KR102067275B1 - Composition for protection of brain neuronal cells and improvement of memory with 5-(3',4'-dihydroxyphenyl)-gamma-valerolactone - Google Patents

Abstract

The present invention improves brain neuron protection and memory containing 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone (5- (3', 4'-dihydroxyphenyl) -gamma-valerolactone) A composition for use, comprising a 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone (5- (3', 4'-dihydroxyphenyl) -gamma-valerolactone) of the present invention Is a natural product with little side effects and good effect, and exhibits the prevention and treatment of degenerative neurological diseases.

Description

Composition for protection of brain neuronal cells and improvement of memory with 5- (3 ', containing 5- (3', 4'-dihydroxyphenyl) -gamma-valerolactone , 4'-dihydroxyphenyl) -gamma-valerolactone}

The present invention relates to a composition for protecting brain neurons and improving memory, and more particularly 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone (5- (3', 4'-). It relates to a composition for protecting brain neurons and improving memory containing dihydroxyphenyl) -gamma-valerolactone).

Polyphenols are known to be beneficial to the human body because they have various physiological activities and are present in many food ingredients. Typical foods high in polyphenols include cacao, green tea, oolong tea and black tea, and red wine. The findings of polyphenol intake are beneficial to the human body, leading to a study of metabolic pathways in the body when ingesting polyphenolic mono- or polyphenol-rich foods. Most polyphenols in nature exist in the form of sugars or organic acids and are classified into flavonoids and non-flavonoids. Flavonoids are again divided into flavonols, flavones, flavones, flavanols, flavan-3-ols, flavonones, anthocyanidins, flavanones, polyphenols This rich food is particularly high in flavanols.

As the medical technology in the modern society has developed, the '100-year-old' has arrived, and as the aging accelerates, the proportion of the elderly population in the total population is increasing. As the aging population progressed, chronic diseases gradually increased, and according to the health insurance statistics published by the National Health Insurance Corporation in 2015, among the chronic diseases, hypertension patients accounted for 5.7 million people, and neurological disease patients were 2.71 million. Followed by commands. In particular, among the chronic diseases, treatment costs increased by about 12% compared to the previous year, showing the highest increase rate. As the aging society enters the most problematic neurological disease is degenerative neurological disease, and dementia is a representative disease. The number of dementia patients continues to increase, and by 2024, the population of dementia is expected to reach 1 million people.

Degenerative neurological disease, a representative senile disease, is caused by brain nerve cell damage, which is caused by various factors such as genetic factors, decreased immunity, and inflammatory reactions. The main cause is oxidative stress. have. Oxidative stress occurs as toxic free radicals in nerve cells increase and damage DNA, protein, and lipid membranes, thereby degrading cell function.

Currently, various degenerative neurological disease treatments have been developed, but stability is not yet secured, and side effects according to individual or intake periods have been reported. Therefore, there is a need for a study on securing the efficacy and finding a composition for preventing and treating degenerative neurological diseases of natural materials with low side effects and good effects.

Korean Patent Registration No. 10-17168010000 (2017.03.09) discloses a food composition for preventing metabolic diseases and treatment of metabolic diseases containing 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone or derivatives thereof. It is described with respect to the pharmaceutical composition.

The present invention relates to a composition for protecting brain neurons and improving memory, and 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone (5- (3', 4'-dihydroxyphenyl) -gamma By developing a composition containing -valerolactone), it is intended to provide a composition for the prevention and treatment of degenerative cerebral nervous system disease, which is a natural material with little side effects and good effect.

Brain neurons characterized by containing 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone (5- (3', 4'-dihydroxyphenyl) -gamma-valerolactone) It provides a food composition for improving protection and memory degradation.

In the food composition for improving brain neurons protection and memory degradation of the present invention, the 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone (5- (3', 4'-dihydroxyphenyl) -gamma-valerolactone) preferably has a concentration of 1 µM to 1 mM.

In addition, the present invention is characterized in that the brain contains 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone (5- (3', 4'-dihydroxyphenyl) -gamma-valerolactone) Provided is a pharmaceutical composition for protecting neurons and preventing or treating memory loss.

In the pharmaceutical composition for brain neurons protection and memory loss prevention or treatment of the present invention, the 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone (5- (3', 4'- dihydroxyphenyl) -gamma-valerolactone) preferably has a concentration of 1 µM to 1 mM.

The composition containing 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone (5- (3', 4'-dihydroxyphenyl) -gamma-valerolactone) of the present invention as an active ingredient has side effects. This small, high-efficiency natural product exhibits preventive and therapeutic effects for degenerative neurological diseases.

1 is a graph showing whether cell death by hydrogen peroxide (H ₂ O ₂ ) is inhibited by treatment of 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone.
Figure 2 is a representative representative of cell death showing the effect of inhibiting neuronal cell death by oxidative stress induced by hydrogen peroxide (H ₂ O ₂ ) of 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone The results were confirmed using biomarkers caspase-3 and PARP-1.
Figure 3 is representative of apoptosis, showing the effect of inhibiting neuronal cell death by oxidative stress induced by hydrogen peroxide (H ₂ O ₂ ) of 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone Results confirmed using biomarkers Bcl-2 and Bcl-xL.

The present invention is characterized by containing 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone (5- (3', 4'-dihydroxyphenyl) -gamma-valerolactone) or derivatives thereof Provided are food compositions for protecting brain neurons and improving memory degradation.

Flavanol is less absorbed by the body and is converted to other substances (metabolites) by the gut microbiota in the large intestine before most of it is absorbed. A representative metabolite among them is 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone (5- (3', 4'-dihydroxyphenyl) -gamma-valerolactone). 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone is one of the metabolites produced by intestinal bacteria when ingested with polyphenol-rich foods such as cacao, green tea, red wine and grapes. It is the substance with the highest increase.

That is, when a human ingests a food rich in polyphenols, procyanidin or catechin contained in the food is metabolized by the intestinal microorganisms and thus 5- (3 ', 4'-dihydroxyphenyl) Gamma-valerolactone (5- (3 ', 4'-Dihydroxyphenyl) -gammavalerolactone, Structural Formula: C ₁₁ H ₁₂ O ₄ ), 3- (3', 4'-dihydroxyphenyl) -delta-valero Lactone (3- (3 ', 4'-dihydroxyphenyl) -delta-valerolactone, formula: C ₁₁ H ₁₂ O ₅ ), 5- (3', 4 ', 5'-trihydroxyphenyl) -gamma-valero Lactone (5- (3 ', 4', 5'-trihydroxyphenyl) -gamma-valerolactone, formula: C11H12O5) and 5- (3-methoxy-4-hydroxyphenyl) -gamma-valerolactone (5- ( 3-methoxy-4-hydroxyphenyl) -gamma-valerolactone, a structural formula: C ₁₂ H ₁₄ O ₄ ).

The concentration of 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone or a derivative thereof contained in the food composition for protecting brain neurons and improving memory degradation of the present invention is preferably 1 μM to 1 mM. It is good and, more preferably, it is 5-40 micrometers.

In the food composition for improving brain neurons protection and memory degradation of the present invention, the 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone or derivatives thereof is preferably for brain neurons protection and memory 0.00001 to 50% by weight relative to the food composition for improving degradation is preferably included. If the amount is less than 0.00001% by weight, the effect is insignificant, and if the amount exceeds 50% by weight, the effect increase compared to the amount of use is insignificant.

The composition for improving brain nerve cell protection and memory degradation of the present invention is, for example, meat, cereals, caffeine beverages, general beverages, chocolate, breads, snacks, confectionery, candy, pizza, jelly, noodles, gums, dairy products, ice cream , Alcoholic beverages, alcohol, vitamin complexes and other health supplements may be any one selected from, but is not necessarily limited thereto.

The present invention is also characterized by containing 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone (5- (3', 4'-dihydroxyphenyl) -gamma-valerolactone) or derivatives thereof. Provided is a pharmaceutical composition for protecting brain neurons and preventing or treating memory loss.

The concentration of 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone or a derivative thereof contained in the pharmaceutical composition for protecting brain neurons and preventing or treating memory loss of the present invention is preferably 1 μM to 1 mM. It is good to be, More preferably, it is good that it is 5-40 micrometers.

On the other hand, the pharmaceutical composition for preventing or treating brain neurons and memory loss prevention of the present invention is a pharmaceutically acceptable carrier, diluent or excipient other than 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone It may further include. Carriers, excipients or diluents which may be used include lactose, dextrose, sucrose, sorbitol, mannitol, ziitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oils, one or more of which may be selected. In addition, the pharmaceutical composition, which is a pharmaceutical composition for preventing or treating brain nerve cell protection and memory loss, may further include one or more selected from fillers, anti-coagulants, lubricants, wetting agents, flavors, emulsifiers, or preservatives.

On the other hand, the formulation of the pharmaceutical composition for brain neuron protection and memory loss prevention or treatment of the present invention may be in a preferred form depending on the method of use, in particular to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. It is recommended to formulate by adopting a method known in the art to be able to. Examples of specific formulations include PLASTERS, GRANULES, LOTIONS, LINIMENTS, LIMONADES, AROMATIC WATERS, POWDERS, and Syrups. SYRUPS, OPHTHALMIC OINTMENTS, LIQUIDS AND SOLUTIONS, AEROSOLS, EXTRACTS, ELIXIRS, OINTMENTS, FLUIDEXTRACTS, Emulsion SUSPENSIONS, DECOCTIONS, INFUSIONS, OPHTHALMIC SOLUTIONS, TABLETS, SUPPOSITIES, INJECTIONS, SPIRITS, CATAPLSMA ), Capsules (CAPSULES), creams (CREAMS), troches (TROCHES), tinks (TINCTURES), pasta (PASTES), pills (PILLS), soft or hard gelatin capsules may be any one selected.

On the other hand, the dosage of the pharmaceutical composition for the prevention and treatment of brain nerve cell protection and memory degradation of the present invention is preferably determined in consideration of the administration method, the age, sex, weight and severity of the disease. For example, the pharmaceutical composition for brain neurons protection and memory loss prevention or treatment of the present invention can be administered once or more at 0.00001 to 100 mg / kg (body weight) per day based on the active ingredient. However, the above dosage is just one example to illustrate, and may be changed by a doctor's prescription according to the condition of the taker.

Hereinafter, the configuration of the present invention will be described in detail through the following examples. However, the scope of the present invention is not limited only to the following examples, but includes modifications of equivalent technical ideas.

< Example 1: 5 -(3 ', 4'- Dihydroxyphenyl )-gamma- Valerolactone  Neuroprotective Effect>

Cell survival rate using MTT assay to determine whether 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone has neuroprotective activity against oxidative stress induced by hydrogen peroxide (H ₂ O ₂ ) (cell viability) was confirmed by measuring.

5 × 10 primary cortical neurons of the frontal lobe of the 14-day-old ICR mouse E14-day-old embryo in 96-well-plates coated with poly-D-lysine. Incubated at 37 ℃, 5% CO ₂ conditions at a density of ⁵ cell / ml. After seeding the cells, a week later, the primary neurons were differentiated through a microscope, and then the cells were starvated for 24 hours. Thereafter, 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone was pretreated at a concentration of 5-40 μM for 1 hour and 50 μM hydrogen peroxide for 22 hours.

When the reaction was completed, the reaction was treated with MTT (3- [4,5-dimethyl-thiazol] -2,5-diphenyl-tetrazolium bromide) reagent at a concentration of 5 mg / ml by 10 μl / well for 2 hours. Thereafter, 150 μl / well of DMSO (dimethyl sulfoxide) was treated, and then the 96 well-plate was placed on a shaker for about 15 minutes while blocking light, and the absorbance was measured at 570 nm (FIG. 1).

1 is a graph showing whether cell death by hydrogen peroxide (H ₂ O ₂ ) is inhibited by treatment of 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone.

The relative cell viability of each treatment group was expressed in% based on the control. In the hydrogen peroxide treatment group, the cell survival rate was reduced to 50%, indicating that 50 μM of hydrogen peroxide was the proper inducer concentration. In addition, when 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone was pretreated at 5-40 µM, cell death caused by hydrogen peroxide treatment was significantly reduced in a concentration-dependent manner (cell Increase in viability). At 40 μM treatment concentration, the cell viability was about 80%.

Through this, it can be confirmed that the 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone of the present invention has an effect of inhibiting and protecting apoptosis of primary neurons caused by oxidative stress in a concentration-dependent manner. there was.

< Example 2: 5 -(3 ', 4'- Dihydroxyphenyl )-gamma- Valerolactone  ( DHPV Inhibition of Neuronal Cell Death)

Western blots show that 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone is effective in inhibiting neuronal cell death by oxidative stress induced by hydrogen peroxide (H ₂ O ₂ ). Confirmed.

Caspase-3, PARP-1 (Poly (ADP-ribose) polymerase), Bcl-2 (B-cell lymphoma 2), and Bcl-xL (B-cell lymphoma-extra large), which are representative biomarkers showing apoptosis ) Protein expression change was confirmed.

Primary neurons in the frontal lobe of the 14-day-pregnant ICR mouse E14-day-old embryos in a 60-mm cell culture dish coated with poly-D-lysine were treated with 4 × 10 ⁶ cells / ml. Incubated at 37 ° C., 5% CO ₂ . After seeding the cells, a week later, the primary neurons were differentiated through a microscope, and then the cells were starvated for 24 hours. Thereafter, 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone was pretreated at 5 μM, 20 μM for 1 hour, and 50 μM hydrogen peroxide for 22 hours.

Primary neurons were lysed with cell lysate (Cell signaling # 9803), left on ice for 30 minutes, and centrifuged (14,000 rpm, 4 ° C, 10 minutes) to separate supernatants containing proteins. Protein concentration was measured using a Biorad protein assay reagent, proteins were separated using SDS-PAGE, and then the proteins were transferred to PVDF membranes. The membranes were treated with 5% skimmed milk for 1 hour at room temperature to reduce nonspecific binding, and Caspase-3, PARP-1, Bcl-2, Bcl-xL and β-actin were 1: 1000 at 4 ° C. After reacting overnight at (Overnight), it was measured by chemiluminescence using ECL (Health Care) (Fig. 2, Fig. 3).

Figure 2 is a representative representative of cell death showing the effect of inhibiting neuronal cell death by oxidative stress induced by hydrogen peroxide (H ₂ O ₂ ) of 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone It was confirmed using the biomarker caspase-3 and PARP-1, Figure 3 is induced by hydrogen peroxide (H ₂ O ₂ ) of 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone The effect of inhibiting neuronal cell death caused by oxidative stress was confirmed using Bcl-2 and Bcl-xL, which are representative biomarkers indicating cell death.

2 and 3, it was confirmed that apoptosis occurred in the hydrogen peroxide treatment group, neuronal cell death in the group pre-treated with 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone. Inhibitory effect was confirmed. Treatment with hydrogen peroxide alone significantly increased the expression of cleaved-caspase-3 and cleaved-PARP, pro-apoptotic proteins that induce apoptosis, while hydrogen peroxide and 5- (3 ', 4) When '-dihydroxyphenyl) -gamma-valerolactone was treated together, it was confirmed that apoptosis was reduced in a concentration-dependent manner.

In addition, when the expression levels of anti-apoptotic proteins Bcl-2 and Bcl-xL, which are involved in the permeability of mitochondrial cell membranes, were measured, 5- (3 ', 4') for Bcl-2 The concentration increased in the group treated with -dihydroxyphenyl) -gamma-valerolactone, but there was no significant difference in the case of Bcl-xL. Through this, it was confirmed that 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone has an effect of inhibiting apoptosis of primary neurons induced by hydrogen peroxide.

Taken together, 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone inhibits or blocks neuronal cell death caused by oxidative stress, thereby protecting brain neurons and improving memory. I could confirm it.

5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone is a form that is actually absorbed by the body and has a physical and chemical component that satisfies the conditions for passing the blood brain barrier. In addition, it is believed to be safer and more effective than functional foods or therapeutics for protecting brain neurons and improving memory loss.

Claims (4)

Brain neuronal cell protection and memory characterized by containing 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone (5- (3', 4'-dihydroxyphenyl) -gamma-valerolactone) Degradation improvement food composition.
The method of claim 1,
5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone (5- (3', 4'-dihydroxyphenyl) -gamma-valerolactone),
Concentration is 1μM ~ 1mM food composition for brain neurons protection and memory degradation improvement.
Prevention of degenerative neurological diseases characterized by containing 5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone (5- (3', 4'-dihydroxyphenyl) -gamma-valerolactone) or Therapeutic pharmaceutical composition.
The method of claim 3,
5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone (5- (3', 4'-dihydroxyphenyl) -gamma-valerolactone),
Pharmaceutical composition for the prevention or treatment of degenerative neurological diseases, characterized in that the concentration is 1μM ~ 1mM.

Images