate-induced apoptosis in HT22 cells.

6) Protection against glutamate-induced cytotoxicity of HT22 cells by MLG through HO-1 induction

To investigate the antioxidant effect of MLG, the mRNA levels of HO-1 and GST, which play key roles in the clearance of oxidized macromolecules, were evaluated. The mRNA level of HO-1 increased noticeably in a dose-dependent manner, whereas the mRNA level of GST did not significantly increase (Fig. 6A).

Previous studies have reported that HO-1 is of considerable interest for its potential neuroprotective effects in apoptosis models. In this study, treatment with MLG slowly increased HO-1 expression in HT22 cells. Using the HO-1 inhibitor SnPP, we investigated whether HO-1 is involved in glutamate-induced cytotoxicity in HT22 cells. As shown in Figure 6A and B, the combined treatment with MLG and SnPP reduced the viability of HT22 cells compared to MLG alone treatment, which involved HO-1 in the MLG-mediated cytoprotective effect on glutamate-induced cytotoxicity. indicates

In the present invention, the neuroprotective effect of isoflavonoids obtained from L. bicolor on HT22 hippocampal cells was investigated.

Among all the compounds, MLG having a part of pterocarpans effectively exhibited a protective effect on HT22 cells even at low concentrations ( FIG. 2B ). As revealed by microscopic analysis, MLG significantly restored glutamate-induced morphological alternation and apoptosis (Fig. 2C).

Glutamate increased intracellular ROS and mitochondrial dysfunction, leading to AIF-dependent apoptosis in neurons.

Since glutamate-induced HT22 apoptosis has typical features of oxidative stress-induced programmed cell death, annexin V/PI staining was used to investigate the relevant apoptosis mechanism in time course experiments.

As a result, it was shown that MLG significantly prevented glutamate-induced cell death (FIG. 3A). The intrinsic apoptosis pathway is mitochondrial-dependent and responds to a variety of stressful conditions, such as excitotoxicity and oxidative stress. Thus, mitochondria play an important role in the regulation of apoptosis.

Low MMP was observed in HT22 cells treated with glutamate according to morphological changes and apoptosis progression. However, MLG prevented degradation of MMP by glutamate in a time- and dose-dependent manner ( FIG. 4B ).

In addition, the expression levels of Bcl-2, PARP, AIF, IF and MAPK were determined by Western blot analysis to study whether caspase regulation is involved in glutamate-induced apoptosis.

MLG treatment resulted in an increase in the level of the antiapoptotic protein Bcl-2 and a decrease in the levels of cleaved PARP and AIF that promote apoptosis ( FIG. 3B ). In addition, glutamate-induced phosphorylation of MAPKs including pp38, pERK and pJNK was significantly reduced by pretreatment with MLG. Previous studies have reported that ROS generation can induce apoptosis through early mitochondrial membrane hyperpolarization that resulted in the disruption of MMPs.

To evaluate the effect of MLG on glutamate-induced oxidative stress, intracellular ROS were measured using MitoSOXTM Red staining and flow cytometry. (Fig. 4A). MLG significantly inhibited the accumulation of intracellular ROS increased by glutamate treatment; Therefore, MLG prevented intrinsic mitochondrial apoptosis in glutamate-treated HT22 cells by regulating ROS and caspase-dependent pathways.

Mitochondrial cell death was assessed by JC-1 assay (Fig. 4C). JC-1 forms aggregates with red fluorescence in the mitochondria of normal cells and remains as a green fluorescent monomer in the cytoplasm of apoptotic cells, the latter showing a decrease in MMP. As expected, MLG significantly restored the mitochondrial red/green fluorescence intensity ratio.

HO-1 is a major antioxidant enzyme that plays an important role in protecting cells from oxidative stress through reduced ROS production. Since the induction of HO-1 by several phytochemicals isolated from natural products has been widely recognized as an effective neuroprotective strategy, HO-1 expression by pharmacological modulators may be a useful target for therapeutic intervention. In the present invention, MLG treatment progressively increased the expression of HO-1 in HT22 cells. In addition, co-treatment of MLG and SnPP reduced the viability of HT22 cells, suggesting that the cytoprotective effect of MLG may be due to the induction of HO-1 expression.

Taken together, the present invention provides insight into the underlying mechanisms for MLG-induced neuroprotection in glutamate-induced apoptosis in HT22 cells. It was demonstrated that MLG suppressed glutamate-induced cell death by abrogating ROS accumulation. In addition, the cytoprotective effect of MLG in glutamate-treated cells was accompanied by a decrease in HO-1 induction and MARK activity. As ROS overproduction becomes increasingly important as a contributing factor in the development of neurological diseases, MLG may serve as a promising natural substance to treat neurodegenerative diseases.

Although the embodiments of the present invention have been described above, the present invention is not limited to the above embodiments, but may be manufactured in a variety of different forms, and those of ordinary skill in the art to which the present invention pertains will appreciate the technical spirit of the present invention. However, it will be understood that the invention may be embodied in other specific forms without changing essential features. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (20)

A pharmaceutical composition for preventing or treating dementia comprising, as an active ingredient, a compound represented by a formula selected from the following Chemical Formulas 1 to 8, or a pharmaceutically composition thereof.
[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

The method of claim 1,
The compound is a pharmaceutical composition for preventing or treating dementia, characterized in that it is derived from an extract of Lespedeza bicolor Turcz. 3. The method of claim 2,
The extract is a crude extract, a polar solvent-soluble extract or a non-polar solvent-soluble extract of C. siri, a pharmaceutical composition for preventing or treating dementia. 4. The method of claim 3,
The preparation is a pharmaceutical composition for preventing or treating dementia, characterized in that it contains an extract soluble in a solvent selected from water including purified water, lower alcohols having 1 to 4 carbon atoms, or a mixed solvent thereof. 4. The method of claim 3,
The non-polar solvent-soluble extract is a pharmaceutical composition for preventing or treating dementia, characterized in that it comprises an extract soluble in methylene chloride, hexane, chloroform, dichloromethane or ethyl acetate. The method of claim 1,
The compound is a pharmaceutical composition for preventing or treating dementia, characterized in that it inhibits the neurotoxicity of glutamate that causes oxidative stress or cell death in nerve cells. The method of claim 1,
The compound is a pharmaceutical composition for preventing or treating dementia, characterized in that alleviating the accumulation of reactive oxygen species (ROS) due to oxidative stress in nerve cells. The method of claim 1,
The compound is a pharmaceutical composition for preventing or treating dementia, characterized in that it modulates the mitochondrial membrane potential (mitochondrial membrane potential, MMP, ΔΨm) collapse. The method of claim 1,
The compound is a pharmaceutical composition for preventing or treating dementia, characterized in that it inhibits phosphorylation of mitogen-activated protein kinases (MAPK). The method of claim 1,
The compound is a pharmaceutical composition for preventing or treating dementia, characterized in that it increases the expression of HO-1 (heme oxygenase 1) in nerve cells. A health functional food for preventing or improving dementia comprising a compound represented by a formula selected from the following formulas 1 to 8 or a pharmaceutically composition salt thereof as an active ingredient.
[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

12. The method of claim 11,
The compound is a health functional food for preventing or improving dementia, characterized in that it is derived from an extract of Lespedeza bicolor Turcz. 13. The method of claim 12,
The extract is a health functional food for preventing or improving dementia, characterized in that it is a crude extract, a polar solvent soluble extract, or a non-polar solvent soluble extract of serrata. 14. The method of claim 13,
The extract is a health functional food for preventing or improving dementia, characterized in that it contains an extract soluble in a solvent selected from water including purified water, lower alcohols having 1 to 4 carbon atoms, or a mixed solvent thereof. 14. The method of claim 13,
The non-polar solvent-soluble extract is a health functional food for preventing or improving dementia, characterized in that it comprises an extract soluble in methylene chloride, hexane, chloroform, dichloromethane or ethyl acetate. 12. The method of claim 11,
The compound is a health functional food for preventing or improving dementia, characterized in that it inhibits the neurotoxicity of glutamate that causes oxidative stress or cell death in nerve cells. 12. The method of claim 11,
The compound is a health functional food for preventing or improving dementia, characterized in that alleviating the accumulation of reactive oxygen species (ROS) due to oxidative stress in nerve cells. 12. The method of claim 11,
The compound is a health functional food for preventing or improving dementia, characterized in that it regulates the mitochondrial membrane potential (MMP, ΔΨm) collapse. 12. The method of claim 11,
The compound is a health functional food for preventing or improving dementia, characterized in that it inhibits phosphorylation of mitogen-activated protein kinases (MAPK). 12. The method of claim 11,
The compound is a health functional food for preventing or improving dementia, characterized in that it increases the expression of HO-1 (heme oxygenase 1) in nerve cells.

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