KR20180094208A - Preparing method of fermentative metabolites from stachys sieboldii having imrpoving cognitive fuctions - Google Patents

Abstract

The present invention relates to a stachys Stachys fermentation metabolome having an activity of preventing of deterioration of a cognitive function and improving of the cognitive function, a manufacturing method thereof, and a composition for preventing or reducing the deterioration of a cognitive function containing the same. The food composition of the present invention comprises all types of natural materials such as functional foods, nutritional supplements, health foods and food additives. The food composition of the above type can be prepared in a variety of forms according to conventional methods known in an art.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for producing a ground-state fermentation metabolite having improved cognitive function,

TECHNICAL FIELD [0001] The present invention relates to a cornerstone permanent-fermentation metabolism body having cognitive function improving activity, a method for producing the same, and a composition for preventing and improving a cognitive dysfunctional disease containing an anticancer body as a active ingredient.

Cornerstone sleep ( Τυαγθψτ τιεβομδιι Miq.) is a perennial plant of Lamiaceae and is used as a rootstock or a plant of foundation stone sleeping. In other words, in China, it is used as a ground, a sweet potato, It is recorded in the Chinese medicinal texts such as 甘 幼 兒, toe yong, 寶塔 菜, wajae), tochusho, 地 紐, etc. In Japan, Elder 喜) is called. The scientific name of foundation stone sleep is the foundation stone sleep ( Τυαγψτ τιεβομδιι Miq.) Of Lamiaceae stingray ( Τυαγθψτ Linne). There are about 300 species of Lepidoptera (Labiatae) stingray ( Τυαγθψτ Linne) in the world, and four varieties are growing in our country.

It is grown in China, Japan, and Russia. Recently, it has grown to have excellent pharmacological efficacy and ease of cultivation. Which is cultivated on a large scale in the Sri Chan area of Korea and the Jiri area.

It is used for the treatment of various adult diseases and chronic diseases in China, and it is a typical longevity vegetable in China. According to the Chinese medicine section, cerebral infarction, memory improvement, improvement of cognitive function, senile dementia and bowel function It is used as a useful material to promote.

Studies on cornerstone sleep were most actively carried out in Japan and China. In Korea, anticancer and immunological effects of primordial asparagus extract, antimicrobial activity of leaf extract (non-patent document 1), effect of active oxygen scavenging, characteristics of stem and root extract Analysis, nitrite scavenging ability, antioxidant activity (non-patent document 2, non-patent document 3, non-patent document 4), acetylchoilne esterase and monoamine oxidase activity inhibitory effect were reported.

In the foreign countries, 9 polyphenyl alcohol glycosides were isolated from ground stone sleep, separation and structure determination of new glycosides, quantification of stachyose using HPLC, improvement of anaerosis, inhibition of hyaluronidase activity, immunosuppressive action and hormone and mutagenic induced hydrogen peroxide spectrum were reported There is a bar. In recent years, acetooside has been found to be very effective in improving glomerulonephritis through immunosuppression, which is attributed to inhibiting the number of white blood cells in the glomeruli.

In addition, methanol extracts from cornstarch were found to improve anoxia in KCN-induced anoxia mice model, and these substances were found to be acteoside, stachysoside, and phenylethanoid glycoside.

Recently, studies on the morphological indicators of extracorporeally regenerating substances in cornerstone sleep have been reported. It has been found that various physiological activities are excellent because it contains a large amount of natural oligosaccharides such as stachyose, saponin, iridoidic fractions, polyphenyl alcohol glycoside, aceteoside, choline and alginate.

In recent years, acetooside, a derivative of corn starch, has been shown to be effective in improving glomerular nephritis by suppressing immune suppression, ie, leukocyte count. And stachyose, which is a natural oligosaccharide which is contained in a large amount of foundation stone sleep, is active in growth of lactic acid bacteria, activating intestinal motility, promoting bowel movement and suiting.

main ingredient efficacy Stachyose Promoting the growth of bacteria in the intestines, promoting bowel movement, promoting absorption of useful substances, inhibiting the absorption of triglycerides and cholesterol, reducing blood sugar Stachydrine Improving sweetness, vision and liver function, improving blood circulation Phenylethanoid  glycoside Direct activation of brain cells to activate brain cells Alginate Promotes dietary fiber, harmful metal emissions, and decreases blood cholesterol Choline The substances of the brain nerve cell, the improvement of the body's lipid metabolism Vitamin B ₄ Fat metabolism essential vitamins, muscle maintenance and development Aceteoside Cerebral infarction, nephritis improvement and treatment (urination and swelling improvement), glomerulonephritis treatment, excessive immunosuppression

On the other hand, biotransformation technology is also used as bioprocessing, bioconversion, biosynthesis, biocatalysis, etc., and the technology to produce desired products from precursors using microorganisms . In the modern society, chemical synthesis technology has been used for industrial development. However, these chemical synthesis technologies have caused severe environmental problems by using high temperature, high pressure, highly toxic reagents and solutions.

Bio-conversion technology is expected to increase by 10 ~ 20% in 2010, and about 50% by 2050, due to its energy consumption and environmental friendliness compared to chemical synthesis technology in many fields such as food, medicine and cosmetics . Among the biotransformation technologies, researches have been made on methods of increasing biologic activity by converting glycosides to aglycone through microbial methods and enzyme conversion. Among the biotransformation techniques, there are liquid culture and solid culture methods for synthesizing substances using microorganisms. Although the technology is stabilized in the liquid culture, it is difficult to improve the productivity through improvement. However, the solid culture is difficult to massify and standardize Low energy consumption and high productivity have recently been attracting attention and research is being conducted. Fermentation using microorganisms can be expected to increase the absorption of the active substance as a result of the low molecular weight of the active ingredient, and to exhibit the same effect normally to a person who does not have a specific intestinal bacteria. Research through biotransformation techniques has been carried out in many places, mainly through the saponin of ginseng. Among them, ginseng contains saponin called gisenoside, and research has been conducted to devise a method to increase the yield of compound K, which is a strong physiologically active ingredient. Studies on the possibility of developing functional food materials using bioconversion technology for flavonoids, which are known to have excellent antioxidant activity, and studies on inhibition of melanin synthesis by using biotransformation technology have been conducted. And so on. Research on the development of new foodstuffs through biotransformation technology is also being carried out, and research using biotransformation technology is expected to proceed in a variety of ways.

However, there are few studies using biotransformation technology of cornstarch sleep, and most of the studies reported through biotransformation technology are high-cost, low-rate liquid fermentation, and studies using low-cost, high-yield solid fermentation are insignificant .

Korean Patent Registration No. 10-1394533 discloses a technique for the production of a crude persimmon fermented metabolite. However, the above-described technology focuses on the preparation of a fermentation metabolite that enhances the prevention and improvement of inflammatory diseases, There has been no known technology for producing a base-lock fermentation metabolite capable of maximizing the effect of improving the deterioration of cognitive function.

With the rapid aging of modern society, the number of senile dementia is increasing rapidly. Considering the growth rate of the dementia population, the world population is estimated to reach 35.6 million in 2010 and triple in 2050, reaching 115.4 million. In Korea alone, the number of demented people in Korea is 535,000 (9.1% of the total elderly population) in 2012, 424,000 (up 26.8%) in 2008, and this trend is expected to increase to more than 1 million in 2025 do.

The socioeconomic burden of dementia in the world is estimated to be $ 604 billion in 2009 (1% of global GDP), and the economic cost of dementia , Heart disease, and stroke. The socioeconomic costs associated with domestic dementia are estimated to be 8,770 million won per year, almost doubling every 10 years (2020: 18.9 trillion won → 2030: 38.9 trillion won) Source: .

In this background, social interest in early detection of dementia is increasing both personally and socially. The prevalence of dementia is expected to decline to 80% in 20 years after delaying the onset of dementia by early detection of dementia with high risk of dementia. The cost of medical treatment after 5 years of drug treatment from early stage is estimated to be 5,174 billion won It is estimated to be reduced.

In fact, dementia is a disease that can be alleviated at the early stage of the disease or it is a stagnant disease. However, there is no proper early diagnosis and treatment method. Currently, Korea is rapidly aging and the population of dementia is rapidly increased. Is a reality.

It is also expected that the number of patients with degenerative neurological diseases will continue to increase due to changes in the global population structure, and that the incidence of Alzheimer's disease (AD) will continue to grow at an annual average rate of about 10%

In Korea, the aging of the world is the fastest pace in the world, and it is expected that the demand for health functional foods and preventive and therapeutic agents for degenerative neurological diseases, which are representative geriatric diseases, will surge.

Degenerative neuropathy is a disorder in which the function of the brain or spinal cord is paralyzed due to abnormal neuronal death, unlike normal aging process. This is one of the leading causes of degenerative neurology. It is an oxidative stress caused by reactive oxygen species (ROS).

In addition, glutamate, which acts as a central excitatory neurotransmitter in neurons, accumulates in the brain, leading to the death of neurons when individuals become diseased such as ischemia and nutritional imbalance.

The role of brain-derived neurotrophic factor (BDNF), one of the neurotrophic factors in neuronal death control, is attracting attention. BDNF is an endogenous protein belonging to the neurotrophin family and has been reported to protect neurons from various neurotoxic factors and plays an important role in maintenance of nerve development, central and peripheral nerve cells.

Korean Patent Registration No. 10-1394533

 Moon, Yoon - Hee, Park, Jong - ok, Song, Seung - Gu. 2002. Effect of active oxygen scavenging on cornerstone sleep. Korean Society for Biological Science. 122-122.  Hong-Seuk Baek. Beung-Ho Ryu, and Seung-Koo Song. 2003. Total polyphenols and antioxidant activities of the extracts from Stachys sieboldii MIQ. Korean Journal of Biotechnology and Bioengineering. 563-566.  Hong-Seuk Baek, Young-Soo Na, Do-Han Kim, Chang-Han Lee, Beung-Ho Ryu and Seung-Koo Song. 2004. Antioxidant Activities of Stachys sieboldii MIQ Roots. Journal of Life Science. 14 (1): 1-7.  Hong-Seuk Baek, Young-Soo Na, Beung-Ho Ryu and Seung-Koo Song. 2003. Antioxidant Activities of Stachys sieboldii MIQ. Stalks. Korean Journal of Biotechnology and Bioengineering. 18 (4): 266-271.

It is an object of the present invention to provide a cornerstone permanent fermentation metabolism body having cognitive function improving activity.

It is still another object of the present invention to provide a composition for preventing or ameliorating a cognitive dysfunctional disease comprising the above-mentioned dead-lock fermented metabolite.

A method for producing a ground-based permanent-fermentation metabolite according to the present invention comprises:

a) cutting the dry crumb stone to an average particle size of 5 to 20 mm to obtain a foundation stone raw material;

b) 10 to 15 times by weight of water is added to the base stone base material and the mixture is refluxed at 95-100 ° C for 9-11 hours, filtered and concentrated under reduced pressure at 50 to 55 ° C to obtain a concentrate adjusted to a concentration of 15 to 20 brix ;

c) 0.5% yeast extract and 1% sugar as a carbon source were added to the concentrate as a nitrogen source, the pH was adjusted to 6.5 ~ 7.0, sterilized at 121 ° C and 1.5 atm for 10 ~ 15 minutes, ;

d) Lactobacillus plantarum and Leuconostocenteroides ( Lactobacillus plantarum ) cultured in the MRS medium at 30 DEG C for 1 day, mensenteroides ) in a concentration of 2% relative to the raw material and incubating at 30 ° C for 4 days;

e) The culture solution was sterilized at 100 ° C for 10 minutes, and the same amount of ethanol was added to the filtrate. The mixture was dipped at 4 ° C for 24 hours, centrifuged at 6,000 rpm for 30 minutes, lyophilized, .

On the other hand, the present invention is characterized in that it is produced by the above method. It was confirmed that the crude polysaccharides contained 3.85% of total polyphenol, 20.23% of stachyose and 112 mg% of crude saponin in the crude polysaccharide freeze-dried powder obtained by the above method.

In addition, the composition for preventing and improving the lowering of the cognitive function of the present invention is characterized by containing the above-ground permanent magnet fermented metabolite as an active ingredient.

More preferably, the composition for preventing and reducing cognitive dysfunction according to the present invention is a freeze-dried powder of a crude crystalline active substance prepared by the above-mentioned method as a granular persimmon fermented metabolite, the same method as in step b) And a lyophilized powder obtained by lyophilization of the crude extract of the crude persimmon hot water obtained by the step of lyophilization to obtain a powder, Dried powder of crude persimmon fermentation metabolism body, lyophilized dry powder of crude persimmon hot water extract and lyophilized powder of Gilkyung hot-water extract were mixed at a ratio of 5: 3: 2.

The freeze-dried powder of the Gyungyang hot-water extract can be obtained by any method known to those skilled in the art, but it is most preferable to add 10-20 times the weight of water to the lyophilized gill size, reflux-extract at 80 ° C for 1 hour, 1 μm) to obtain an ultrafiltrate (5,000 Daltons). The nanofiltration residue was obtained, sterilized at 95 ° C. for 40 minutes, and lyophilized at -40 ° C. for 12 hours and at 40 ° C. for 36 hours.

The present invention relates to a functional food composition or a pharmaceutical composition for improving or preventing cognitive dysfunction, which is useful for maintaining and improving cognitive function as a general processed food composition. It can be used as an additive raw material.

The food composition of the present invention includes processed forms of all natural materials such as functional foods, nutritional supplements, health foods and food additives. Food compositions of this type may be prepared in a variety of forms according to conventional methods known in the art. For example, they may be prepared in the form of tea, juice, extract pouches, and drinks, and may be ingested, granulated, encapsulated and powdered. In addition, in order to use the fermented metabolite and the composite composition of the present invention in the form of a food additive, they may be prepared in liquid or powder form. When used as a food additive, it may be added in an amount of 0.01 to 10% by weight, preferably 0.1 to 5% by weight based on the raw material. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). However, in the case of long-term consumption intended for health and hygiene purposes or for health control purposes, the amount may be less than the above range, and since there is no problem in terms of stability, the active ingredient may be used in an amount exceeding the above range. There is no particular limitation on the kind of the food. Examples of the food to which the extract can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, , An alcoholic beverage and a vitamin complex, and includes all the health foods in a conventional sense.

It has been confirmed that the ground-breaking fermented metabolite of the present invention contains a large amount of active substance which prevents deterioration of cognitive function. According to the present invention, it is possible to provide natural medicines including health functional foods and nutrients that help prevent and improve diseases caused by lowering of cognitive functions such as dementia and Alzheimer's.

FIG. 1 is a graph showing MTT analysis results showing glutamate-treated neuroprotective activity of the ground-root fermented metabolite of the present invention and a composition containing the same,
FIG. 2 is a graph showing the results of LDH analysis showing the neuroprotective activity of glutamate-treated granulomatic fermented metabolites and compositions containing the same,
FIG. 3 is a graph showing inhibition of the production of reactive oxygen species (ROS) after glutamate treatment of the granular activated carbon metabolite of the present invention and a composition containing the same,
FIG. 4 is a graph showing the brain-derived neurotrophic factor BDNF expression level of the ground-glass permanent fermentation metabolite of the present invention and a composition containing the same.

The present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example 1: Preparation of cornstal bed fermentation metabolite

The dried ground stone bed was cut into 1 × 2 cm size, and 15 times of water was added to the raw material weight, and the mixture was refluxed at 95 ° C. for 10 hours, filtered and concentrated under reduced pressure at 55 ° C. for 15 to 20 brix. After adding 0.5% of nitrogen source yeast extract and 1% of sugar as carbon source to the obtained crude extract, the pH was adjusted to 6.5 ~ 7.0 and then sterilized at 121 ℃ and 1.5 atm for 15 minutes to prepare a groundmagnification fermentation medium. The cultures of Lactobacillus plantarum and Leuconostoc mensenteroides cultured in the MRS medium at 30 DEG C for 1 day were inoculated into the above ground permanent fermentation broth with 2% of each of the raw materials, and cultured at 30 DEG C for 4 days. Then, After sterilization for 10 minutes, the same amount of ethanol was added to the filtrate, and the mixture was dipped at 4 ° C for 24 hours at 4 ° C, centrifuged at 6,000rpm for 30 minutes, and lyophilized to prepare a frozen dried powder of the crude persimmon fermented metabolism.

The slurry was cut into 1 × 2 cm pieces and 15 times of water was added to the raw material weight. The mixture was refluxed at 95 ° C. for 10 hours, filtered, and concentrated under reduced pressure at 55 ° C. to adjust the concentration to 15 to 20 brix. The obtained crude extracts of the basal hydrothermal extract were sterilized at 100 ° C for 10 minutes, filtered, and lyophilized to prepare lyophilized powders of base-coenzyme hydrolyzate.

Example 2: Preparation of Composite Composition Containing Tungsten Paste Fermentation Metabolite

Freeze-dried powder of Gilyung hot-water extract was prepared to be mixed with the lyophilized frozen-dried granular powder obtained in Example 1 and the lyophilized dry powder of cornstarch hydrolysis extract (freeze-dried bloom → 15 times water → 80 ° C, refluxing for 1 hour → Microfiltration (1um) → Ultrafiltrate (5,000Dalton) → Nano filtration residue → Packaging (3kg each) → Sterilization (95 ℃, 40min) → Freeze drying (-40 ℃, 12 hours / 40 ℃, 36 hours )).

50% by weight of the frozen-dried crude cornstarch supernatant produced in Example 1, 30% by weight freeze-dried powder of persimmon hydrothermal extract, and 20% by weight of freeze-dried powder of Gilyung hot-water extract were mixed, A composite composition for improvement was prepared.

Experimental Example 1: Confirmation of the activity of nerve cell protection against glutamate excitotoxicity

When glutamate accumulates excessively in the brain, it degrades the function of the antioxidant system in the body, resulting in an imbalance between the oxidant system and the antioxidant system, causing excessive excitement of the nerve, resulting in excitotoxicity, which eventually leads to apoptosis . Therefore, a substance capable of inhibiting cytotoxicity induced by oxidative stress and excitotoxicity induced by glutamate may be helpful in improving cognitive function.

In this experiment, the cornstarch suspension fermented product (cornstarch fermentation product) according to Example 1 and the composition according to Example 2 were pretreated with 50, 100 and 200 ug / ml for 24 hours respectively, and 5 mM glutamate After 24 hours of treatment, the neuroprotective activity of glutamate which induces neurotoxicity was measured by MTT and LDH assay.

The experimental results are shown in Figs. 1 and 2. Fig . As shown in FIGS. 1 and 2, the neuronal cell protective activity was induced in a concentration-dependent manner in the composition of the ground stone fermented product and the composition.

Experimental Example 2: Changes in oxidative stress caused by glutamate-induced nerve excitotoxicity

Degenerative neuropathy is a disorder in which the function of the brain or spinal cord is paralyzed due to abnormal neuronal death, unlike normal aging process. This is one of the leading causes of degenerative neurology. It is an oxidative stress caused by reactive oxygen species (ROS).

The neuronal cell line HT-22 was pretreated with 50, 100, and 200 ug / ml of the composition according to Example 1 for 24 hours, respectively, and the composition according to Example 2 was subjected to neurotoxic toxicity Glutamate, which induces an oxidative stress, was treated with 5 mM for 24 hours. ROS production was determined by measuring the fluorescence of the cells using 10 uM 2 ', 7'-dichlorofluorescein diacetate (DCFDA).

As a result, a significant inhibitory activity of ROS formation was observed at a concentration of 100-200 ug / ml of the crude stone fermented product and the composition thereof (FIG. 3).

EXPERIMENTAL EXAMPLE 3: Expression of brain-derived neurotrophic factor (BDNF)

Brain-derived neurotrophic factor (BDNF), one of the neurotrophic factors, is expressed in the hippocampus, cortex and basal forebrain of the central nervous system (CNS) BDNF is known to be a major regulator of synaptic plasticity and cognition, which is the neurobiological basis of synaptic transmission and learning and memory capacity.

BDNF is not only depressed by stress, but also significantly reduces BDNF expression in degenerative brain diseases such as Alzheimer's disease, Parkinson's disease, stroke, Huntington's chorea, cerebral ischemic disease, and neurodegenerative diseases. This may be a major target for BDNF to improve neuronal damage and brain cognitive function.

The nerve cell line HT-22 according to Example 1 was treated with each of the granular activated carbon metabolites (crude stone fermented product) and the composition according to Example 2 at 50, 100 and 200 ug / ml, respectively, cDNA was prepared and the expression of BDNF, a representative neurotrophic factor, was measured using real-time PCR. As a result, an increase in the gene expression of BDNF in a concentration-dependent manner in the cornstarch-free fermentation product and composition was confirmed (FIG. 4).

Claims (4)

a) cutting the dry crumb stone to an average particle size of 5 to 20 mm to obtain a foundation stone raw material;
b) 10 to 15 times by weight of water is added to the base stone base material and the mixture is refluxed at 95-100 ° C for 9-11 hours, filtered and concentrated under reduced pressure at 50 to 55 ° C to obtain a concentrate adjusted to a concentration of 15 to 20 brix ;
c) 0.5% yeast extract and 1% sugar as a carbon source were added to the concentrate as a nitrogen source, the pH was adjusted to 6.5 ~ 7.0, sterilized at 121 ° C and 1.5 atm for 10 ~ 15 minutes, ;
d) 2% Lactobacillus plantarum and Leuconostoc mensenteroides cultured in the MRS medium at 30 ° C for 1 day were inoculated in the cornstarch supernatant, Culturing at 30 DEG C for 4 days;
e) The culture broth was sterilized at 100 DEG C for 10 minutes, and the same amount of ethanol was added to the filtrate. After 24 hours of immersion at 4 DEG C, the suspension was centrifuged at 6,000 rpm for 30 minutes and lyophilized, Wherein the freeze-dried powder of the present invention is obtained by a method comprising the steps of:
A ground-stone permanent fermentation metabolite produced by the method of claim 1. A composition for prevention and improvement of a cognitive dysfunctional disease containing as an active ingredient the ground-root fermentation metabolite of claim 2. In claim 3, the present invention provides a method for prevention and improvement of a cognitive dysfunctional disease containing 5: 3: 2 by weight of freeze-dried powder, freeze-dried powder of base stone hydrothermal extract and freeze- Composition.

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