KR20220092472A - Composition for prevention or treatment of neurological disorder comprising extract of red ginseng and method for manufacturing the same - Google Patents

Abstract

The present invention relates to a composition for preventing or treating degenerative neurological diseases containing a red ginseng extract, and a method for manufacturing the same. Specifically, the present invention relates to a composition for preventing or treating degenerative neurological diseases with improved NO production inhibitory activity induced by lipopolysaccharide (LPS) in BV2 cell line without cytotoxicity, and a method for manufacturing the same.

Description

Composition for prevention or treatment of neurological disorder comprising extract of red ginseng and method for manufacturing the same

The present invention relates to a composition for preventing or treating degenerative neurological disease comprising a red ginseng extract, and a method for preparing the same, and specifically, to a degenerative agent with improved NO production inhibitory activity induced by LPS (Lipopolysaccharide) in BV2 cell line without cytotoxicity. It relates to a composition for preventing or treating cranial nerve disease and a method for manufacturing the same.

With the advancement of medicine, the average lifespan is extended, and thus the proportion of the elderly population has also increased significantly. A representative degenerative cranial nerve disease is senile dementia, which is characterized by loss of memory and deterioration of learning ability. On the other hand, the quality of human life has improved thanks to the development of life sciences and science and technology, but the incidence of adult diseases is also increasing. As such, the invention of degenerative neurological diseases continues to increase due to various causes, but drugs capable of effectively preventing or treating these diseases have not yet been developed.

Degenerative brain and nervous system diseases are secondary symptoms caused by other adult diseases such as structural degeneration of cranial nerve cells due to aging, circulatory disorders, or damage to the brain due to various physical and chemical factors such as traffic accidents, industrial accidents, and carbon monoxide poisoning. (Rothman, S. M. (1984) Synaptic release of excitatory amino acid neurotransmitter mediates anoxic neuronal death. J. Neurosci. 4: 1884-1891, and Weiloch, T. (1985) Hypoglycemia-induced neuronal damage prevented by an NMDA antagonists. Science 230: 681-683).

If the blood flow to the brain is reduced or the blood supply to the brain is cut off for the same reasons as above, and the brain tissue is in a state of hypoxia and low glucose, the brain cannot perform normal metabolism and eventually suffers irreparable damage. (Stroke), trauma (trauma) and ischemic injury (ischemic injury) as well as degenerative brain nervous system disease is very common.

For these degenerative cranial nerve diseases, there is still a lack of a detailed understanding of the onset or progression mechanism, so a fundamental treatment has not been found. Various factors, such as genetic factors, oxidative stress, excessive inflammatory response, and abnormal accumulation of peptides, are known as the causes of degenerative brain disease by related studies to date.

Degenerative brain diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, and Amyotrophic Lateral Sclerosis (ALS), which is well known as Lou Gehrig's disease.

First, Alzheimer's disease is a degenerative brain disease in which the cerebral gyrus in the frontal and temporal lobes of the cerebrum is atrophied or reduced due to the death of neurons in the cerebral cortex. one At first, it starts as forgetfulness, and in the later stages, personality is disorganized and psychotic symptoms appear. The exact cause is not yet known, but it is presumed to be genetic, structural changes in the nervous system, inflammatory mediators, cholinergic nervous system, and other neurotransmitters abnormalities.

Parkinson's disease (Parkinson's disease) is a degenerative neurological disease in which the arms, legs, or whole body become trembling and stiff and unable to focus. This is a problem in the brain nerve cells, which causes abnormal secretion of dopamine, a neurotransmitter, and gradually destroys certain nerve cells in the brain.

Typical symptoms of Parkinson's disease are tremors, muscle stiffness, and movement disorders that slow body movements. Because this disease progresses slowly, it is difficult to know exactly when the disease started, and when symptoms are found and the disease is suspected, the nerve cells in the hump in the brain have already changed considerably. In addition, facial expressions may become hardened, gait length may be shortened, and depression or urinary disorders may appear.

Huntington's disease is caused by the degeneration of neurons in the caudate nucleus and putamen of the brain. Huntington's disease is accompanied by uncontrollable body movements, particularly in the arms and legs. This disease can get worse and lead to cognitive and emotional disorders, and eventually death occurs within about 10 to 15 years after symptoms appear. Huntington's disease is a hereditary disease caused by a dominant gene on chromosome 4. Huntington's disease usually occurs in the 30s to 40s, earlier than other degenerative brain diseases, and sometimes appears in the early 20s.

Amyotrophic Lateral Sclerosis (ALS), better known as Lou Gehrig's disease, is a disease that selectively kills only motor neurons. All of the lower motor neurons appear to be progressively destroyed.

Clinical symptoms begin with progressive weakness and atrophy of the extremities, and as the disease progresses, respiratory muscle paralysis eventually leads to death within a few years.

The cause of the disease is not yet known, but several hypotheses have been raised so far. About 5-10% of all amyotrophic lateral sclerosis patients are known to be due to a family history, and mutations in the causative gene on chromosome 21 are confirmed in about 20% of families.

In addition, as a non-hereditary cause, apoptosis by excitotoxicity is considered to play an important role.

Alzheimer's, Parkinson's, Huntington's, and Lou Gehrig's disease are all caused by damage to brain nerve cells, and it is known that oxidative stress accompanied by radical formation is the main cause of damage to brain nerve cells.

Oxidative stress refers to damage to cells or tissues by toxic free radicals, and in particular, brain neurons are sensitive to oxidative stress.

The process by which the brain oxidizes sugars for energy consumes large amounts of oxygen through highly aerobic metabolism. Although the brain is only about 2% of body weight, it consumes enough oxygen to use 20% of total oxygen consumption. Therefore, brain nerve cells are damaged by a lot of free radicals generated along with consumption.

In addition, brain neurons contain a high concentration of polyunsaturated fatty acids, which are easily oxidized, are rich in metal ions (Fe ²⁺ , Cu ²⁺ ) used as catalysts for radical formation, and lack antioxidant enzymes such as catalase compared to other tissues. Therefore, it is more vulnerable to oxidative stress because the defense mechanism against oxidative stress is not sufficient.

Another cause of brain nerve cell damage is damage caused by an inflammatory response. Inflammatory reaction is one of the pathological phenomena that appear in most degenerative brain diseases such as Alzheimer's disease and Parkinson's disease. In the case of degenerative brain disease, unlike general infectious diseases, it is known that various factors contribute little by little to cause and progress the disease.

In particular, according to Kreutzberg, microglial cells among cranial nerve cells are major immune cells involved in brain inflammation, and play a major role in defense from the host and tissue regeneration in the cranial nervous system.

However, according to a study by Chao et al., hyperactivation of microglia occurs rapidly after brain damage such as inflammation, and is a cause of degenerative neurological diseases. Excessive activation of microglia is known to occur by substances such as lipopolysaccharides (LPS), β-amyloid related protein, gp120, an external protein of human immunodeficiency virus (HIV), and nitric oxide (NO) , inflammatory mediators such as prostaglandin E ₂ (PGE ₂ ), interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and reactive oxygen species ( It induces neurotoxicity by promoting the secretion of reactive oxygen species (ROS).

Therefore, in order to prevent damage to the cranial nerve cells, the development of a composition for prevention and treatment capable of suppressing oxidative stress applied to the cranial nerve cells is required.

KR 10-2016-0052824 (2016.05.13.)

The present invention has been devised to solve the above-described problems, and the first object of the present invention is to provide a composition having improved efficacy of inhibiting NO production by LPS in BV2 cell line without cytotoxicity, and second solution The object to be addressed is to provide a method for preparing such a composition.

In order to solve the above-described first problem, the present invention is a powder obtained by drying and pulverizing red ginseng ( Panax ginseng CA Meyer ) root and Saururus chinensis leaves with water (H ₂ O), C ₆ or less lower alcohol or water and the above It provides a composition having LPS-induced NO inhibitory activity, characterized in that it comprises a mixture of the extract extracted with an extraction solvent containing a mixed solution of a lower alcohol.

In a preferred embodiment of the present invention, the extraction solvent may be a 50% to 90% by weight aqueous solution of ethanol (C ₂ H ₅ OH).

In a preferred embodiment of the present invention, the mixture of extracts may further include an extract obtained by extracting the dry pulverized powder of the roots of Paeonia lactiflora Pallas with the extraction solvent.

In a preferred embodiment of the present invention, the red ginseng root may be more than 5 years old.

In a preferred embodiment of the present invention, the mixture of extracts is a powder mixture of red ginseng root, peony root, and ginseng root whose composition satisfies the following condition 1) and further satisfies one of conditions 2) and 3) It may be extracted from

1) 10wt% ≤ C _{red ginseng} ≤ 90wt%

2)

3)

In the above conditions 1) to 3), C _{red ginseng} , C _peony , and C _trifolieae refer to the content by weight of red ginseng root, peony root, and trifolium leaf powder in the powder mixture, respectively.

In a preferred embodiment of the present invention, the extract mixture may be extracted with an extraction solvent 10 to 20 times the weight of the powder mixture.

In order to solve the second problem described above, the present invention comprises the steps of (1) preparing a powder mixture obtained by drying and pulverizing red ginseng ( Panax ginseng CA Meyer ) root and Saururus chinensis leaves; and (2) the powder Extracting the mixture with an extraction solvent comprising water, a lower alcohol of C ₆ or less, or a mixed solution of water and the lower alcohol; provides a method for producing a composition having an effect of inhibiting LPS-induced NO activity, comprising.

In a preferred embodiment of the present invention, step (2) may be reflux extraction twice to 4 times at a temperature of 70 ° C. to 120 ° C. for 30 minutes to 2 hours.

In addition, the present invention comprises the steps of: (i) preparing a powder obtained by drying and pulverizing red ginseng roots and leaves of ginseng; (ii) extracting the powder with an extraction solvent including water, a lower alcohol of C ₆ or less, or a mixed solution of water and the lower alcohol, respectively; And (iii) mixing the extract obtained in the extraction step; provides a method for preparing a composition for preventing or treating degenerative neurological disease comprising.

According to the composition for the prevention and treatment of degenerative cranial nerve disease according to the present invention, the inhibitory activity of neuroinflammation induction by LPS is remarkably improved while having little toxicity to nerve cell lines, so that it has an excellent effect in preventing and inhibiting the progression of degenerative cranial nerve disease.

Figure 1a is a graph showing the amount of NO generated by LPS in the BV2 cell line according to each concentration when extracts extracted from 6-year-old red ginseng roots and leaves of ginseng root with 70% aqueous ethanol solution were treated alone.
Figure 1b is a graph showing the amount of NO generated by LPS in the BV2 cell line according to the concentration of each extract when each extract extracted from peony roots with 70% ethanol aqueous solution was treated alone.
Figure 2 shows the composition of the examples in which the content of each component is changed in the ternary mixture of red ginseng, peony and ginseng. Each dot represents the composition of the examples.
4 is a graph showing the amount of NO generation in the BV2 cell line according to the concentration of the composition extracted by changing the composition of the mixture in the preferred embodiments of the present invention.
5a is a graph showing cell viability during LPS treatment after treatment with BV2 cell line at a concentration of 100 μg/ml of a composition extracted by changing the composition of a mixture in preferred embodiments of the present invention.
5B is a graph showing cell viability during LPS treatment after treatment with BV2 cell line at a concentration of 200 μg/ml of a composition extracted by changing the composition of a mixture in preferred embodiments of the present invention.
6 is a graph showing the cell viability of the BV2 cell line according to each concentration when the extracts extracted from Rehmannia glutinosa (Gaertner) Libosch) roots and peony roots with 70% ethanol aqueous solution were treated alone.
7 is a graph showing the cell viability of the BV2 cell line according to the treatment concentration of each extract when extracts obtained by extracting Rehmannia glutinosa Liboschitz ex Steudel roots and leaves of trifolieae with 70% ethanol aqueous solution were treated alone.
8 is a graph showing the cell viability of the BV2 cell line according to the concentration of each extract when the extracts obtained by extracting Angelica gigas Nakai root and Schisandra chinensis fruit with 70% ethanol aqueous solution were treated alone.
Figure 9 shows the cell viability of the BV2 cell line according to the treatment concentration of each extract when extracts extracted from Lvcium Chinense Miller fruit and Gilgyeong ( Platycodon grandiflorum A. De Candolle) roots with 70% ethanol aqueous solution were treated alone; This is the graph shown.
Figure 10 shows the cell viability of the BV2 cell line according to the treatment concentration of each extract when extracts extracted from red ginseng (4-year-old) roots and black ginseng ( Panax ginseng CA Meyer, Black Ginseng) roots with 70% ethanol aqueous solution were treated alone. This is the graph shown.
11 is a graph showing the cell viability of the BV2 cell line according to the concentration of the extract when the extract obtained by extracting red ginseng (6-year-old root) with 70% ethanol aqueous solution is treated alone.
12 is a BV2 cell line of extracts obtained by extracting Geonjihwang root, peony root, red ginseng 4-year-old root, Sukji-hwang root, Sambaekcho leaf, black ginseng root, Angelica asiatica, Omija fruit, red ginseng 6-year-old root, Goji berry fruit, and Gilgyeong root with 70% aqueous ethanol solution. It is a graph showing cell viability when treated with LPS after treatment.

As described above, it is known that oxidative stress applied to cranial nerve cells acts as a factor in degenerative cranial nervous system diseases. The present invention has the activity of inhibiting the generation of inflammatory nitric oxide (NO, nitric oxide) induced by LPS in BV2 cells, which are microglia, thereby protecting brain cells and preventing and treating degenerative cranial nerve diseases such as memory improvement. It provides a composition having.

Specifically, the present invention is an extraction solvent containing a powder obtained by drying and pulverizing red ginseng ( Panax ginseng CA Meyer ) roots and leaves of Saururus chinensis with water, a lower alcohol of C ₆ or less, or a mixed solution of water and the lower alcohol. To provide a composition for preventing or treating degenerative cranial nerve disease, characterized in that it comprises a mixture of the extracted extract to protect the cranial nerve cells from oxidative stress as described above.

Here, the mixture of the extracts may be an extract obtained by extracting a powder mixture of each raw material, that is, dry-pulverized powder of red ginseng root and ginseng leaf, with the extraction solvent, and an extract obtained by extracting the dry-pulverized powder of the raw material with the extraction solvent, respectively. It may be a mixture of extracts mixed with That is, the raw material may be mixed before extraction and then extracted, or the extract after extraction may be mixed.

Preferably, it is good to extract after mixing the powdered raw material before extraction. Doing so facilitates control of the composition.

The composition according to the present invention has an improved activity to inhibit the generation of NO induced by LPS in BV2 cells, which are microglia, and thus is effective in suppressing memory decline and preventing degenerative neurological diseases.

Red ginseng root contains a large amount of ginsenoside, and ginsenoside is known to have excellent NO inhibitory activity in BV2 cells. The present invention includes a mixture of extracts of red ginseng root and leaf powder of ginseng and can realize better NO inhibitory activity compared to the case of extracting only red ginseng root.

Preferably, the composition for preventing or treating degenerative brain disease of the present invention may further include an extract of dry powder of the root of the peony ( Paeonia lactiflora Pallas). By including the ternary mixture of extracts as described above, the NO inhibitory activity can be more clearly improved compared to the therapeutic composition including the extract of red ginseng alone.

According to a preferred embodiment of the present invention, the mixture of extracts extracts the active ingredient at a high concentration compared to the case of using the raw material root or leaf as it is by extracting the dried and pulverized powder of red ginseng root, ginseng root, and peony root. This can be done, and there is an advantage in that the yield is increased and the productivity is improved.

Preferably, the mixture comprises a drying step of a) removing foreign substances, etc. from the raw materials such as red ginseng root, peony root, and ginseng leaf through a screening process, and b) removing moisture through freeze-drying or natural drying; and c) a pulverizing step of finely dicing or pulverizing the raw material using a pulverizer; it may be used as a dry pulverized powder.

At this time, if moisture is removed through freeze drying in step b), preferably at a temperature of -60°C to -80°C, it can be carried out for 12 hours to 48 hours, and in the case of natural drying, 12 hours in a cool shade It can be dried by carrying out for 48 hours. This drying method is only an example and is not necessarily limited thereto.

According to a preferred embodiment of the present invention, the extraction solvent may be one or more selected from water, ethanol (ethanol), methanol, and butanol.

The extraction solvent may be preferably a mixed solution of water and the lower alcohol of C ₆ or less, and more preferably an aqueous solution containing 50 to 90% by weight of the lower alcohol. By using an aqueous alcohol solution as the extraction solvent, the index component content and yield can be improved compared to using other solvents.

The lower alcohol may be preferably ethanol (ethanol, C ₂ H ₅ OH). Ethanol has less toxicity to the human body when ingested than other alcohols, so the extract can be obtained without a separate washing or dilution process, so it has the most economical advantage.

The extraction solvent may be preferably a 50 wt% to 90 wt% aqueous solution of ethanol (alcohol), more preferably a 60 wt% to 80 wt% aqueous solution of ethanol, more preferably 65 wt% ethanol to 75% by weight. When extracting with an aqueous alcohol solution within this range, the yield is excellent, contains a lot of physiologically active substances, and there is an advantage in that the extraction time can be relatively shortened.

The red ginseng root may preferably be used for 5 years or more. In the case of using less than 5-year-old root, the inhibitory efficacy of LPS-induced NO production in the concentration range (about 25 to 200 μg/ml) in which the extract does not show cytotoxicity is relatively low compared to the case of using 5-year-old or more root.

In a preferred embodiment of the present invention, the mixture of extracts may be extracted with the extraction solvent 10 to 20 times the weight of the powder mixture. In the case of extraction with an extraction solvent less than 10 times the weight of the mixture, the extraction rate may decrease, resulting in reduced productivity.

The powder mixture may preferably have a composition satisfying the following condition 1).

1) 10% by weight ≤ C red ginseng ≤ 90% by weight

In the above condition 1), C _{red ginseng} refers to the content by weight of red ginseng root powder in the powder mixture.

If C _{red ginseng} < 10% by weight, there may be a problem in that the content of ginsenoside, an active ingredient of red ginseng, is lowered in the extract mixture, and when C _{red ginseng} > 90% by weight, peony root or tribaekcho leaf There may be little synergistic effect by mixing with

When the mixture of extracts further comprises an extract of peony root, the mixture of extracts may include a mixed extract obtained by extracting a ternary mixture of red ginseng root, ginseng root, and dry pulverized powder of peony root with the extraction solvent. In this case, the ternary powder mixture may further satisfy the following conditions 2) or 3).

2)

3)

*92In the conditions 2) and 3) above, C _peony and C _trifoliea mean the content by weight of peony root and trifolium leaf in the ternary mixture of red ginseng root, peony root, and trifolium leaf, respectively.

Here, the meaning that red _ginseng C is the content based on the weight of red ginseng root in the ternary powder mixture means that the powder mixture may further include other ingredients in addition to the ternary mixture of red ginseng root, peony root, and ginseng leaf powder. it means.

Next, a method for preparing a composition for preventing or treating degenerative brain nervous system disease according to the present invention will be described.

The composition for preventing or treating degenerative brain system disease according to the present invention comprises the steps of: (1) preparing a powder mixture obtained by drying and pulverizing red ginseng ( Panax ginseng CA Meyer ) root and Saururus chinensis leaves; and (2) extracting the mixture with an extraction solvent containing water, a lower alcohol of C ₆ or less, or a mixed solution of water and the lower alcohol.

In addition, the composition comprises the steps of: (i) preparing a powder obtained by drying and pulverizing red ginseng roots and leaves of ginseng; (ii) extracting the powder with an extraction solvent including water, a lower alcohol of C ₆ or less, or a mixed solution of water and the lower alcohol, respectively; and (iii) mixing the extract obtained in the extraction step;

That is, the composition for preventing or treating degenerative brain system disease according to the present invention is obtained by extracting a mixture of dry and pulverized powder of red ginseng root and trifolium leaf with the extraction solvent to obtain a mixed extract, or by changing the order, each raw powder is used as the extraction solvent It is prepared by a method of obtaining a mixture of extracts by mixing extracts extracted with

Of these, as described above, the method of preparing the powder mixture in step (1) includes: a) removing foreign substances from the raw materials such as red ginseng root, peony root, and ginseng root through a screening process; and b) freeze-drying or natural drying. a drying step to remove moisture through; and c) a pulverizing step of finely dicing or pulverizing the raw material using a pulverizer;

In this case, since the content regarding the drying conditions is the same as described above, it is omitted.

In addition, at this time, the mixture may include peony root powder obtained by selecting peony roots to remove foreign substances, and then pulverizing after removing moisture through freeze-drying or natural drying.

In addition, in the case of preparing a composition for preventing or treating degenerative brain disease including steps (i), (ii) and (iii), in step (i), a powder of peony root may be further prepared.

In this case, in step (ii), the process of extracting the powder of the peony root through the extraction solvent may be performed, and in step (iii), the extract may be further mixed with the extract.

The effect according to this is to prepare a composition for preventing or treating degenerative neurological disease comprising a mixture of ternary extracts, and the effect thereof is the same as that of the composition described above.

Step (2) may preferably be to reflux extraction twice to 4 times at a temperature of 70° C. to 120° C. for 30 minutes to 2 hours, and the same applies to step (ii).

Hereinafter, the present invention will be described in more detail through examples. However, the examples and manufacturing examples described in the present specification are only examples of the most preferred embodiment of the present invention, and do not represent all of the technical spirit of the present invention, so various equivalents that can replace them at the time of the present application It should be understood that there may be water and variations.

Examples 1 to 10

6-year-old red ginseng root (purchased at Geumsan Herb Market in 2018), peony root (purchased at Geumsan Herb Market in 2018) and Sambaekcho leaf (purchased at Geumsan Herb Market in 2018), which are distributed in Korea, are individually selected through a selection process. After removing foreign substances and the like, it was pulverized using a pulverizer and used as a powdery sample.

In this example, the powdered sample was mixed and used in the raw material state of the red ginseng root, peony root, and leaf of ginseng.

100 g of mixed powder was prepared by mixing the obtained 6-year-old red ginseng root, peony root, and ginseng leaf powder at the weight ratios shown in Table 1 below.

About 100 g of the mixed powder was placed in each of the herbal nonwoven fabrics, placed in an extraction container, and 70% ethanol (alcohol) aqueous solution was added thereto. Extraction under reflux at a temperature of 100° C. in the extraction vessel was repeated twice in total for 1 hour at a time. After the extraction, the ethanol aqueous solution was filtered under reduced pressure using a filtration device (vacum pump, GAST).

The obtained extract was concentrated using a reduced pressure concentrator equipped with a cooling condenser (EYELA rotary evaporation system, Tokyo, Japan). After freeze-drying (Freeze dryer, IilshinBioBase) or vacuum-drying (Vaccum dry oven, WiseVen) to remove the remaining moisture or ethanol, it was homogenized with a mortar and powdered to prepare a composition for preventing or treating degenerative brain disease.

division designation Content (total 100) extraction yield
(%) red ginseng
(6 years old) peony three hundred seconds Example 1 RPS-1 10.00 10.00 80.00 10.38 Example 2 RPS-2 21.67 56.67 21.67 30.63 Example 3 RPS-3 10.00 45.00 45.00 40.61 Example 4 RPS-4 80.00 10.00 10.00 29.10 Example 5 RPS-5 45.00 45.00 10.00 21.32 Example 6 RPS-6 45.00 10.00 45.00 20.74 Example 7 RPS-7 10.00 80.00 10.00 19.42 Example 8 RPS-8 21.67 21.67 56.67 14.01 Example 9 RPS-9 33.33 33.33 33.33 28.27 Example 10 RPS-10 56.67 21.67 21.67 25.01

Preparation Examples 1 to 11

An extract was prepared using the materials shown in Table 2 below as raw materials by the same method as in the above Examples.

division Content (total 100g) extraction yield
(%) Preparation Example 1 4 years old red ginseng 38.24 Preparation 2 black ginseng 42.59 Preparation 3 6 years old red ginseng 41.10 Preparation 4 three hundred seconds 25.14 Preparation 5 Schisandra 49.15 Preparation 6 donkey 54.71 Preparation 7 peony 33.71 Preparation 8 Suk Sook-hwang 60.33 Preparation 9 Guernsey Hwang 72.87 Preparation 10 Gilkyung 49.38 Preparation 11 Goji berries 54.74

Experimental example

Microglia BV2 was obtained by adding 100 U/ml penicillin and 100 mg/ml streptomycin to DMEM medium containing inactivated 5% (v/v) fetal bovine serum (FBS) to maintain temperature and humidity at 37°C and 5 It was cultured in an incubator under % CO2 conditions. After culturing each cell in a 96-well plate for 24 hours, single extracts and mixed extracts were treated at concentrations of 25, 50, 100, 200, and 500 μg/mL, and then cultured for 24 hours. After adding 10 μL of Cell Counting Kit (Dojindo, Kumamoto, Japan) solution and reacting for 2 hours, absorbance was measured at 450 nm using a microplate reader (Bio-Rad, California, USA). The average absorbance value for each sample group was obtained.

In the following experiment, the cell viability was calculated as a relative cell viability (% of control) by comparing the absorbance of each experimental group with the absorbance value of the control group. In the cell activity experiment, each extract was treated within a concentration range that did not show cytotoxicity.

Experimental Example 1: Cytotoxicity test

A. Sole extract

1) The single extracts prepared according to Preparation Examples 1 to 11 were treated in the BV2 cell line at 25, 50, 100, 200, and 500 μg/mL by concentration, and cell death was observed after 24 hours. The cell line viability according to the treatment concentration of each extract is shown in the graphs of FIGS. 6 to 11 .

2) In addition, the effect of a single extract prepared according to Preparation Examples 1 to 11 on LPS-stimulated BV2 cells on cell survival was confirmed. A single extract of microglia BV2 was applied to BV2 cells at a concentration of 25, 50, 100, 200, and 500 μg/mL, respectively, incubated in a CO ₂ incubator for 1 hour, and then treated with LPS (100 ng/ml). After incubation for 24 hours, cell death was observed. The viability of the cell line according to the treatment concentration of each extract in LPS-stimulated BV2 cells is shown in the graph of FIG. 12 .

6 to 12, the 70% ethanol extract prepared according to Preparation Examples 1 to 11 was treated on BV2 cells by concentration. As a result, 7 extracts based on 100% cell viability of the control group not treated with the extract 25-500 μg/mL for Geonjihwang, Sukjihwang, Angelica, Omija, Goji, Gilgyeong, red ginseng (4-year-old root), and black ginseng), and 25-200 μg/mL for 3 types of extracts (peony, ginseng plant, and red ginseng (6-year-old root)). It showed more than 80% cell viability in the concentration range of mL.In addition, as a result of confirming the toxicity of each extract to LPS-stimulated BV2 cells, it did not show cytotoxicity in the concentration range of 25 to 200 μg/mL, confirming the safety. could

B. Mixed Extracts

In addition, when the extracts of Examples 2, 4, 6 and 9 among the compositions of Examples 1 to 10 were treated in the BV2 cell line at concentrations of 100 μg/mL and 200 μg/mL by the same method as above, each Cell viability is shown in the graphs of Figures 5a (100 μg / mL) and 5b (200 μg / mL) for the Example.

5A and 5B, in all compositions comprising the extracts of Examples 2, 4, 6 and 9 (represented by RPS-2, PRS-4, RPS-6 and RPS-9, respectively), 100 μg/mL and It was confirmed that good cell viability was exhibited at all of 200 μg/mL.

Experimental Example 2: LPS-induced NO inhibitory activity test

1) The concentration of NO generated from the BV2 cells activated by LPS was detected using a Griess reagent reagent (Promega NO assay kit) that recognizes NO ₂ ⁻ present in the cell culture medium. To this end, after culturing BV2 in a 96-well plate for 24 hours, the extracts prepared according to Preparation Examples 3, 4 and 7 were treated at concentrations of 25, 50, 100 and 200 μg/mL, and LPS (100 ng /ml) was re-treated. After culturing for 24 hours, the cell culture supernatant was recovered. Equal amounts of cell culture supernatant and Griess reagent (Sulfanilamide Solution: NED Solution (volume ratio) = 1:1) were mixed and reacted for 10 minutes, then absorbance was measured at 540 nm using a microplate reader, and a standard concentration curve The amount of nitrate was calculated according to

The generation amount was measured, and the results are shown in FIGS. 1A (Preparation Examples 3 and 4) and 1B (Preparation Example 7), respectively.

1A and 1B, extracts made from peony (root), ginseng (leaf), and red ginseng (six-year-old root) as raw materials were produced by LPS without toxicity to BV2 cells within a concentration range of 25 to 200 μg/mL. It tends to decrease the induced NO production. It was confirmed that the extracts of these three substances can be used as an active ingredient in a composition for the treatment of degenerative cranial nerve disease.

Among the compositions of Examples 1 to 10, when the extracts of Examples 2, 4, 6 and 9 were treated in the BV2 cell line at concentrations of 100 μg/ml and 200 μg/ml (LPS is 100 ng/mL), the amount of NO generated was shown in FIG. shown in The NO generation amount measurement method is the same as the NO emission amount measurement method for Preparation Examples 3, 4 and 7.

That is, the extracts of Examples 2, 4, 6 and 9 in microglia BV2 were respectively treated in BV2 cells at a concentration of 100 and 200 μg/mL and incubated in a CO ₂ incubator for 1 hour, followed by LPS (100 ng / ml) and cultured for 24 hours, and then the supernatant was recovered. Using Griess Reagent reagent, the cell culture supernatant and Griess reagent (Sulfanilamide Solution: NED Solution (volume ratio)=1:1) were mixed in equal amounts and reacted for 10 minutes, then absorbance was measured at 540 nm using a microplate reader, standard concentration The amount of nitrate was calculated according to a standard curve.

Referring to FIG. 4 , it was confirmed that the amount of NO generated was significantly reduced when the compositions of Examples 2, 4, 6 and 9 were added together compared to the cell line to which only LPS was added without adding any composition, and in particular, Example 6 It was confirmed that the most excellent NO inhibitory activity was shown in the composition of

Table 3 below is a table showing the measurement of the inhibition rate of NO production by LPS when the compositions prepared according to Examples 2, 4, 6 and 9 were treated at a concentration of 100 μg/mL.

Example 2 Example 4 Example 6 Example 9 Component mixing ratio (weight ratio) 21.7:56.7:21.7 80:10:10 45:10:45 33:33:33 NO production inhibition rate (%) relative to control 50 54 70 54

Referring to Table 3, it can be seen that the compositions of Examples 2, 4, 6 and 9 have excellent NO production inhibition rate, and in particular, the NO production inhibition rate of Example 6 is the highest compared to the control group.

Claims (6)

Red ginseng (Panax ginseng CA Meyer) root, peony (Paeonia lactiflora Pallas) root and Saururus chinensis leaf are dried and pulverized to 10 to 20 times the weight of the powder with water (H ₂ O) and C ₆ Includes a mixture of extracts extracted with a mixed solution of the following lower alcohols,
The mixture of extracts is a composition having an LPS-induced NO inhibitory activity, characterized in that it is extracted from a powder mixture of red ginseng root, peony root, and trifolium leaf, the composition of which satisfies the following conditions 1) to 3):
1) 10% by weight ≤ C _{red ginseng} ≤ 90% by weight
2) C _{red ginseng} ≥ C _{300 seconds} + C _peony
3) 1/ _3≤ C 300 sec /C _Peony ≤ 5
In the above conditions 1) to 3), C _{red ginseng} , C _peony , and C _trifolieae refer to the content by weight of red ginseng root, peony root, and trifolium leaf powder in the powder mixture, respectively. The method of claim 1,
The extraction solvent is a composition having an LPS-induced NO inhibitory activity, characterized in that 50% to 90% by weight of an aqueous solution of ethanol (C ₂ H ₅ OH). The method of claim 1,
The composition with LPS-induced NO inhibitory activity, characterized in that the red ginseng root is more than 5 years old. (1) preparing a powder mixture of dry and pulverized red ginseng (Panax ginseng CA Meyer) root, peony (Paeonia lactiflora Palls) root and Saururus chinensis leaf; and
(2) extracting the powder mixture with a mixed solution of water (H ₂ O) and C ₆ or less lower alcohol of 10 to 20 times the weight of the powder mixture;
The powder mixture is a method for preparing a composition having an LPS-induced NO inhibitory activity, characterized in that it satisfies the following conditions 1) to 3):
1) 10% by weight ≤ C _{red ginseng} ≤ 90% by weight
2) C _{red ginseng} ≥ C _{300 seconds} + C _peony
3) 1/ _3≤ C 300 sec /C _Peony ≤ 5
In the above conditions 1) to 3), C _{red ginseng} , C _peony , and C _trifolieae refer to the content by weight of red ginseng root, peony root, and trifolium leaf powder in the powder mixture, respectively. 5. The method of claim 4,
The step (2) is a method for producing a composition having an LSP-induced NO inhibitory activity, characterized in that the extraction is refluxed 2 to 4 times at a temperature of 70 ° C. to 120 ° C. for 30 minutes to 2 hours. (i) preparing a powder obtained by drying and pulverizing red ginseng root, peony root, and ginseng root, respectively;
(ii) extracting the powder with a mixed solution of 10 to 20 times the weight of water (H ₂ O) and C ₆ or less lower alcohol relative to the weight of the powder, respectively; and
(iii) mixing the extract obtained in the extraction step; including,
The red ginseng root, peony root, and powder obtained by drying and pulverizing the leaves of ginseng root, the method for preparing a composition having LPS-induced NO inhibitory activity, characterized in that the following conditions 1) to 3) are satisfied:
1) 10% by weight ≤ C _{red ginseng} ≤ 90% by weight
2) C _{red ginseng} ≥ C _{300 seconds} + C _peony
3) 1/ _3≤ C 300 sec /C _Peony ≤ 5
In the above Conditional Formulas 1) to 3), C _{red ginseng} , C _ginseng plant and C _peony represent the content based on the weight of each powder relative to the total weight of the red ginseng root, ginseng leaf and peony root powder, respectively.

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