KR101801295B1 - Composition of memory improvement and preparation method thereof using ultrafiltration - Google Patents

Abstract

The present invention relates to a composition for enhancing memory effect using ultrafiltration from red ginseng, and a method for preparing the same. The external fluid fraction of red ginseng extract obtained by the ultrafiltration obtained through the present invention contains many more hydrophobic substances And is expected to be utilized as a new substance capable of alleviating cognitive disorders and cholinergic disorders of degenerative diseases such as Alzheimer's disease.

Description

TECHNICAL FIELD The present invention relates to a composition for improving memory performance using ultrafiltration,

The present invention relates to a composition for enhancing memory effect using ultrafiltration from red ginseng, and a method for producing the composition.

Alzheimer's disease (AD) is the most common age-related neurodegenerative disease characterized by the formation of a senile plaque in the brain that contains amyloid-beta (Aβ). Alzheimer's disease represents behavioral and cognitive impairment in daily life. According to the cholinergic hyphothesis, cholinergic disorders in the brain are associated with decreased cognitive function in the elderly and Alzheimer's disease. Inhibition of acetylcholinesterase (AChE) is currently the primary treatment strategy for Alzheimer's disease. Although four AChE inhibitors have been approved for the treatment of Alzheimer's disease to date, they cause side effects such as diarrhea, nausea, vomiting and bradycardia. Thus, much attention has been focused on finding new substances from natural products by researchers around the world. As a result, many plants tested for anti-cholinesterase activity measurement as memory enhancing factors.

Ginseng ( Panax ginseng Meyer) has been used as a traditional herb medicine in Asian countries. This study confirmed various preventive effects of ginseng on memory impairment and oxidative stress. In particular, it has been reported that cognitive abilities are improved through the treatment of ginsenosides Rb ₁ and Rg ₁ in animal models. Despite its attractive features as a potentially functional food for Alzheimer's disease in ginsenosides, its use has been limited for several reasons, including high production costs and low bioavailability.

 Shen Z et al., Two-way shuttle box avoidance conditioning and brain NADH in rats. Physiol Behav. 48: 515-7,1990.

In order to solve the above problems, in the present invention, when studying effective fractions of red ginseng as a functional food for improving memory performance and improving memory performance with safety, it was found that the memory enhancing substance of Korean red ginseng is not ginsenosides Substance. In the present invention, the hydrogen peroxide-induced cells were treated with ultrafiltration system and chromatography to determine whether or not the water-soluble amyloid-beta was reduced and the memory inhibition of the experimental group was resolved by treating the fraction with or without ginsenoside Respectively.

The present invention relates to a process for producing a red ginseng extract, comprising the steps of: a) extracting and extracting red ginseng with an extraction solvent; b) diluting the extract concentrated in step a) with water and ultrafiltering with a molecular cut-off of 2 kDa to 6 kDa to obtain a filtered extraneous liquid; And c) further fractionating the external liquid obtained in the step b) by adding an extraction solvent.

The present invention also relates to a composition for improving memory performance produced by the above-mentioned production method.

The extracellular fluid fraction of red ginseng extract obtained through the present invention contains many hydrophobic substances that are more effective for improving memory and is a novel substance that can attenuate cognitive disorders and cholinergic disorders of degenerative diseases such as Alzheimer's disease It is expected to be utilized.

Figure 1 relates to measurement results of cell viability.
Figure 2 relates to the acetylcholinesterase test results.
Figure 3 relates to the inhibition concentration according to statistical inference in the acetylcholinesterase assay.
Figure 4 relates to passive avoidance test results.
Figure 5 relates to the collection and Western blot analysis of mouse brain cells.

The present invention relates to a process for producing a red ginseng extract, comprising the steps of: a) extracting and extracting red ginseng with an extraction solvent; b) diluting the extract concentrated in step a) with water and ultrafiltering with a molecular cut-off of 2 kDa to 6 kDa to obtain a filtered extraneous liquid; And c) further fractionating the external liquid obtained in the step b) by adding an extraction solvent.

Ginseng is a perennial plant belonging to the genus Panax. The genus Panax is a perennial perennial perennial plant belonging to the genus Araliaceae. Representative species include Korean ginseng (Panax ginseng), hwagisam (Panax quinquefolia), jeoncheolsam (thirty-seven, Panax notoginseng), jukjeol three (Panax japonica), three yeopsam (Panax trifolia), Himalayan ginseng (Panax pseudoginseng), Vietnamese ginseng (Panax vietnamensis ), etc. (Understanding of ginseng, Korean Ginseng Society, p.9, 1995; Advances in Ginseng Research, Korea Ginseng Society, p.127-137, 1998).

In the present invention, the extraction solvent in step a) may be any one or a mixture of two or more selected from the group consisting of water and lower alcohols having C1 to C4 carbon atoms.

In the present invention, the dilution of step b) is not limited, but may be carried out by adding 3 to 20 times water to the total weight of the red ginseng extract.

In the present invention, the molecular weight cut in step b) may be 3 kDa or 5 kDa although not limited thereto. When ultrafiltration is performed using the membrane having the molecular weight cut, there is an advantage that a substance effective for improvement of cognitive disorder, Alzheimer's disease, and memory improvement can be effectively separated as an external fluid.

In the present invention, the ultrafiltration in step b) is not limited, but it may have a ratio of the inner liquid to the outer liquid of 75 to 85:25 to 15. When the concentration of the red ginseng extract is performed up to the above ratio, there is an advantage that a substance effective for memory improvement is effectively separated into an external fluid.

In the present invention, the extraction solvent in step c) may be 20 to 40% by weight ethanol, though not limited thereto. According to one embodiment of the present invention, the ginsenoside component was not detected in the fraction eluted with ethanol at the concentration, and the hydrophobic component fraction eluted with ethanol had a higher rate of apoptosis than the hydrophilic fraction fractioned with purified water Which is a significant reduction in the number of newborns. In addition, the fraction was significantly better in reducing cell death by hydrogen peroxide treatment compared with red ginseng extract (RE), and showed the best effect among REUO which is a hydrophilic and hydrophobic mixture of the composition of the present invention .

The present invention also relates to a composition for improving memory performance produced by the above-mentioned production method.

But are not limited to, the present invention may include food additive additives that are further acceptable to the composition. The composition of the present invention may further include additives commonly used in this field, but is not limited thereto. As the above additives, minerals, vitamins, spices, antioxidants, stabilizers, coloring agents, sweeteners and nutritional enhancers can be used, and they can be added for the purpose of improving the negative sign composition, .

In the present invention, the formulation of the composition is not limited, but may be in the form of powders, granules, tablets, capsules, pills, suspensions, emulsions, syrups or aerosols or beverage forms.

Examples of 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, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. 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 ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . 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 the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

In addition, since the composition of the present invention has little toxicity and side effects, it can be safely used for prolonged use for the purpose of prevention or treatment.

The composition of the present invention is not particularly limited to the other components and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. 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 polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. As natural flavors other than those described above, natural flavoring agents (tau martin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 mL of the composition of the present invention.

In addition to the above-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the extracts of the present invention may contain flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the content of the present invention will be described in more detail with reference to Examples. It is to be understood that the scope of the present invention is not limited to these embodiments and that variations, substitutions, and insertions conventionally known in the art can be carried out, And this is included in the scope of the present invention.

[Materials and reagents]

Red ginseng ( Panax ginseng CA Meyer) was purchased from Geumsan ginseng cooperative (Geumsan, Korea). The red ginseng was deposited with the International Ginseng Herbal Research Institute (No. GS201105).

All other chemical reagents were analytical grade and purchased from Sigma.

The Tg2576 mouse used in the experiment was purchased from Taconic Laboratories as a species overexpressing a modified form of human amyloid precursor protein (APP) 695. All animal testing procedures were conducted in accordance with the guidelines of the Animal Experiment Ethics Committee of Seoul National University, Republic of Korea.

[Statistical analysis method]

The measured data were expressed as mean ± standard error (SE). one-way ANOVA and Dunnett's post hoc test (SPSS version 21). At this time, p <0.05 was used as a confidence interval.

[HPLC analysis of ginsenoside]

10 mg of red ginseng extract was dissolved in 1 mL of methanol (MeOH), sonicated for 2 hours, filtered through a 0.45 μm membrane filter, and analyzed by HPLC (Waters, USA). The HPLC was carried out using a PDA detector (Water, 2998), a Waters Xbridge ^占 C18 column (250 mm x 4.6 mm, 5 占 퐉; Waters, USA). The detection wavelength, flow rate, injection volume and column oven temperature were set at 203 nm, 1.0 mL / min, 20 μL and 40 ° C, respectively. The mobile phase was used as shown in Table 1 below.

[Table 1]

[Example 1]

300 g of the purchased dried red ginseng was extracted with 80 mL of 80 wt% ethanol at 80 ° C using a round bottom flask equipped with a condenser. The resulting ethanol extract (RE) was concentrated and dried in vacuo. The dried ethanol extracts were dissolved in 1 L distilled water and concentrated to 20% of the extraction volume in an ultrafiltration system equipped with capillary cartridges of membranes with a molecular weight cut of 3 kDa and area of 6 m ² . The concentrated inner fraction (REUI) and the filtered outer fraction (REUO) were dried in a freeze dryer. The outer liquid was subjected to final fractionation by Diaion HP-20 column chromatography using a sequential elution process of distilled water, 30 wt% ethanol, 50 wt% ethanol and 70 wt% ethanol. The four fractions obtained through this process were named REUO-00, REUO-30, REUO-50 and REUO-70, respectively.

The ginsenoside content of the red ginseng extract and the fractions obtained from the fraction was measured and the results are shown in Table 2 below.

[Table 2]

* PPT: protopanaxatriol, PPD: protopanaxadiol

From the above results, it was confirmed that the sum of ginsenosides of REUI and REUO was 4.4 times and 9.9 times higher than that of ginseng extract of red ginseng extract, respectively. In particular, it was confirmed that the ginsenoside of the extracellular fluid was a protopanaxatriol (PPT) type, and the ginsenoside of the inner fluid was a protopanaxadiol (PPD) type and was more hydrophobic.

The molecular weight of the PPD type ginsenoside is slightly larger than that of the PPT type, but the molecular weight of the ginsenoside is 1,110 or less. The molecular size of the ginsenoside is smaller than that of the ultrafiltration membrane having the 3 kDa molecular weight cutoff. However, the PPD type ginsenoside having a relatively hydrophobic property in the red ginseng extract does not pass through the hydrophilic membrane having a molecular weight cut of 3 kDa, Re and Rg ₁ , which are hydrophilic PPT type ginsenosides, slowly pass through the membrane in a continuous flow system. Thus, the outer fluid (REUO) will contain the ginsenosides of the PPT type and the smaller dissolved molecules that have passed through the membrane. Surprisingly, however, no ginsenosides were detected in the REUO-30 fraction, as can be seen in Table 2 above.

[Test Example 1] Measurement of cell viability

SH-SY5Y (neuroblastoma (neuroblastoma); ATCC, USA) at a density of 7 x 10 ³ cells / well was fed to a 96-well plate. Each fraction of Example 1 and the prodrug 50 μM DHED (10β, 17β-dihydroxyestra-1,4-dien-3-one) were each treated with MEM (minimum essential medium) for 4 hours before treatment with 750 μM hydrogen peroxide . Cell viability was determined using WST-1 metabolic activity according to the manufacturer's instructions (Roche, Indianapolis, IN, USA). The absorbance of the reaction product was finally determined at 450 nm using an ELISA reader (Bio-Rad, Munich, Germany).

As a result, it was confirmed that SH-SY5Y was exposed to hydrogen peroxide (vehicle) and showed marked neurotoxicity compared to the negative control (control) (FIG. 1). As a result of treatment with hydrogen peroxide after pretreating each fraction of 50 μM DHED and red ginseng extracts, the cell death by hydrogen peroxide during treatment with DHED and external fluid (REUO) was found to be decreased compared with the case of treatment with vehicle or internal fluid (REUI) (Fig. 1A). According to the description in Table 2, since the content of ginsenoside of REUI is about 10.7%, while the content of ginsenoside of REUO is only about 1.1%, the decrease of cell death by hydrogen peroxide treatment is influenced by the content of ginsenoside It was found that the inhibitory effect was due to other unknown components contained in the non-REUO.

The results of the additional fractionation tests conducted to identify these unknown components are as follows. REUO-00, a hydrophilic component fraction eluted with purified water, contains a hydrophilic component such as sugar. According to Table 2, the ginsenoside component was not detected in the REUO-30 fraction eluted with 30% ethanol. The hydrophobic component fractions REUO-30, REUO-50 and REUO-70 significantly reduced cell death by hydrogen peroxide treatment compared to the vehicle or hydrophilic fraction (FIG. 1B).

In addition, the REUOEtOHs fraction mixed with the hydrophobic REUO-30, REUO-50 and REUO-70 was compared with the RE (red ginseng extract) and REUO, which is a mixture of hydrophilic and hydrophobic. As a result, (Fig. 1C). In particular, in the case of the hydrophobic fraction REUO-EtOHs, it was confirmed that cell death was reduced depending on the dose (Fig. 1D).

[Test Example 2] Acetylcholine esterase test

Acetylcholinesterase (AChE) test was performed by colorimetric method using acetylthiocholine iodide as a substrate. In the experimental group, 37.8 μM DHED and 0.5 mg / mL AIE were used as the positive control, and 250 μM Rb ₁ , Rg ₁ , ginsenoside, red ginseng extract (RE), external fluid (REUO) and REUO-EtOHs Respectively.

Absorbance was measured at 410 nm immediately after adding 100 μL of acetylcholinesterase to each experimental group and repeatedly measured at intervals of 30 seconds for 5 minutes.

The acetylcholinesterase activity was calculated with an absorbance coefficient of 1.36 L / mmol / min.

The half-maximal inhibitory concentration (IC ₅₀ ), a half-peak inhibitory concentration of the sample, was calculated from a linear estimate of the enzyme inhibition dose response curve.

The measurement results for each experimental group are shown in Fig.

The result shows that REUO-EtOHs, which is a hydrophobic fraction component, exhibits at least three times superior inhibitory effects against ginsenoside, red ginseng extract (RE) and external fluid (REUO) The effect of treating the hydrophobic fraction was remarkable (FIG. 2). For reference, the 50% inhibitory concentration of REUO is approximately 2.358 mg / mL (FIG. 3). From this standard, the acetylcholinesterase inhibitor properly maintains the concentration of acetylcholine in the brain, It can function as an appropriate indicator material.

[Test Example 3] External fluid treatment for animals

The 50 mg / kg extracellular fluid (REUO) or 50 mg / kg AIE (Artemisia iwayomogi) extract of the ultrafiltered red ginseng extract and the standard laboratory food were mixed and administered to the 9-month old Tg2576 mouse for 3 months. Food intake and mouse weights were measured and recorded weekly.

[Test Example 4] Passive avoidance test

The effects of learning and memory of REUO and AIE in Test Example 3 were evaluated using a step-through type passive avoidance reaction tester (Model PACS-30, Columbus Instruments Int., USA) Shen et al., 1990).

As a result, in the case of the untreated Tg2576 mouse (37.86 ± 20.53 s), the delayed rate of the wild-type mouse (202.70 ± 42.57 s, p = 0.004) and the wild-type mouse treated with REUO (177.83 ± 40.35 s, p = 0.012), respectively. In contrast, the untreated Tg2576 mice (37.86 ± 20.53 s) showed an increase in delayed rate compared to the REUO-treated Tg2576 mice (177.83 ± 40.35 s, p = 0.012). It was also found that the effect of AIE was less than that of REUO (Fig. 4).

[Test Example 5] Collection of mouse brain cells and western blot analysis

After the behavioral testing of the mice, the experiment was terminated at the 13th month. The mouse brain was harvested and used for further work.

Protein concentrations were quantified using Bio-Rad protein assay reagent (Bio-Rad, USA). The brain tissues were homogenized using a 10-fold volume of homogenization buffer (12.5 mM sodium phosphate, pH 7.0, 400 mM sodium chloride) and then centrifuged at 1,000 x g for 10 minutes. The supernatant was loaded with homogenization buffer containing 0.5% Triton X-100, and the mixture was continuously stirred and centrifuged again at 10,000 x g for 10 minutes. Proteins were dissolved in 16.5% Tris / glycine gel, loaded with 40 mg protein per lane, electrophoresed, and transferred to nitrocellulose membrane (Amersham Pharmacia, Buckinghamshire, UK).

Western blot was identified as a primary antibody to 6E10 for total soluble amyloid-beta protein and secondary antibody (HRP, horadish-peroxidase) was detected with a secondary antibody (Amersham Pharmacia, Buckinghamshire, UK). The immunoreactive band was visualized with an enhanced luminescence system (ECL; Amersham Pharmacia, Buckinghamshire, UK). GAPDH (glyceraldehyde 3-phosphate dehydrogenase) antibody was used as a control for protein loading.

The experimental results are as follows.

The amyloid-beta in the mouse brain was consistently higher than vehicle-treated wild-type mice and REUO-treated wild type mice in the vehicle-treated Tg2576 mice as a result of continued administration of the REUO of the present invention Respectively. Furthermore, in the case of AIE, it was found that the effect of treatment was not greater than that of REUO in which the amount of amyloid-beta was remarkably decreased after treatment (Fig. 5A).

The brain is available for moderate oxidative stress because of its high oxygen consumption, its high polyunsaturated fatty acid content and its low antioxidant defense. It is known that the production of reactive oxygen species and the inhibition of choline intake are important factors in relation to the invention of Alzheimer's disease. The REUO of the present example improves memory decay in Tg2576 mouse, and the whole amyloid-beta And the like. Particularly, in the case of the fraction not saponin in the additional fraction of REUO from the present red ginseng extract of the present invention, cholinergic dysfunction, oxidative stress, and food additives for improving cognition such as the production of amyloid-beta It is expected to be useful.

Claims (9)

a) adding to the red ginseng either one or a mixture of two or more selected from water or lower alcohols having a carbon number of from C1 to C4, and concentrating;
b) diluting the extract concentrated in step a) with water and ultrafiltering with a molecular cut-off of 2 kDa to 6 kDa to obtain a filtered extraneous liquid; And
c) further fractionating the external liquid obtained in the step b) by adding 20 to 40% by weight of ethanol;
&Lt; / RTI &gt; delete The method according to claim 1,
Wherein the dilution of step (b) is performed by adding 3 to 20 times water to the total weight of the red ginseng extract. The method according to claim 1,
Wherein the molecular weight cut in the step b) is 3 kDa or 5 kDa. The method according to claim 1,
Wherein the ultrafiltration in the step b) has a ratio of an inner liquid to an outer liquid of 75 to 85:25 to 15. delete A composition for improving memory performance, which is prepared by the method according to any one of claims 1 and 3 to 5. 8. The method of claim 7,
A composition for improving memory, comprising a food-aid additive which is further admissible in said composition. 9. The method of claim 8,
Wherein the formulation of the composition is in the form of a powder, granule, tablet, capsule, pill, suspension, emulsion, syrup or aerosol, or beverage form.

Images