KR20170023512A - Composition for improvement of learning and memory function comprising broccoli leaf extract or its fraction as effective component - Google Patents
Composition for improvement of learning and memory function comprising broccoli leaf extract or its fraction as effective component Download PDFInfo
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- KR20170023512A KR20170023512A KR1020150118751A KR20150118751A KR20170023512A KR 20170023512 A KR20170023512 A KR 20170023512A KR 1020150118751 A KR1020150118751 A KR 1020150118751A KR 20150118751 A KR20150118751 A KR 20150118751A KR 20170023512 A KR20170023512 A KR 20170023512A
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- broccoli
- extract
- leaf extract
- chloroform
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Abstract
The present invention relates to a health functional food composition for improving cognitive function or memory ability containing a broccoli leaf extract or a fraction thereof as an active ingredient, and a pharmaceutical composition for preventing or treating degenerative brain disease, and more particularly, The extract or fraction thereof has excellent antioxidative, lipid peroxidation inhibitory and acetylcholinesterase (AChE) inhibitory effects, thus effectively protecting the brain nerve cells and having an excellent recovery effect on the learning and learning ability in the Alzheimer- It can be usefully used in a composition for preventing, ameliorating or treating cognitive function or memory ability.
Description
The present invention relates to a composition for improving cognitive function or memory ability containing a broccoli leaf extract or a fraction thereof as an active ingredient, and more particularly to a composition for improving cognitive function or memory ability comprising a broccoli leaf extract or a fraction thereof, which can be used as a health functional food or a pharmaceutical composition A composition for improving cognitive function or memory ability.
With advances in medical technology and improved living standards, the average life expectancy of human beings has doubled over the past half century, resulting in a rapid increase in the proportion of the elderly population to the total population. According to the 2005 data released by the National Statistical Office, the average life expectancy of people in Korea is 78.5 years, and in 2000, the proportion of people over 65 years old has already reached 7%. In addition, it is predicted that Korea will enter an aging society with an elderly population of 20% or more in 2028 and an elderly population in 2018. In 2004, 12.1% of elderly dependents (population aged 65 and over / population aged 15 ~ 64) are expected to increase to 21.3% in 2020 and 35.7% in 2030. In 2004, If you support it, you will be supporting one elder per person in 6.8 in 2010, one elder per 4.7 people in 2020, and one elder per 2.8 in 2030. This implies that the possibility of social burden due to the deterioration of economic growth, national competitiveness,
As reflected in these social prospects and realities, attention has recently been focused on the diseases that afflict the elderly. In particular, the incidence of dementia is significantly higher in older adults, so it is known that the incidence rate of elderly people aged 75 to 84 is about 20% and that of elderly people aged 85 and older is about 50% It is becoming a serious social concern because it must be absolutely dependent. In addition, the economic loss due to dementia is becoming very large, and the social cost of treatment for dementia in Korea is estimated to be 3.4 trillion to 4.4 trillion won in consideration of all aspects such as work loss. In the United States, the cost of treating dementia is estimated to be in the range of $ 1,000 to $ 120 billion per year. In other words, as society becomes more and more aged, senile dementia is emerging as the greatest health problem that humanity needs to solve in the 21st century. Accordingly, there is a growing demand for the development of functional materials and foods capable of preventing and treating cognitive dysfunction, including dementia.
Alzheimer's disease is a neurodegenerative disorder that causes gradual loss of cognitive function and memory ability. It is a result of studies that acetylcholine neurons in the cerebral basement are damaged. Muscarinic acetylcholine receptor drugs capable of enhancing the function of acetylcholinergic neurons have been developed before various functional groups such as agonist, acetylcholine production promoter and acetylcholinesterase (AChE) inhibitor for muscarine acetylcholine receptor . In fact, most of the drugs currently used in Alzheimer's disease are acetylcholinesterase inhibitors, including tacrine and donepezil. In addition, rivastigmin (rivastigmin), galatamin (galatamine) are being used.
Currently, there is a strong demand for the development of more effective drugs (including health functional food materials) for improving cognitive function or memory capacity as a degenerative brain disease. In conclusion, it is necessary to develop a new multi-functional effect introducing the mechanism of neuronal cell for the development of dementia.
Meanwhile, broccoli (broccoli, Brassica oleracea var italica ) is a part of the cruciferous vegetables, also known as cauliflower, and is a type of cabbage that is made from edible buds and stems. Broccoli is a nutritionally important crop, cultivated and edible all over the world, and contains excellent antioxidant dithiolthione, organosulfuric compounds such as isothiocyanate, beta carotene β-carotene, rutin, vitamin C, selenium, quercetin, and glutathione. Current related researches have been reported on the reduction of blood cholesterol by water-soluble dietary fiber, the physiological effects such as cancer prevention and mutation inhibition, and the antioxidative and antimicrobial effects of broccoli. Although the demand for broccoli is increasing due to this physiological effect, only the bud portion of broccoli is used for edible purposes, and more than 70% of leaves and stems that can be utilized as other edible resources are abandoned .
Korean Patent Publication No. 2012-0054947 discloses a glucosinolate compound derived from broccoli and a composition having antioxidant and antimicrobial activity containing it. Korean Patent Laid-Open Publication No. 2013-0087174 discloses a composition containing an extract of Broccoli hot water as an active ingredient Lt; RTI ID = 0.0 > inflammatory < / RTI > However, a composition for preventing, improving or treating cognitive function or memory ability containing the broccoli leaf extract of the present invention or a fraction thereof as an active ingredient is not known.
The present invention has been made in view of the above-mentioned needs, and it is an object of the present invention to provide a composition for preventing, improving or treating cognitive function or memory ability containing Broccoli leaf extract or a fraction thereof as an active ingredient. The broccoli leaf extract or its fractions according to the present invention are excellent in acetylcholinesterase (AChE) inhibitory effect, brain nerve stress inhibitory effect and neuronal cell protection ability and can be used for prevention, improvement or treatment of cognitive function or memory ability By confirming, the present invention has been completed.
In order to achieve the above object, the present invention provides a health functional food composition for improving cognitive function or memory ability containing a broccoli leaf extract or a fraction thereof as an active ingredient.
The present invention also provides a pharmaceutical composition for preventing or treating a degenerative brain disease comprising an extract of Broccoli leaf or a fraction thereof as an active ingredient.
The broccoli leaf extract or its fractions of the present invention have excellent antioxidative, lipid peroxidation and acetylcholinesterase (AChE) inhibitory effects, thus effectively protecting brain nerve cells, and exhibiting excellent recovery in cognition and learning ability in an Alzheimer- And thus can be usefully used in compositions for preventing, improving or treating cognitive function or memory ability.
FIG. 1 shows the results of measuring the total phenol contents of fractions obtained from broccoli leaf extract according to an embodiment of the present invention.
FIG. 2 shows the results of measurement of ABTS radical scavenging activity of the fractions obtained from broccoli leaf extract according to an embodiment of the present invention.
FIG. 3 shows the malondialdehyde (MDA) inhibitory activity of the lipid peroxidation intermediate during the treatment of the fractions obtained from the broccoli leaf extract according to an embodiment of the present invention.
FIG. 4 shows the oxidative stress inhibitory activity of the chloroform fraction obtained from the broccoli leaf extract according to an embodiment of the present invention when treated with hydrogen peroxide.
FIG. 5 shows cell viability of PC12 neuronal cells treated with the concentration of chloroform fraction obtained from broccoli leaf extract according to an embodiment of the present invention when hydrogen peroxide was treated.
FIG. 6 is a graph showing the protective effect of the neurotropic membrane when treated with the concentration of the chloroform fraction obtained from the broccoli leaf extract according to an embodiment of the present invention when treated with hydrogen peroxide.
FIG. 7 shows the effect of treating the amyloid beta with the concentration of the chloroform fraction obtained from the broccoli leaf extract according to an embodiment of the present invention to inhibit oxidative stress formation.
FIG. 8 shows cell viability of PC12 neuronal cells treated with the concentration of chloroform fraction obtained from broccoli leaf extract according to an embodiment of the present invention when amyloid beta was treated.
FIG. 9 is a graph showing the effect of treatment with amyloid beta on brain cell membrane protection upon treatment of the concentration of chloroform fraction obtained from broccoli leaf extract according to an embodiment of the present invention.
FIG. 10 shows acetylcholinesterase (AChE) inhibitory activity of the chloroform fraction obtained from the broccoli leaf extract according to an embodiment of the present invention.
FIG. 11 shows the results of a test for evaluating the recovery ability of the chloroform fractions obtained from the broccoli leaf extract for the mouse with reduced spatial cognition using amyloid beta according to an embodiment of the present invention. (A) is an alternative behavior; (B) shows the number of passes through the arm.
FIG. 12 is a result of evaluation test of recovery ability of chloroform fractions obtained from Broccoli leaf extract for short-term memory and learning-deficient mice using amyloid beta according to an embodiment of the present invention.
FIG. 13 is a graph showing the results of a test for evaluating the recovery ability of the chloroform fractions obtained from the broccoli leaf extract for long-term memory and learning-deficient mice using amyloid beta according to an embodiment of the present invention. (A) shows the time required for the mouse to find the platform; (B) shows the measurement of the time that the platform has passed (W zone) after the platform is removed on the 5th day of experiment.
14 shows the results of measurement of acetylcholinesterase (AChE) activity in brain tissues of mice that were fed with the chloroform fraction obtained from the broccoli leaf extract according to an embodiment of the present invention.
FIG. 15 is a graph showing the results of measurement of superoxide dismutase (SOD) content in brain tissues of mice fed with the chloroform fraction obtained from broccoli leaf extract according to an embodiment of the present invention.
FIG. 16 is a graph showing the results of measurement of oxidative glutathione (GSSH) / total glutathione content in brain tissue of mice fed with the chloroform fraction obtained from broccoli leaf extract according to an embodiment of the present invention.
FIG. 17 is a graph showing the results of measurement of malondialdehyde (MDA) content in brain tissue of mice fed with the chloroform fraction obtained from broccoli leaf extract according to an embodiment of the present invention.
In order to accomplish the object of the present invention, the present invention provides a health functional food composition for improving cognitive function or memory ability containing a broccoli leaf extract or a fraction thereof as an active ingredient.
In the health functional food composition for improving cognitive function or memory ability of the present invention, the broccoli leaf extract may be an extract of a solvent selected from the group consisting of water, a lower alcohol having 1 to 4 carbon atoms, and a mixed solvent thereof, May be, but not limited to, ethanol extracts.
The broccoli leaf extract may contain any one of the extract obtained by the extraction treatment, the diluted or concentrated liquid of the extract, the dried product obtained by drying the extract, or the adjusted product or the purified product.
In addition, in the health functional food composition for improving the cognitive function or memory ability of the present invention, the broccoli leaf extract can be extracted by various extraction methods such as cold extraction, reflux cooling extraction, heat extraction, ultrasonic extraction, Preferably, reflux cooling extraction is preferred. In the above extraction method, fractions having high activity from the broccoli leaf extract can be obtained without breaking the active ingredient.
In the health functional food composition for improving cognitive function or memory ability of the present invention, the fraction may be obtained by fractionating the ethanol extract of broccoli leaves with hexane, chloroform, ethyl acetate, butanol or water, preferably chloroform But is not limited thereto.
The functional food composition for improving cognitive function or memory ability of the present invention may inhibit the activity of acetylcholinesterase, but is not limited thereto.
The health functional food composition for improving the cognitive function or memory ability of the present invention can be manufactured by any one of powder, granule, ring, tablet, capsule, candy, syrup and beverage, but is not limited thereto.
The health functional food composition is not particularly limited as long as it can be ingested to improve cognitive function or memory ability.
When the health functional food composition of the present invention is used as a food additive, the health functional food composition may be added as it is, or may be used together with other food or food ingredients, and suitably used according to a conventional method. The active ingredient may be suitably used depending on its intended use (prevention or improvement). Generally, the health functional food composition of the present invention is added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight based on the raw material, when the food or beverage is produced. However, in the case of long-term intake intended for health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount of more than the above range.
There is no particular limitation on the kind of the health functional food. Examples of the foods to which the health functional food composition can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen and other noodles, gums, ice cream, soups, Drinks, alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.
In addition, the health functional food composition of the present invention can be produced as a food, particularly a functional food. The functional food of the present invention includes components that are ordinarily added in food production, and includes, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, in the case of a drink, a natural carbohydrate or a flavoring agent may be included as an additional ingredient in addition to the active ingredient. The natural carbohydrate may be selected from the group consisting of monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g., dextrin, cyclodextrin, For example, xylitol, sorbitol, erythritol, etc.). The flavoring agent may be a natural flavoring agent (e.g., tau Martin, stevia extract, etc.) and a synthetic flavoring agent (e.g., saccharin, aspartame, etc.).
In addition to the above health functional food composition, it is also possible to use various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusters, stabilizers, preservatives, glycerin, A carbonating agent used in beverages, and the like. Although the ratio of the above-mentioned ingredients is not critical, it is generally selected in the range of 0.01 to 0.1 part by weight based on 100 parts by weight of the health functional food composition of the present invention.
The present invention also provides a pharmaceutical composition for preventing or treating a degenerative brain disease comprising an extract of Broccoli leaf or a fraction thereof as an active ingredient.
In the pharmaceutical composition for preventing or treating neurodegenerative diseases of the present invention, a neurodegenerative disease caused by degeneration of cognitive function or memory ability is a degenerative brain disease, and typical degenerative brain diseases include Alzheimer's disease Alzheimer's disease, Progressive supranuclear palsy, Striatonigral degeneration, Huntington's disease, Amyotrophic lateral sclerosis (ALS), Cortical-basal ganglia (Cortico but are not limited to, basal ganlionic degeneration, Diffuse Lewy body disease, Parkinson-ALS-dementia complex of Guam, or Pick's disease.
The pharmaceutical composition for preventing or treating the degenerative brain disease of the present invention may comprise 0.02 to 80% by weight, preferably 0.02 to 50% by weight of the extract or fraction thereof, based on the total weight of the pharmaceutical composition.
The pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients or diluents conventionally used in the manufacture of pharmaceutical compositions.
The pharmaceutical dosage forms of the compositions according to the invention may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable set.
The pharmaceutical composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols or the like, oral preparations, suppositories or sterilized injection solutions according to a conventional method . Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention include lactose, textol sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, and methylhydroxybenzoate. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, . In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of liquid formulations for oral use include suspensions, solutions, emulsions and syrups. In addition to water and liquid paraffin which are commonly used diluents, various excipients such as wetting agents, suspending agents, emulsions, freeze- And the like. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of suppository bases include tapestry, macrogol, tween 61, cacao paper, laundromat, glycerogelatin and the like.
The preferred dosage of the composition of the present invention may vary 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, and may be appropriately selected by those skilled in the art. However, for the desired effect, the fraction of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg, on a daily basis. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.
The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.
In the pharmaceutical composition for preventing or treating a degenerative brain disease of the present invention, the broccoli leaf extract may be an extract of a solvent selected from the group consisting of water, a lower alcohol having 1 to 4 carbon atoms, and a mixed solvent thereof, May be, but not limited to, an ethanol extract.
In the pharmaceutical composition for preventing or treating degenerative brain disease according to the present invention, the fraction may be fractionated from the ethanol extract of broccoli leaves using hexane, chloroform, ethyl acetate, butanol or water, But is not limited to,
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited thereto.
Manufacturing example 1. Broccoli leaf extract or its Fraction Produce
The broccoli leaves used in the present invention were supplied from Lombom Farm, Yeoju, Gyeonggi Province and used as a material. 40 g of broccoli leaves were lyophilized, pulverized, and 2 l of 95% (v / v) ethanol was added thereto, followed by reflux cooling for 2 hours to obtain an ethanol extract. Next, the ethanol extract was treated with No. 2 filter paper (Whatman plc., Kent, UK) and concentrated. The broccoli leaves ethanol extracts n - hexane and water to 1: into a fraction funnel 1 ratio n - was fractionated with hexane layer and an aqueous layer, the aqueous layer re-chloroform in a 1: into a fraction funnel 1 ratio fractions of chloroform and an aqueous layer Respectively. Ethyl acetate was added in the same manner as described above, followed by water layer and fractionation, followed by concentration and lyophilization using a freeze dryer.
Example 1. Solvent from broccoli leaf extract Of fractions Determination of total phenolic compounds
9 ml of distilled water was added to 1 ml of the fraction (hereinafter referred to as "sample solution") obtained from the broccoli leaf extract prepared in Preparation Example 1, and 1 ml of the Folin-Ciocalteau's phenol reagent was added thereto The mixture was reacted at room temperature for 5 minutes. To the reaction solution, 10 ml of a 7% sodium carbonate (Na 2 CO 3 ) solution was added and mixed again, followed by diluting with 25 ml of distilled water. The mixed solution was allowed to stand at room temperature for 2 hours and absorbance was measured at 760 nm. The total absorbance of the phenolic compound was calculated from the calibration curve using gallic acid. As a result, the total phenolic compound content of the Broccoli leaf (BL) as shown in Fig. 1 was 95% (v / v) ethanol 51.75, 48.67 n- hexane in dried broccoli leaf, The highest total phenolic compound content in the chloroform fractions was found to be in chloroform 206.75, ethyl acetate 175.50, butanol 111.75, and distilled water 20.50 mg GAE / g. This indicates that the total phenolic compound content was increased about four times in the chloroform fraction when compared to the fraction before (95% ethanol extract).
Example 2. Solvent from broccoli leaf extract Of fractions ABTS Radical Measurement of scavenging activity
And 2.5 mM ABTS (2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt) and 1.0 mM AAPH (2,2'-azobis- (2-amidinopropane) hydrochloride) The mixture was mixed with 100 mM phosphate buffer (pH 7.4) and adjusted at 734 nm so that the absorbance value of the control group became 0.70 ± 0.02. 20 μl of the sample solution and 980 μl of the ABTS solution were mixed and reacted for 10 minutes and the radical scavenging activity was calculated by measuring the absorbance at 734 nm using a UV-fluorescence photometer (UV-1601, Shimadzu Co., Japan). As a result, as shown in FIG. 2, the solvent fraction of broccoli leaves showed the highest ABTS radical scavenging activity in the chloroform fraction, and in particular, vitamin C (1,000 μg), which is a positive control in 1,000 μg / ml of broccoli leaf chloroform fraction, / Ml), which showed no statistically significant difference.
Example 3. Solvent from broccoli leaf extract Of fractions MDA Production inhibitory effect
In order to examine the lipid peroxidation inhibitory activity of mouse brain membrane, the activity of inhibiting the production of malondialdehyde (MDA), a lipid peroxidation product, was measured by the following method. The mouse brain tissue was homogenized in 10 times volume of Tris-HCl buffer (20 mM, pH 7.4) and centrifuged at 12,000 g for 15 minutes at 4 ° C. To 0.1 ml of the supernatant, 0.1 ml of 10 μM iron sulfate, 1 ml of 0.1 mM vitamin C and 0.2 ml of the sample solution were added and cultured at 37 ° C. for 1 hour. The reaction was terminated by the addition of 0.1 ml of 30% trichloroacetic acid to the reaction solution, and 0.3 ml of 1% thiobarbituric acid was added. The mixture was heated at 80 ° C for 30 minutes and then absorbed at 532 nm Were measured.
As a result, as shown in FIG. 3, the concentration of the fraction of broccoli leaf extract was increased in a manner similar to the ABTS radical scavenging activity pattern, thereby suppressing MDA production. In particular, the chloroform fraction showed statistically significant results at a concentration of 500 μg / ml or more when catechin used as a positive control was compared with 75.31% inhibition of MDA production. As a result, the following experiment was conducted using chloroform fractions having excellent antioxidative activity among the solvent fractions of broccoli leaves.
Example 4. Chloroform from broccoli leaf extract Fraction Hydrogen peroxide-induced Oxidative Nerve cell protection effect on stress
To measure the effect of the chloroform fraction obtained from leaf extract of broccoli leaves on the oxidative stress damage of PC12 neurons by hydrogen peroxide (H 2 O 2 ), DCF-DA (2 ', 7'-dichloro-fluorooleate diacetate) Method. First, the cells were divided into 96-well plates at 2.5 × 10 4 cells / well. Broccoli-leaf chloroform fractions were treated at different concentrations and cultured at 37 ° C. and 5% CO 2 for 48 hours. After incubation, 200 μM hydrogen peroxide (H 2 O 2 ) was added and incubated for 3 hours. 10 μM DCFH-DA was added. After 50 minutes, the medium was removed and washed with phosphate buffered saline (PBS) (
Example 5. Chloroform from broccoli leaf extract Fraction Hydrogen peroxide-induced Oxidative About stress MTT Measurement of cell viability by analysis
The protective effect on PC12 cells induced by hydrogen peroxide (H 2 O 2 ) was measured by MTT (3- (4,5-dimethyldiazol-2-yl) -2,5- . The chloroform fraction obtained from broccoli leaf extract was treated with PC12 cells at different concentrations and preincubated for 48 hours. Hydrogen peroxide was then dissolved in distilled water at a concentration of 200 μM, and then cultured for 3 hours. PC12 cells were treated with MTT storage solution (10 μl / well) and incubated at 37 ° C. for 3 hours. Then, the medium was removed and 100 μl of MTT solubilization solution (DMSO) was added to terminate the reaction . Finally, the absorbance was measured at 570 nm (determination wave) and 630 nm (reference wave) in a microplate reader (680, Bio-rad, Japan). Vitamin C (200 μM) was used as a positive control and cell viability was expressed as a% concentration relative to the control group. The survival rate of PC12 neurons in the oxidative stress induced by hydrogen peroxide was measured by MTT assay. As shown in FIG. 5, the survival rate of the treated plants treated with hydrogen peroxide was 77% as compared with the control (control) And vitamin C at the same time, the survival rate was about 29% (106%). The survival rate of 5 and 10 ㎍ / ㎖ of broccoli leaves treated with chloroform fraction showed a significant survival rate compared to the positive control, vitamin C (200 μM) above 20 ㎍ / ㎖, And the survival rate was increased with increasing concentration in the above treatments.
Example 6. Chloroform from broccoli leaf extract Fraction Hydrogen peroxide-induced Oxidative Suppression of cell membrane damage by stress
The extracts were centrifuged for 4 hours, followed by centrifugation (400 g) for 5 minutes, and 100 μl of each of the extracts was added to the wells. After the pre-incubation for 48 hours, the cells were treated with 200 μM hydrogen peroxide (H 2 O 2 ) The supernatant was transferred to a new well and the effect of cell membrane damage was measured with an LDH (lactate dehydrogenase) assay kit (Sigma Chemical Co, St. Louis MO., USA). In order to confirm the inhibitory effect of the chloroform fraction of broccoli leaves on the damage of nerve cell membrane induced by hydrogen peroxide (H 2 O 2 ), cytoplasmic component LDH analysis was performed. As a result, as shown in FIG. 6, The amount of LDH emission was about 35%, while the amount of LDH released by hydrogen peroxide was about 17% increased by hydrogen peroxide. In the control group, vitamin C (200μM) treated group showed about 35% LDH release. Broccoli leaf chloroform fraction treated with 5, 10, 20, 25, 50 and 100 μg / (100 μg / ㎖), similar to the treatment with vitamin C (200 μM).
Example 7. Chloroform from broccoli leaf extract Fraction Amyloid beta Amyloid beta, Aβ) One -42 Induced Oxidative Nerve cell protection effect on stress
DCF-DA (2 ', 7'-dichloro-p-dioleate diacetate) method was used to measure the effect of chloroform fraction obtained from leaf extract of Broccoli leaf on the oxidative stress damage of PC12 neurons by Aβ 1-42 Respectively. First, the cells were dispensed into a 96 well plate at 2.5 × 10 4 cells / well, and the broccoli-leaf chloroform fraction was treated for each concentration and cultured at 37 ° C. and 5% CO 2 for 3 hours. After culturing, 100 μg / ml of Aβ 1-42 was added, and the cells were cultured for 24 hours. 10M DCFH-DA was added thereto, and after 50 minutes, the medium was removed and washed with phosphate buffered saline (PBS) (
As a result of measuring the inhibitory effect of the chloroform fraction of broccoli leaves on the induction of oxidative stress formation induced by A? 1-42 in PC12 neuron cells by the DCF-DA assay method, it was found that Aβ 1-42 alone treatment In the group treated with broccoli leaf chloroform fraction, the oxidative stress was increased by about 31% compared to the control (131.l%), whereas it was about 78.75% at 10, 20, 50 and 100 ㎍ / ㎖ Aβ 1-42- treated group, the inhibition of oxidative stress-induced neuronal cell death was confirmed.
Example 8. Chloroform from broccoli leaf extract Fraction Amyloid beta Amyloid beta, Aβ) One -42 Induced Oxidative About stress MTT Measurement of cell viability by analysis
The protective effect on PC12 cells induced by A [beta] l- 42 was measured by the MTT (3- (4,5-dimethyldiazol-2-yl) -2,5-depenetrazolium promeptide) . The chloroform fraction obtained from the broccoli leaf extract was treated with PC12 cells at different concentrations and preincubated for 3 hours. 100 μg / ml of Aβ 1-42 was added thereto, followed by culturing for 24 hours. PC12 cells were treated with MTT storage solution (10 μl / well) and incubated at 37 ° C. for 3 hours. Then, the medium was removed and 100 μl of MTT solubilization solution (DMSO) was added to terminate the reaction . Finally, the absorbance was measured at 570 nm (determination wave) and 630 nm (reference wave) in a microplate reader (680, Bio-rad, Japan). As a result of measuring the survival rate of PC12 neuron cells in the oxidative stress state induced by A? 1-42 , as shown in FIG. 8, it was found that 69% of the cells treated with A? 1-42 alone And survival rate of Aβ 1-42 and vitamin C was 94%, which was about 25%. (5, 10, 20, 50, and 100 ㎍ / ㎖) of broccoli leaves treated with chloroform fraction showed a higher survival rate compared to the control (contorl). In particular, the cell viability was higher than that of vitamin C (200 μM) at 5 μg / ml.
Example 9 Inhibitory Effect of Chloroform Fractions from Broccoli Leaf Extracts on Cell Membrane Damage
After the concentration-specific extract was pre-incubated for 3 hours, 100 μg / ml of Aβ 1-42 was treated and cultured for 24 hours, followed by centrifugation (400 g) for 5 minutes to precipitate 100 μl of The supernatant was transferred to a new well and the effect of cell membrane damage was measured with an LDH (lactate dehydrogenase) assay kit (Sigma Chemical Co, St. Louis MO., USA). Results of the LDH assay method of the cellular component to determine broccoli inhibiting cell membrane damage in leaf chloroform fraction effect on the nerve cell membrane damage induced by Aβ 1-42, LDH emission amount of control (control), as described in FIG. 9 11%, whereas Aβ 1-42 showed a 37% emission, and Aβ 1-42 increased LDH emission by about 26%. The vitamin C (200μM) treated group, which was used as a positive control, showed about 28% LDH release. Broccoli leaf chloroform fraction showed excellent protective effect against neuronal cell membrane damage with increasing concentration, and treated with 100μg / (24%) LDH release and showed a superior cell membrane damaging inhibitory effect than the vitamin C (200 μM) treatment, which is a positive control.
Example 10. Chloroform from broccoli leaf extract Fraction AChE Inhibition activation
Measurement of acetylcholinesterase inhibition activity was performed using acetylcholine iodide as a substrate. 5 ml of a solution for homogenization (1M NaCl, 50 mM MgCl 2 , 1% Triton X-100 mixed solution, adjusted to pH 7.2 with 10 mM Tris-HCl) was added to 1 ml of the PC12 cell culture medium to obtain a glass- -Col homogenizer), and the homogenized cell culture was centrifuged at 10,000 rpm for 30 minutes. The supernatant was used for the enzyme assay. All extraction procedures were performed at 4 ° C and protein content of the extracted enzyme was measured using Quant-iT ™ Protein Assay Kit (Invitrogen, St. Oregon, Eugene, USA). 10 μl of the extract was added to 10 μl of the purified enzyme and preincubated at 37 ° C for 15 minutes. Then, 70 μl of Ellman's reaction mixture [0.5 (0.5 μl) dissolved in 50 mM sodium phosphate buffer (pH 8.0) mM acethylthiocholine, 1
Based on the hypothesis that various cognitive disorders such as loss of memory and learning ability caused by Alzheimer's dementia are caused mainly by damage of the acetylcholinergic neurons of the cerebral basal part, in the present invention, the acetylcholinesterase of the chloroform fraction obtained from the broccoli leaf extract (AChE ). As shown in FIG. 10, the acetylcholinase inhibitory activity of the group treated with 1 μM tacrine as a positive control was about 73% as shown in FIG. 10, and the chloroform fraction of broccoli leaf extract showed AChE inhibition And the activity increased as the concentration increased. The AChE inhibitory activity (IC 50 ) of the chloroform fraction of broccoli leaves was 75.01 / / ㎖.
Example 11. Amyloid Beta (Amyloid beta, A [beta]) One -42 because of Spatial cognitive ability Chloroform from broccoli leaf extract against degradation Fraction Assessment of recovery ability
The animals used in the experiment were a 4-week-old ICR-male mouse, which was allowed to undergo a 5-day adaptation period. The mice were housed in one breeding case for 2 days at room temperature (22 ± 2 ° C) ) Were maintained, and were raised in the same laboratory environment with alternating day and night at 12-hour intervals. These rats were divided into 5, 10 and 20 mg / kg body weight (
Replacement behavior (%) = (actual change / maximum change) × 100, (maximum change = total number of passes - 2)
Y-maze was performed on mouse models in which memory and learning ability were deteriorated using Aβ 1-42 . The results are shown in FIG. Observation of the behavior of the mice in Y-maze for 8 minutes showed that the A? 1-42 group had a memory loss of spatial cognitive function by 47% compared with the control (62%) (Fig. 11A). On the other hand,
Example 12. Amyloid Beta (Amyloid beta, A [beta]) One -42 Of short-term memory and learning impairment due to chlorella leaf extract from broccoli leaves Fraction Assessment of recovery ability
A passive avoidance test was performed to confirm cognitive function and short-term memory recovery ability of mice consuming the chloroform fraction obtained from leaf extract of Broccoli leaf. The manual evasive instrument is divided into two sections, a bright chamber with a light and a dark chamber, and the bottom is made of wire mesh. A 1-42 After 4 days of injection, passive avoidance training trial was conducted. Each mouse was illuminated in a lighted chamber for 1 minute without illumination, then illuminated and adapted for 2 minutes and then applied an electric shock for 0.5 seconds, 3 seconds, as the mouse moved into the dark chamber. On the day following the learning test, each mouse was subjected to a test trial and the latency time (retention time) of the mouse's four feet in the illuminated chamber was measured up to 300 seconds.
The results of passive avoidance learning test are shown in Fig. 12 for the measurement of cognitive function and short-term memory ability of the mice ingested with the broccoli chloroform fraction. The Aβ 1-42 group showed a remarkable short-term memory loss (88% reduction) in 38 seconds compared to the control (300 seconds). In contrast, CBL5, 10 and 20 were significantly improved memory when compared to the Aβ 1-42 group at 72, 91, and 229 seconds, respectively. Especially
Example 13 Amyloid beta (Aβ) 1-42 And chloroform obtained from broccoli leaf extracts against long-term memory and learning impairment due to Fraction Assessment of recovery ability
(23 ± 2 ℃) in a circular water tank (150cm in diameter and 60cm in height) and set up a platform on one side of the water tank on the 4th floor and add edible squid ink (Cebesa, Valencia, Spain) . On the first day of the experiment, the experimental animals were allowed to swim freely for 60 seconds without escaping from the water tank, and after training for adaptation, the esophagus was placed 1cm below the water surface for 4 days. , E, and W), and were trained repeatedly four times a day and recorded using a Smart 3.0 video tracking system (Panlab, Barcelona, Spain) (hidden trial). The animals were allowed to stay in the esophagus for 10 seconds if they reached the esophagus within 60 seconds. If the esophagus was not found, they were guided to the esophagus by hand for 20 seconds. On the 5th day of the experiment (probe trial), a probe trial was conducted to record the time (sec) staying in the area (W area) where the escape zone was located for 60 seconds to remove the escape hatch and to measure long- .
As a result of Morris water maze experiment to examine the long-term memory and learning ability recovery ability of the chloroform fraction obtained from the leaf extract of Broccoli, in the case of the Hidden trial as shown in Fig. 13, the chloroform fraction obtained from the broccoli extract It was confirmed that the time to visit the esophagus was decreased in all the groups of the fractions. In the last 4 days, the Aβ 1-42 group showed a remarkable long-term memory and learning ability as 41 seconds compared to the control (29 seconds). In contrast, the
Example 14. Chloroform from broccoli leaf extract The fraction In the ingested mouse brain tissue AChE Active measurement
After the completion of the Morris water maze experiment, the brains of the mice were extracted and homogenized with a 10-fold volume of phosphate buffered saline (PBS), followed by homogenization with a glass-col homogenizer Centrifuged at 12,000 rpm for 30 minutes, and the supernatant was used for enzyme experiments. All extraction procedures were carried out at 4 ° C and the protein content of the extracted enzyme was measured using Quant-iT ™ Protein Assay Kit (Invitrogen Co., Carlsbad, Calif., USA). After adding 50 mM sodium phosphate buffer (65 캜) to 5 효 of the enzyme and preincubating the mixture at 37 캜 for 15 minutes, 70 E of Ellman's reaction mixture was added to the reaction mixture, The absorbance was measured at intervals of 2 minutes for 10 minutes. AChE activity in mouse brain tissue was expressed as% activity relative to control per mg of protein.
As shown in FIG. 14, the AChE activity in the A? 1-42 group was 121% as compared with the control (100%), and A? 1-42 was present in mouse brain tissue Lt; RTI ID = 0.0 > AChE. ≪ / RTI > In contrast,
Example 15. Chloroform from broccoli leaf extract The fraction The amount of SOD ( superoxide dismutase ) Content measurement
The cells were homogenized with a glass-col homogenizer (Phosphate Buffered Saline, PBS) at 10 times the brain weight of the extracted mice, and centrifuged at 12,000 rpm for 30 minutes to discard the supernatant. Respectively. 1X cell extract solution (1 ml of 10X SOD solution, 0.2 ml of 20% triton X-100, 8.8 ml of distilled water, and 10 ml of 200 mM PMSF) was vortexed every 5 minutes for 30 minutes and then incubated at 1,000 rpm for 10 minutes After centrifugation, the supernatant was used for the experiment. All procedures were carried out at 4 ° C and Quant-iT protein assay kit (Invitrogen Co) was used to measure the protein content of the supernatant. SOD activity was measured using an SOD measurement kit (Sigma-Aldrich Chemical Co.), and the measured absorbance value was expressed as SOD (U / mg of protein).
Peroxide anion (Superoxide anions) and a result of measuring the content of SOD of antioxidant to protect the tissue by changing to the hydrogen peroxide free radicals, such as, control group (control) as described in Figure 15 was 1.77 U per protein mg, Aβ 1 The -42 group showed a decrease in SOD content in brain tissue by 0.91 U per mg protein. On the other hand,
Example 16. Chloroform from broccoli leaf extract The fraction In the ingested mouse brain tissue GSSG / total GSH Content measurement
5% metaphosphoric acid was added to a certain amount of mouse brain and homogenized. Then, the mixture was centrifuged (14,000 g) for 15 minutes to obtain supernatant. 10 μl of 2M 4-vinylpyridine was added thereto, Were used for glutathione (GSSG) measurement. All procedures were maintained at 4 ° C. Quant-iT protein assay kit (Invitrogen) was used to measure the protein content of extracted GSSG. The content of glutathione was measured using a glutathione (GSSG / GSH) kit (Enzo Diagnostics, Farmingdale, NY, USA) and the total glutathione (GSH), oxidative glutathione Absorbance values were assigned to each standard curve and expressed as GSSG / total GSH.
Mitochondrial oxidative glutathione (GSSG) is associated with oxidation of mitochondrial DNA and is known to cause cell senescence or apoptosis. That is, as a result of measuring the GSSG / total GSH content as an oxidative index against oxidative stress of brain tissue, the control (control) was 19% and the A? 1-42 group was 28% The relative GSSG content was increased and it was shown to induce oxidative stress in brain tissue by Aβ 1-42 . On the other hand, in the
Example 17. Chloroform from broccoli leaf extract The fraction In the ingested mouse brain tissue MDA Content measurement
After adding 960 μl of 1% phosphoric acid to 160 μl of mouse brain tissue homogenate extracted with phosphate buffered saline, 320 μl of 0.67% thiobarbituric acid was added, and the mixture was reacted at 95 ° C. for 1 hour . The reaction solution was centrifuged at 5,000 rpm for 10 minutes, and the absorbance of the supernatant was measured at 532 nm.
As a result of measuring the content of MDA, an intermediate of lipid peroxidation in brain tissue, the control (cotnrol) was 3.72 nmole / mg protein and the Aβ 1-42 group was 4.47 nmole / mg, The injection of 1-42 was found to increase the MDA content in brain tissue. 4.38, 4.26 and 3.33 nmole / mg protein in the
Comparing the results of biochemical analysis (SOD, GSSG / total GSH and MDA) in these brain tissues, it was found that even in mouse brain tissues obtained from the chloroform fraction obtained from broccoli leaf extract, the superiority of Aβ 1-42 -induced oxidative stress It seems to be antioxidant activity.
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| CN109674055A (en) * | 2019-01-18 | 2019-04-26 | 温州科技职业学院 | A kind of extraction process of broccoli sprout extract |
| WO2020179982A1 (en) * | 2019-03-04 | 2020-09-10 | 주식회사 비케이바이오 | Composition containing broccoli sprouts as active ingredient for improving cognitive functions or memory |
| KR20210118492A (en) * | 2020-03-23 | 2021-10-01 | 주식회사 네이처센스 | A composition for improving memory, cognitive function, or preventing and improving brain neurological diseases |
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| US20210353698A1 (en) * | 2018-10-01 | 2021-11-18 | Astrogenesis Co., Ltd. | Pharmaceutical composition for preventing or treating neurodegenerative diseases, containing angelica gigas nakai extract or mixed extract of angelica gigas nakai and broccoli |
| KR20200066120A (en) | 2018-11-30 | 2020-06-09 | 푸른여름팩토리 주식회사 | Composition comprising red ginseng extraction, Acorus gramineus extraction and Polygala tenuifolia extraction for improvement in cognitive dysfunction |
| KR20220017060A (en) | 2020-08-04 | 2022-02-11 | 대구한의대학교산학협력단 | Composition comprising the extract of Gossypium nanking seed for preventing or treating neuro degenerative disease |
| KR20230059207A (en) | 2021-10-26 | 2023-05-03 | 이호규 | Composition for inner beauty cosmetics from broccoli leaves fraction extract and manufacturing method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109674055A (en) * | 2019-01-18 | 2019-04-26 | 温州科技职业学院 | A kind of extraction process of broccoli sprout extract |
| WO2020179982A1 (en) * | 2019-03-04 | 2020-09-10 | 주식회사 비케이바이오 | Composition containing broccoli sprouts as active ingredient for improving cognitive functions or memory |
| KR20200106296A (en) * | 2019-03-04 | 2020-09-14 | 주식회사 비케이바이오 | Composition for improvement of memory or cognitive ability comprising broccoli sprouts |
| KR20210118492A (en) * | 2020-03-23 | 2021-10-01 | 주식회사 네이처센스 | A composition for improving memory, cognitive function, or preventing and improving brain neurological diseases |
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