KR20120060125A - A composition for prevention and treatment of dementia comprising extracts of Morus alba as an active ingredient - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing Morus alba extract is provided to protect cell from toxicity or thapsigargin. CONSTITUTION: A pharmaceutical composition for preventing and treating dementia contains Morus alba extract as an active ingredient. The extract is prepared using water, low carbon number alcohol, or mixture thereof. The dementia is Alzheimer's disease. A health functional food for preventing and treating dementia contains the extract as an active ingredient. A method for preparing the extract comprises: a step of adding extraction solvent to dry Morus alba and extracting; a step of filtering the extract; and a step of decompressing the extract.

Description

A composition for prevention and treatment of dementia comprising extracts of Morus alba as an active ingredient

The present invention is heartache (scientific name: Morus alba ) relates to a pharmaceutical composition or health functional food for the prevention and treatment of dementia containing the extract as an active ingredient.

Looking at the percentage of Korea's aging population, announced by the National Statistical Office in October 2003, Korea entered the aging society as the share of the total population aged 65 and over reached 7.2% in 2000, which is 14% in 2019. It is expected to enter aging society beyond. As the aging issue becomes a social issue, the public's interest in the characteristics of the elderly population, the elderly, such as housing, health, culture, leisure, etc., is increasing, and the demand for statistics is increasing. The key to this change is that chronic degenerative diseases are becoming more of an issue than acute infectious diseases, which have been the leading cause of death for the last 50 years, due to the aging population. In particular, the death from cerebrovascular disease among chronic degenerative diseases is a very important disease that ranks second in the mortality rate from a single disease.

Dementia is dementia when one or more of four, including memory disorders, disorientation, decreased computing power, and changes in personality and emotions, are memory impaired to the extent that they impair normal daily life in occupation, social life, and relationships. Diagnosed with. Dementia is a pathological condition that should be distinguished from normal aging, depending on the cause of Alzheimer's disease, vascular dementia, other alcoholism, trauma, and dementia caused by Parkinson's disease. do. In addition, recent epidemiologic studies have reported that risk factors for cerebrovascular diseases such as hypertension, diabetes, hyperlipidemia, and heart disease increase the incidence of Alzheimer's as well as vascular dementia. It is true.

Alzheimer's disease (AD) is characterized by neuronal loss and extracellular senile plaques, whose major component is amyloid-beta (Aβ), a 39-43 amino acid peptide derived from amyloid precursor protein. senile plaque). In vitro and in vivo studies have shown that Aβ or Aβ peptide fragments have toxic effects, suggesting that Aβ plays an important role in the development of AD (Butterfield et al. al . , Free Radical Biology and Medicine , 2002 , 32: 1050-1060; ButterfIeld et al ., Free Radical Biology and Medicine , 2007, 43: 658-677). In culture, Aβ directly induces neuronal death and renders the neurons vulnerable to excitatory toxicity and oxidative damage. N-methyl-D-aspartate receptor (NMDA) receptors act as selective substrates of Aβ binding or mediators of Aβ-induced glutamate excitatory toxicity. NMDA receptors are highly transmissive in particular ligand in Ca ^{2 +} - gate / overvoltage - is sensitive cation channels. Extensive increase in [Ca ^{2 +]} _i will lead directly to cell dysfunction, over-excitement or death. Thus, the neurotoxic effect of Aβ is (5R.10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo [a, d] cyclohepten-5,10, a non-competitive NMDA receptor antagonist. Reduced by imine maleate (MK-801) [5R.10S]-(+)-5-methyl-10,11-dihydro-5H-dibenzo (a, b) cyclohepten-5,10-imine maleate] as will be demonstrated by the report, Ca ^{+ 2} influx through NMDA receptors by exposure Aβ plays a crucial role in the neurotoxicity induced Aβ-. The formation of reactive oxygen species (ROS) is also believed to be involved in the development of degenerative brain diseases. Some evidence supports the involvement of oxidative stress as an active factor in Aβ-mediated neuropathy by triggering or facilitating neurodegeneration through a wide range of molecular shapes that interfere with neuronal homeostasis. However, the clinical benefits of NMDA receptor antagonists and direct blockers of neuronal channels are controversial, as they lack the identifiable efficacy or have serious side effects.

Heartache (scientific name: Morus alba , English name: white Mulberry) is a deciduous tree belonging to the Mulberry family (Moraceac), when the fruit of mulberry radish is magenta, it is harvested and used as a medicinal herb, and it is known to be used for the treatment of dizziness, tinnitus, browning, and beef Byung-Soo Kang et al., Herbology, Younglimsa, Seoul, Korea 2000), and Japan, it is known to apply to the treatment of tonic, pain medication, insomnia, tinnitus, dizziness, back pain, constipation, etc. (Namba, T., The Encyclopedia of Wakan-Yaku with Color Pictures Vol. 1 Hoikusha, Osaka, Japan, 1993). Dongbogam writes that it governs Sogal, benefits the five chapters, and gathers the mulberry tree's tablets. (Engbobogam Regional Committee, Dongbobogam, Namsan Party, Seoul, Korea, 2000). Heartache has been reported with various vitamins, organic acids, sugars, etc., and research reports on active ingredients are not known.

Therefore, the present inventors are trying to develop a natural material having a therapeutic and prophylactic effect on dementia, while the extract of the heart is a cell against amyloid-β (Abeta) -induced toxicity in cortical and hippocampal cells Protective effect, ER stress (Endoplasmic reticulum stress) toxic to Thapsigargin, mitochondrial membrane potential, reactive oxygen species (ROS), bcl-2, bax and caspase-3 (Caspase-) 3) Completion of the present invention by revealing that the anti-apoptosis effect through measurement and the protective effect on neurons through MAP-2 positive cell count can be useful in the prevention and treatment of dementia. It was.

An object of the present invention is upset (scientific name: Morus alba ) to provide a pharmaceutical composition for the prevention and treatment of dementia containing the extract as an active ingredient, and a dietary supplement for the prevention and improvement of dementia.

In order to achieve the above object, the present invention is the heart (morus name: Morus alba ) provides a pharmaceutical composition for preventing and treating dementia containing the extract as an active ingredient.

In addition, the present invention provides a health functional food for preventing and improving dementia containing the extract of the heart as an active ingredient.

Upset of the present invention (the scientific name: Morus alba ) extracts have been shown to protect the cells against beta amyloid (Abeta) -induced toxicity and cytoprotective effects against Thapsigargin, and by confirming the anti-self killing effect and protection against neurons. The extract may be effectively used in the development of a pharmaceutical composition for preventing and treating dementia or a health food for the purpose.

1 is a heartache (scientific name: Morus alba ) is a graph showing the results of the passive avoidance task after the extract was treated.
Figure 2 is a diagram showing the change in neuroprogenitor cells (doublecortin; DCX) after treating the heart extract.
Figure 3 is a diagram showing the change in the growth of neurites (neurite) after processing the heart extract.
Figure 4 is a graph measuring the level of NGF (nerve growth factor; nerve growth factor) after treatment of the heart extract.
5 is a graph showing the cell viability after treatment of the heart extract.
Figure 6 is a graph showing the cell viability of the heart beat extract pretreatment for beta amyloid (Abeta) toxic treatment.
Figure 7 is a graph showing the anti-self killing effect of the lettuce extract.
8 is a graph showing the number of MAP-2 positive cells.
Figure 9 is a graph showing cell viability using tapsigargin for the heart extract.

Hereinafter, the present invention will be described in detail.

The present invention is heartache (scientific name: Morus alba ) provides a pharmaceutical composition for preventing and treating dementia containing the extract as an active ingredient.

The heart can be used without limitation, such as cultivated or commercially available.

The dementia is preferably Alzheimer's disease, but is not limited thereto.

The lettuce extract is preferably prepared in the following steps, but is not limited thereto.

1) extracting the dried heart by adding an extraction solvent;

2) filtering the extract of step 1); And

3) concentrating the filtered extract of step 2) under reduced pressure.

In the above method, the extraction solvent of step 1) is preferably a solvent selected from water, an alcohol or a mixture thereof, preferably a C ₁ to C ₂ lower alcohol or a mixed solvent thereof, and a 70% aqueous ethanol solution is used. It is more preferable to use, but is not limited thereto. The amount of the extraction solvent is preferably 5 to 15 times the dry weight of the heart, and more preferably 7 to 10 times, but not always limited thereto. The extraction method may be an extraction method such as hot water extraction, immersion extraction, reflux cooling extraction or ultrasonic extraction, but is not limited thereto. The extraction temperature is preferably 10 ℃ to 100 ℃ and more preferably room temperature. The extraction time is preferably 30 minutes to 3 hours, more preferably 1 to 2 hours, but is not limited thereto. The number of extraction is preferably 1 to 5 times, more preferably 3 times, but is not limited thereto.

In the above method, the reduced pressure concentration in step 3) is preferably a vacuum rotary evaporator, but is not limited thereto. In addition, the drying is preferably lyophilized, but is not limited thereto.

The present inventors conducted a passive avoidance test to confirm the simple cognitive function of the heart extract extract, it was confirmed that showing the significant memory enhancement effect when administering the heart extract (see Fig. 1).

In addition, in order to confirm the simple cognitive function of the extract of the heart, extracts were measured by immunohistochemical staining of the changes in the neural precursor cells (doublecortin) and neurites (neurite). It was confirmed that it showed a significant increase in the projections (see FIGS. 2 and 3).

In addition, in order to confirm the effect of improving the simple cognitive function of the lettuce extract, as a result of measuring the NGF (nerve growth factor; nerve growth factor) level, it was confirmed that the NGF significantly increased when administering the lettuce extract (see Fig. 4). ).

Therefore, the extract of the heart of the present invention shows a significant memory enhancing effect, passive increase in neuronal progenitor cells and neurites, and a significant increase in NGF as a result of passive avoidance test, preventing and improving diseases related to cognitive dysfunction Or as an effective ingredient of a therapeutic pharmaceutical composition.

In addition, in order to confirm the protective effect in the cells of the lettuce extract, the cell viability was measured according to the beta amyloid (Abeta) -induced toxicity, the administration of the lettuce extract significantly increased the cell viability compared to the control group It was confirmed that (see Fig. 5 and 6).

In addition, the present inventors measured the cell viability using Thapsigargin, which is toxic to ER stress (Endoplasmic reticulum stress), in order to confirm the protective effect in the cells of the heart beat extract, the cell viability reduced by Topsijin It was confirmed that the dama extract extract was protected by simultaneous treatment (see FIG. 9).

In addition, the present inventors measured the mitochondrial membrane potential, reactive oxygen species (ROS), bcl-2 and bax in order to confirm the anti-apoptosis effect of the lettuce extract, the lettuce extract In the pretreatment group, the inhibition of mitochondrial membrane potential disruption affecting apoptosis signaling, the reduction of ROS that promotes apoptosis, the reduction of bax, a protein that induces apoptosis, and the increase of the anti-apoptosis protein, bcl-2, were confirmed. (See FIG. 7).

In addition, the present inventors measured the number of MAP-2 positive cells in order to confirm the protective effect on the neurons of the heart extract extract, it was confirmed that the MAP-2 positive cell number reduced by Abeta is protected by the heart extract extract pretreatment ( 8).

Therefore, the heart extract of the present invention protects the cells against the toxicity of beta amyloid and top sigajin, has an anti-self killing effect, and has a protective effect on neurons as an active ingredient of the pharmaceutical composition for preventing and treating dementia. It can be usefully used.

The composition of the present invention, preferably containing 0.1 to 90% by weight of the heart extract extract based on the total weight of the composition is not limited thereto.

The composition of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

The composition of the present invention may be administered orally or parenterally, and when parenteral administration, it is preferable to select external or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection injection method. It is not limited thereto.

The composition of the present invention can be used in the form of oral formulations, external preparations, suppositories, and sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, respectively, according to conventional methods. have. Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may be used in the extract of at least one excipient such as starch, calcium carbonate, sucrose ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The preferred dosage of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the composition is preferably administered at 0.0001 to 1 g / kg, preferably 0.001 to 200 mg / kg, but is not limited thereto. The administration may be administered once a day, or may be divided several times. The dose is not intended to limit the scope of the invention in any way.

In another aspect, the present invention provides a health food for preventing and improving dementia containing the extract of the heart as an active ingredient.

The dementia is preferably Alzheimer's but is not limited thereto.

The extract of the present invention protects the cells against the toxicity of beta amyloid and top siginine, has an anti-self killing effect, and has a protective effect on neurons, so it is usefully used as an active ingredient of health food for preventing and improving dementia. Can be.

There is no particular limitation on the kind of the food. Examples of the foods include drinks, meat, sausages, breads, biscuits, rice cakes, chocolates, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, alcoholic beverages and vitamins. Combinations and the like, and include all of the health foods in the conventional sense.

The lettuce extract of the present invention may be added to a food as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to its use purpose (for prevention or improvement). In general, the amount of the extract in the health food can be added at 0.01 to 15% by weight of the total food weight, the health beverage composition can be added in a ratio of 0.02 to 5 g, preferably 0.3 to 1 g based on 100 ml. have. However, in the case of long-term intake for health and hygiene or health control purposes, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

The health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the above-mentioned lettuce extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. .

In addition to the above, the food of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the extract of the present invention may contain a fruit flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not so critical but is usually selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the extract of the present invention.

Hereinafter, the present invention will be described in detail by Examples, Experimental Examples and Preparation Examples.

However, the following Examples, Experimental Examples and Preparation Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples, Experimental Examples, and Preparation Examples.

< Example  1> Heartache Morus alba ) Preparation of extract

100 mL of 70% ethanol was added to 100 g of the heartbeat purchased from Jeong-Do (Seoul), and leached under stirring for 24 hours, followed by filtering using Whatman filter paper # 2. . The filtrate was concentrated under reduced pressure at 50 ° C. (Rotavapor R-200, heating bath B-490, BUCHI; Flawil, Switzerland). The sample was freeze-dried and stored at -20 ° C., and was used in the preparation of experiments. The yield was 20.53. Was%.

< Experimental Example  1> Confirm the effect of improving the simplicity of heartache

<1-1> Experimental Animal Preparation

Experimental animals used male ICR mice (mice) (25 ~ 30 g, Orient). The wound was used after suspension in 10% dimethyl sulfoxide (DMSO). The drugs were all used on the day of the experiment, and divided into three groups as shown in Table 1. Each group was orally administered with solvents and samples for 7 days. After 1 hour of oral administration on the 6th and 7th days, the training trial and the acquisition trial were performed in a manual avoidance test. On the 8th day, a retention trial was conducted.

group p.o. Control group 10% DMSO ME-100 mg / kg 10% DMSO with 100 mg / kg of heart ME-500 mg / kg 10% DMSO with 500 mg / kg of heart

<1-2> Manual avoidance experiment Passive avoidance task )

Training trials and tests were conducted in two separate and independent bright and dark square boxes. Bright areas (20 × 20 × 20 cm) were illuminated with 50 W incandescent bulbs. The light and dark areas (20 × 20 × 20 cm) were spaced 1 cm apart with 2 mm stainless steel rods and laid out the length of the area. The zone was separated by a guillotine door (5 × 5 cm). The mice are placed in the first bright zone and the door between them opens after 10 seconds. When the mice entered the dark area, the door was automatically closed and immediately subjected to an electric foot shock (0.3 mA) through a stainless steel rod for 3 seconds (Acquisition trial). 24 hours after the accumulation trial, the mice were placed in bright areas to measure the retention trial. The time was measured for both the acquisition trial and the retention trial until the rats had all four feet from the light room to the dark room. The mice were orally administered with solvent and heart beat extract 1 hour before the accumulation trial.

As a result, as shown in Figure 1, it was confirmed that statistically significant memory enhancing (memory enhancing) effect when oral administration of the heart extract extract at 100 and 500 mg / kg for 7 days (Fig. 1).

<1-3> Immunohistochemical Staining

Experimental Example <1-1> For immunohistochemical staining, the animals were perfused with 1X PBS (phosphate buffer saline), fixed with 4% paraformaldehyde, and brains were extracted. It was fixed and put in a 30% squarose solution (sucrose) solution was stored every two days until the frozen section at 4 ℃. Thereafter, brain tissue was frozen in a cryostat by dropping the OCT (optimal cutting temperature) compound at -20 ° C, and then made into sections having a thickness of 30 µm and stored in 4 ° C. Immunohistostaining was performed with the hippocampus, and the tissues washed with PBS were treated with 1% H ₂ O ₂ for 15 minutes to prevent nonspecific reactions. 0.05M PBS, 1.5% normal goat serum , 0.5 mg / ml bovine serum albumin, 0.3% triton X-100 and a primary antibody (goat anti-DCX primary antibody, 1: 500) were reacted at 4 ° C. for 24 hours. After removing the primary antibody, the tissue was reacted with a peroxidase-linked secondary antibody (1: 200) for 90 minutes, and then diluted with ABC in a buffer solution for about 1 hour at room temperature. After washing three times with PBS, and then developed with 0.02% DAB and 0.0 1% H ₂ O ₂ slide samples were made through ethanol and xylene dehydration process.

As a result, as shown in Figs. 2 and 3, neuronal progenitor cells (doublecortin; DCX) were significantly increased and oral neurite was significantly increased after oral administration at 100 and 500 mg / kg for 7 days. Confirmed. Therefore, it was confirmed that the long-term administration of heartache is effective in improving the simple cognitive function (Figs. 2 and 3).

<1-4> NGF ( nerve growth factor ; Neural growth factor) level measurement

NGF levels were measured to determine the effect of cognitive improvement on heartache. In order to measure NGF levels, the brains of the experimental animals of Experimental Example <1-1> were extracted, and the hippocampus was homogenated. Chemikine ^TM purchased from Chemicon International (Temecula, CA, USA) NGF levels were measured using an NGF sandwich enzyme-linked immunosorbentassay (ELISA) kit.

As a result, as shown in FIG. 4, it was confirmed that NGF significantly increased upon oral administration at 100 and 500 mg / kg of heartbeat for 7 days. Therefore, it was confirmed that the long-term administration of heart heart is effective in improving the NGF level and thus simple cognitive function (Fig. 4).

< Experimental Example  2> In vitro ( in vitro Heartache  Extract Anti-alzheimer's  Check the effect

<2-1> cerebral cortex ( Cortical Cytoprotective effect in

Amyloid plaque, produced by the accumulation of beta amyloid-β (Abeta), is known as a substance that accumulates in the brain and kills nerve cells, and is used for Alzheimer's research. Abeta (25-35) artificially aggregated (aggregated) material to produce a toxicity to the cerebral cortical cells by using the MTT assay (assay) to determine the cytoprotective effect of the heart extract. Specifically, first, only the cerebral cortex is isolated from the 18-day-old fetus of Sprague-Dawley rats (Orient Bio, Seoul), and then mechanically decomposed to obtain cells, and then poly-L- Cells were seeded at 1 × 10 ⁴ / well in 96 well plates pre-coated with lysine (Poly-L-lysine) and cultured for 7 days. Subsequently, the beta amyloid (Abeta, 8 μg) was treated with 0.1% 1, 10 or 100 μg / mL of the persimmon extract in 0.1% DMSO / B27 free neuronal media. Or non-treated and incubated for 24 hours, after 3 hours of 1 mg / mL MTT treatment, formazan was dissolved in DMSO and absorbance was measured at 570 nm.

As a result, as shown in Figures 5 and 6, when treated with a concentration of 0.1, 1, 10 or 100 ㎍ / mL in the cerebral cortex cells alone, it was confirmed that does not affect the cell viability. In addition, in the case of Abeta toxicity treatment, the cell viability was reduced to 73.68 ± 1.11% compared to the control group, and in the case of the lettuce extract pretreatment group showed a cytoprotective effect at a concentration of 10 or 100 ㎍ / mL (Figs. 5 and 6). .

<2-2> hippocampus ( hippocampal Cytoprotective effect in

In order to confirm the cytoprotective effect of the heart extract, only the hippocampus was isolated from the 18-day-old fetus of Sprague Dawley rats (Orient Bio, Seoul), and mechanically decomposed to obtain cells, followed by poly-L-lysine. Cells were seeded at 1 × 10 ⁴ / well in 96 well flights precoated with 7 days. Subsequently, after 30 minutes of treatment with 0.1, 1, 10 or 100 μg / mL of the persimmon extract in 0.1% DMSO / B27 free neuronal basal medium, beta amyloid (Abeta, 8 μg) was treated or non-treated in total 24 After incubation for 3 hours, 3 mg of 1 mg / mL MTT treatment, formazan was dissolved in DMSO, and absorbance was measured at 570 nm.

As a result, it was confirmed that the cell viability was increased at 1, 10 or 100 ㎍ / mL concentration when compared to the concentration of 0.1, 1, 10 or 100 ㎍ / mL in the seaweed extract alone in the hippocampal cells. In addition, Abeta inhibited the survival of 52.97 ± 0.97% hippocampal cells as compared to the control group, and showed a statistically significant protective effect in pretreatment concentrations of 0.1, 1, 10 or 100 ㎍ / mL of the heart extract.

<2-3> anti-self killing of the lettuce extract ( anti - apoptosis ) effect

In order to confirm the anti-self killing effect of the extract of the heart, extracts were inoculated in a 60 mm plate coated with PLL-coated hippocampal cells, and cultured for 7 days, and the extract of 10 μg / mL in a 0.1% DMSO / B27 free neuronal base medium was used. After 30 minutes of treatment, Abeta (8 ㎍) treated with a total 18 hours of incubation, mitochondrial membrane potential (mitochondrial by measuring the fluorescence of the red and green wavelengths using mitochondrial-specific dye JC-1 membrane potential) was observed.

As a result, it was confirmed that the mitochondrial membrane potential, which affects apoptosis signal transduction, was maintained by the heart extract extract as shown in FIG. 7 (FIG. 7).

<2-4> Anti-Apoptosis of Lettuce Extracts anti - apoptosis ) effect

In order to confirm the anti-self killing effect of the extract of the heart, extracts were inoculated in a 60 mm plate coated with PLL-coated hippocampal cells, and cultured for 7 days, and the extract of 10 μg / mL in a 0.1% DMSO / B27 free neuronal base medium was used. After 30 minutes of treatment, Abeta (8 ㎍) treated with a total of 18 hours incubation, cells were collected and divided into mitochondrial and mitochondrial (cytosol), the protein was extracted and the change was observed using the antibody.

As a result, the mitochondrial membrane potential, which affects apoptosis signaling, is maintained by the heartworm extract pretreatment, as shown in FIG. 7, whereby the concentration of cytochrome C in the cytosol was compared with the Abeta group. It was confirmed that low in (Fig. 7).

<2-5> Anti-Apoptosis of Lettuce Extracts anti - apoptosis ) effect

In order to confirm the anti-self killing effect of the heart extract, the amount of reactive oxygen species (ROS), bcl-2 and bax produced after drug and toxic treatment of hippocampal cells with DCF-DA fluorescent dye (dye) Was measured.

As a result, as shown in Figure 7 when treated with Abeta compared to the control, an increase in ROS that promotes apoptosis, a decrease in the anti-apoptosis protein (bcl-2) and cells While it was confirmed that bax, which is a pro-atpotosis protein, was increased, pretreatment with the heart beat extract showed a decrease in ROS production, an increase in bcl-2, and a decrease in bax (FIG. 7).

<2-6> Anti-self-killing of Lettuce Extracts anti - apoptosis ) effect

In order to confirm the anti-apoptotic effect of the extracts, the inoculated cells were inoculated into a 60 mm plate coated with PLL-coated hippocampal cells, and cultured for 7 days, and 10 µg / mL of the extracts were added to a 0.1% DMSO / B27 free neuronal base medium. After 30 minutes of treatment, the cells were incubated with Abeta (8 ㎍) for 24 hours, and then the cells were collected to collect caspase-3 activity and cleavaged caspase-3 form. ) Were identified using kit (Bio Vision, USA) and Western blot, respectively.

As a result, caspase-3 activity and cleavaged caspase-3 form, which are involved in apoptosis, were reduced by pretreatment of the wick extract as shown in FIG. 7. By confirming that, the anti-self killing effect of the lettuce extract (Fig. 7) was confirmed.

<2-7> MAP -2 Positive cell count  Confirm

In order to measure the protective effect on neurons using MAP-2 antibody, a neuronal marker, hippocampal cells were inoculated in 24 wells coated with PLL and cultured for 7 days, and 0.1% DMSO / B27 free. After treatment for 10 minutes of 10 ㎍ / mL heart extract extract to the neuronal base medium, Abeta (8 ㎍) was incubated for 24 hours. After the reaction was fixed and stained with MAP-2 antibody (antibody) and the number of positive cells were counted and compared with the control.

As a result, as shown in FIG. 8, it was confirmed that the number of MAP-2 positive cells significantly reduced by Abeta was protected by the heart extract extract pretreatment (FIG. 8).

<2-8> Top City ( Thapsigargin ) For

In order to confirm cell viability using TopSigazine, which is toxic to ER stress (Endoplasmic reticulum stress), inoculated with hippocampal cells into PLL-coated 96 well flight at 1 × 10 ⁴ / well for 7 days, followed by 0.1% DMSO / B27-free neuronal basal medium was treated with 0.1, 1, 10, or 100 μg / mL of the persimmon extract, followed by incubation for 24 hours with or without treatment with Topsigin (25 nM). After 3 hours of 1 mg / mL MTT treatment, formazan was dissolved in DMSO and absorbance was measured at 570 nm.

As a result, as shown in Figure 9, it was confirmed that the cell viability reduced by the top sigajin is protected by the co-treatment extract (Fig. 9).

< Manufacturing example  1> Preparation of Pharmaceutical Formulations

<1-1> Powder  Produce

2 g of lettuce extract of the present invention

1 g lactose

The above components were mixed and packed in airtight bags to prepare powders.

<1-2> Preparation of Tablet

100 mg of lettuce extract of the present invention

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

&Lt; 1-3 > Preparation of capsules

100 mg of lettuce extract of the present invention

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

&Lt; 1-4 >

1 g of lettuce extract of the present invention

Lactose 1.5 g

1 g of glycerin

Xylitol 0.5 g

After mixing the above components, it was prepared to be 4 g per ring according to a conventional method.

<1-5> Preparation of granules

150 mg of lettuce extract of the present invention

Soybean Extract 50mg

Glucose 200 mg

Starch 600 mg

After mixing the above components, 100 mg of 30% ethanol was added and dried at 60 ° C. to form granules, which were then filled into fabrics.

< Manufacturing example  2> Manufacture of food

Foods containing the heart beat extract of the present invention was prepared as follows.

<2-1> Production of flour food

0.5 ~ 5.0 parts by weight of the lettuce extract of the present invention was added to flour, and bread, cake, cookies, crackers and noodles were prepared using this mixture.

<2-2> soup  And juicy ( gravies Manufacturing

0.1 ~ 5.0 parts by weight of the lettuce extract of the present invention was added to soups and gravy to prepare meat products for health promotion, soups and gravy of noodles.

<2-3> ground Beef  Produce

10 parts by weight of the lettuce extract of the present invention was added to the ground beef to prepare a ground beef for health promotion.

<2-4> dairy products ( dairy products Manufacturing

5 to 10 parts by weight of the lettuce extract of the present invention was added to milk, and various milk products such as butter and ice cream were prepared using the milk.

<2-5> Solar  Produce

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and the mixture was granulated to a powder having a particle size of 60 mesh.

Black soybeans, black sesame seeds, and perilla seeds were steamed and dried by a conventional method, and then they were prepared into powder having a particle size of 60 mesh by a pulverizer.

The concentrated heart extract of the present invention was concentrated under reduced pressure in a vacuum concentrator, and the dried product obtained by drying with a sprayer and a hot air dryer was pulverized with a particle size of 60 mesh to obtain a dry powder.

The grains, seeds and the extract of the present invention prepared according to the present invention were prepared by combining the following ratios:

Cereals (30 parts by weight brown rice, 15 parts by weight brittle, 20 parts by weight of barley),

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

Lettuce extract of the present invention (3 parts by weight),

Ganoderma lucidum (0.5 parts by weight), and

Sulfur (0.5 part by weight).

< Manufacturing example  3> Manufacturing of beverage

<3-1> Health drink  Produce

Instant sterilization by homogeneously combining 5 g of the heart extract extract of the present invention with subsidiary materials such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%) and water (75%). Then it was prepared by packaging in a small packaging container such as glass bottles, plastic bottles.

<3-2> Preparation of Vegetable Juice

Vegetable juice was prepared by adding 5 g of the lettuce extract of the present invention to 1,000 ml of tomato or carrot juice.

<3-3> Preparation of Fruit Juice

1 g of the lettuce extract of the present invention was added to 1,000 ml of apple or grape juice to prepare a fruit juice.

Claims (7)

Heartache (scientific name: Morus alba ) pharmaceutical composition for the prevention and treatment of dementia containing the extract as an active ingredient.
The method of claim 1, wherein the extract is water, C ₁ To C ₂ Pharmaceutical composition for the prevention and treatment of dementia, characterized in that extracted with a lower alcohol, or a mixture thereof.
The method of claim 2, wherein C ₁ To C ₂ Lower alcohol is a pharmaceutical composition for preventing and treating dementia, characterized in that ethanol or methanol.
The method of claim 1, wherein the dementia is Alzheimer's (AD; Alzheimer's disease), characterized in that the pharmaceutical composition for preventing and treating dementia.
Health functional food for the prevention and improvement of dementia, containing the extract of the heart as an active ingredient.
6. The dietary supplement for preventing and improving dementia according to claim 5, wherein the extract is extracted with water, ethanol or a mixture thereof.
6. The dietary supplement for preventing and improving dementia according to claim 5, wherein the dementia is Alzheimer's.

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