KR101771788B1 - Herbal composition comprising fermented scutellariae radix and gastrodiae rhizoma for the prevention and improvement of nerologic disease - Google Patents

Abstract

The present invention relates to a composition for preventing and improving neurological disease, and more particularly, to a fermented golden ginseng extract powder composed of fermented golden, ginseng, dermis, ginseng, and ginseng.
The composition for preventing and ameliorating neuropathy according to the present invention has the effect of promoting the expression of mRNA gene and protein expression of beta-III tubulin and GAP-43 in promoting neurite outgrowth of neurons.
It also exhibits pERK1 / 2 protein expression and neural prolene growth promoting activity.
The extract of fermented golden venom of the present invention can be used as a functional food for prevention and improvement of cerebropathy without side effects and cytotoxicity by using a substance derived from a natural origin.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for preparing a fermented golden starch complex solution for preventing or ameliorating neuropathy,

The present invention relates to a fermented golden starch complex solution for preventing and improving neurological disease, and more particularly, to a fermented golden starch complex solution for prevention and improvement of neurological diseases, III tubulin) and GAP-43 mRNA and protein expression, promoting the expression of pERK1 / 2 protein, and promoting the growth of neurite outgrowth .

In normal adult brain, 100 trillion nerve cells are connected to form neural network at the time of birth, and neural network is an important organs that enable high level mental activities such as thinking, memory, and reasoning. The neurons that make up the brain are not born to multiply, but over the age of 30, about 10 - 200,000 cells die each day. A serious problem is that even during normal aging, many nerve cells die and cognitive functions such as memory and judgment are reduced. In particular, due to various causes, rapid death of many neurons in the aging process leads to degenerative brain diseases such as Parkinson 's disease, and dementia resulting in severe cognitive impairment, leading to a disruption in daily life.

Neurons in the brain use glucose as an energy source, so about 30% of blood is supplied from the whole blood that the heart contracts. Even if blood supply is interrupted for a very short time, neuronal cells will undergo irreversible changes and die. Thus, the onset of cerebral stroke, which causes the death of nerve cells due to temporary interruption of blood supply or decline, is a terrible disease that causes a loss of much brain function and makes daily life difficult.

Neurons located in the central nervous system are not regenerated or proliferated. Therefore, once neuronal death occurs, if a functional disorder occurs, the disorder lasts for a long time and most of them are difficult to recover. However, the structural and functional changes of the neural network formed by the surviving neurons can replace the functions of the neurons, which is called neural plasticity of the neural network.

It is known that the dendrites and dendritic spines of nerve cells are important for the formation of neuronal plasticity. Therefore, the development of drugs capable of promoting the plasticity of neural networks by promoting the production of dendrites and dendritic spines of nerve cells is an important parameter for the treatment of nervous system.

PC12 cells (rat adrenal pheochromocytoma cells) are cell lines mainly used for neurodevelopment studies. It originated from the chromium affinity cells of the adrenal gland of the rat, and chromaffin cells originated from neuroectodermal cells. Studies have shown that treatment of these cells with neuronal growth factor (NGF) differentiates into neurons, increases neurotransmitter dopamine, and forms neurites and synaptic vesicles . Therefore, many researchers have used neural progenitor and synaptic vesicle formation of PC12 cells as an experimental model for neuronal cell growth and neuroplasticity in vitro [1].

NGF is a type of neurotrophin that is activated by phosphorylated TrkA (pTrkA, phosphorylated TrkA), which binds to the tyrosine kinase receptor (TrkA). Ras, Raf (protein-serine / threonine kinases ). As Ras and Raf become activated, extracellular signal-regulated kinases (ERK1 / 2), one of the protein kinase aggregates, are activated and converted to pERK1 / 2 through phosphorylation. pERK1 / 2 is involved in cell differentiation and cell survival, and regulates cell growth and has functions related to neural plasticity.

Tubulin is a major component of microtubules and is a GTP-dependent protein involved in mitosis and cytoskeleton of cell organelles. Beta-III tubulin is expressed in early neuroepithelial cells, It is used as a neurospecific marker.

In addition, GAP-43 (growth associated protein 43) is a component of the neurite and pre-synaptic terminals and is highly expressed when neurites grow during embryonic development, and thus is used as a factor involved in neuronal cell growth and neuroplasticity.

Recent research on neurogenesis and research on neurological diseases using stem cells is underway. In other words, neurons generate neurites with various pathways and various factors. Studies are under way to induce neural differentiation by using various substances, so as to prevent, improve and cure neurological diseases. In addition, there have been many experimental studies on the treatment and nerve regeneration of various brain diseases using herbal medicines used in oriental medicine.

The results of experimental studies on the neurological diseases of the herbal medicines, which are described in the present invention, are as follows.

Golden (Scutellariae Radix) is a perennial plant belonging to the family Lamiaceae. It is the dried root of Scutellaria baicalensis GEORGI, which is a plant of the genus Lilium, , Fever (fever), blob (fistula), early anxiety (gestation anxiety), and moist heat (exhaled heat) is known.

In one room, it was used as fever, diuretic, branch, insect, and anti-inflammatory. Experimental studies showed that pretreatment of the methanol extract of gold in cultured PC12 cells inhibited oxidative cell damage by hydrogen peroxide as well as inhibition of intracellular signal transduction related to cell death. In addition, epidural administration of gold methanol extracts in transient precursor ischemia induced significant reduction of neuronal cell death in the hippocampus.

The ethanol extract of gold inhibited the death of hippocampal neurons by ibotenic acid toxicity in rats and controlled the changes of various nerve agents to reduce memory decline. Baikalin, baicalein, wogonin and oroxylin are known to be major flavonoid compounds extracted from gold.

These single substances inhibit oxidative cell damage or inhibit cell death by chemical (amyloid, alpha-synuclein) -induced toxicity, and thus have a powerful nerve cell protection effect.

For example, baicalein inhibits degenerative neurodegenerative damage in cultured neuronal cells and reduces ischemic brain injury in rats, which is due to antioxidant and anti-inflammatory effects.

These antioxidative and antiinflammatory effects were more pronounced in Baicalaine than in Baikalin. Orcoxin improved cognitive function and memory in elderly rats and experimental animals with degenerative brain disease. Ugonin inhibited neuronal death of cerebral cortex in acute regional cerebral ischemic injury model and induced behavioral symptom improvement.

Gastrodia Rhizoma is a dried herbaceous root of perennial parasitic herbaceous plant belonging to the Orchidaceae. It is an herbal medicine used for treating epilepsy, painkillers, dizziness inhibitors and paralysis in one room.

Gastrodin, a phenolic monosaccharide (glucoside) as an active ingredient of chamai, inhibits apoptosis by ischemia and glutamate toxicity in various types of cultured neurons and inhibits intracellular calcium and NO production It is known to activate intracellular PI3K secondary signaling system.

In addition, it temporarily inhibited cerebral cortical neuronal apoptosis by ischemic injury by blocking blood flow of middle cerebral artery in rats, inhibited the increase of glutamate during ischemia in hippocampus, and increased GBAB concentration during reperfusion.

Vanillyl alcohol and 4-hydroxybenzyl alcohol in rats inhibited the development of convulsions induced by ferric chloride in rats or induced ischemic brain injury by the production of free radicals Through inhibition.

Chondroitin ether fraction inhibits neuronal cell death induced by amyloid beta peptide.

Dipper (Citrus unshiu peels) belongs to more than a mountain of blue-green small tree, a fruit shell of a citrus tree, is a sun dried Chinese medicine that is said to be a sun-dried herbal medicine according to Dongbokgam, digestion promotion, cough suppression, vomiting suppression, have. In addition, anti-inflammatory action, anti-ulcer, diarrhea suppression, and improvement of pulmonary edema have been reported by the pharmacological action of dermis (Park Young-soon's pharmacology explanation).

Regarding the pharmacological use of dermis, Korean Patent Registration No. 10-0732564 entitled " Dermatological composition containing dermis extract as a major active ingredient ", Korean Registered Patent No. 10-0665503 " Ischemic diseases and degenerative diseases Compositions for the prevention and treatment of brain diseases ", Korean Patent No. 10-0720671 entitled " Composition for treating type IV allergy and inflammation containing secretion, dermis and Rhizome extracts ", Korean Registered Patent No. 10-20110082040 " A method for producing a dermis fermentation extract having an obesity effect, and a dermis fermentation extract thereof ".

The active ingredients contained in the dermis include flavonoids such as hesperidin, rutin, naringin, neohesperidin and nobiletin, and limonoids Nomilinic acid and the like are known. In addition, it contains vitamin B1, vitamin C, calcium, iron, dietary fiber and so on.

Among them, hesperidin is the most abundant ingredient in the dermis, and it is known that it has various pharmacological actions such as blood pressure lowering, anti-inflammation, blood cholesterol reduction and anti-cancer action.

Experimental studies have also shown therapeutic effects on various diseases of the nervous system. For example, hesperidin reduces the degradation of intracellular glucose utilization by beta amyloid in cultured neurons, inhibiting autophagy and inhibiting oxidative cytotoxicity by H2O2.

It is known that CCl4 toxic substances and transient cerebral ischemia cause neuronal cell damage due to oxidation of lipids and proteins in brain tissue at the level of experimental animals, and that hesperadine inhibits these phenomena. In addition, Nobiletin inhibits the loss of memory by amyloid beta protein in the rat, as well as promotes the stimulation of cAMP / PKA / ERK / CREB signaling system and neuronal cell growth in cultured hippocampal neurons It inhibits cell degeneration.

Puerariae Radix is a dried root of Pueraria lobata (Willd. Ohwi.), A vine of soybeans. It treats cold fever, fever, , Stomach pain, nausea and stiff neck pain, stiffness (diabetes), fatigue, and other detoxification was also effective.

In recent studies, Pueraria has been shown to have hypotensive effect, increase in cerebral blood flow, enhancement of memory, and expansion of coronary artery to mitigate myocardial infarction and improve ischemic response on electrocardiogram (Kim, Ho-Cheol, 94, 2001).

The component of the gruel root contains flavonoid compounds such as puerarin, daizein and daidzin and saponin such as soyasaponin and starch (Genomic Medicine Textbook Compilation Committee, Genomics, Tongmyung, p. 100-102, 2006).

In the case of Puerariae Radix extract, the concentration of norepinephrine and dopamine in the hippocampus, the striatum, and dopamine in the rat induced cerebral ischemia by closing the bilateral carotid arteries, .

It has been studied mainly on the isoflavone components of puerarin and daidzein.

Pseudomonas pseudomonas aeruginosa (Pseudomonas aeruginosa) is a widely used medicinal product for treating acute stroke in China. It has been reported that fuerarin inhibits glutamate, Ca2 + cell entry, and NO production in in vitro studies and protects nerve cells by blocking acid-sensing ion channels.

In the model of cerebral ischemia, which is an animal model of middle cerebral artery occlusion, it has been reported that various effects such as inhibition of NMDA receptor expression, inhibition of intracellular Ca2 + influx, antioxidant, antiinflammation and anti - apoptotic effects protect nerve cells.

On the other hand, ginseng (Radix Alba) belongs to the family Araliaceae and has a peculiar smell.

The active ingredient of ginseng is mainly Dammarane saponin, which contains protopanaxadiol derivatives Ginsenoside Ra1, Ra2, Rb1, Rb2, Rb3, Rc, Rd, (Ginsenoside) Re, Rf, Rg1, Rg2, Rh1 and the like, which are Protopanaxatriol derivatives, and Oleanane saponin and Ginsenoside Ro.

The pharmacological action of ginseng inhibits blood triglyceride production, prevents thrombogenesis, promotes blood circulation by inhibiting platelet coagulation, stimulates the cerebral cortex to promote peripheral blood flow, and improves blood circulation in the gastric wall have.

It also enhances the protein synthesis ability of bone marrow cells and promotes erythropoiesis, accelerates the excitement conduction of the nerves, enhances the condition reflection ability to alleviate fatigue, enhances the secretion of digestive juices, and strengthens appetite And stimulates the peristalsis of the stomach to promote digestion and absorption.

In addition, it has a lipid-decomposing action, and it is known to exhibit prevention and improvement effects of fatty liver due to alcoholic liquor, and to enhance gastrointestinal motility along with tonic action.

Studies on various ginsenosides as an active ingredient of ginseng have been actively carried out. The action of the ginsenosides is actively performed on the central nervous system, immunity enhancing action, action on cardiovascular action, hypoglycemic action, action on lipid metabolism, , Learning ability improvement action, fatigue recovery and stress reduction action, and hair growth promoting action have been reported.

The action of the ginseng extract on the central nervous system is to stabilize the heart at low doses (Tranquilizer effect), to exacerbate the vomiting center and the respiratory center, the ginsenoside Rb group to have central inhibitory action, and the ginsenoside Rg group to have central excitement Lt; / RTI >

In recent years, despite a great deal of interest and research on natural substances that are effective in the prevention and amelioration of neurological diseases, it has been reported that a substance that inhibits neuronal cell death by promoting growth and oxidation of nerve cells through cell biology and animal experiments There is not much development in Korea.

In addition, the development of natural substances that can prevent and improve neurological diseases by promoting the growth of neural cell development genes (mRNA) and promoting the expression of proteins (pERK1 / 2) without side effects and cytotoxicity It is reality that it is not active.

It is an object of the present invention to prevent and improve brain diseases by using herbal medicine powders containing an extract powder of fermented golden ginseng extract as an active ingredient.

The present invention is to prevent and improve neurological diseases by promoting the growth of neurites in the fermented golden ginseng complex extract powder as an active ingredient.

The present invention aims to prevent and improve neurological diseases by promoting the expression of mRNA and protein of beta-III tubulin and GAP-43.

The present invention provides a composition for promoting protein expression of pERK1 / 2 to prevent and improve neuropathy.

The present invention provides a method of safely extracting an effective ingredient from a fermented golden, ginseng, dermis, pupa, and ginseng with high efficiency, thereby ensuring long-term ingestion and lowering the cost of raw materials.

The present invention provides a herbal powder and a health functional food for preventing and improving brain diseases by protecting nerve cell death and enhancing brain function through growth and regeneration of nerve cells.

The present invention relates to a method for pulverizing, washing and mixing a fermented golden, a chamois, a dermis, a brow, and a ginseng; Adding the mixture to an extractor and preparing a complex extract using the extract; A step of preparing a complex concentrate which is obtained by concentrating the complex extract in a concentrator and heating under reduced pressure; And a step of sterilizing and drying the complex concentrate to prepare a powder. The fermentation gold is a fermented golden ginseng extract powder for fermentation and improvement of fermented and aged fermented and matured fermented mung bean It is related.

The present invention relates to a method for preparing a fermented golden ginseng extract powder prepared by the above method.

The present invention relates to a method for preparing a fermented yeast-fermented yeast culture, which comprises the steps of putting a yeast-enzyme complex fermentation broth obtained by mixing a concentrated yeast culture powder and a mash enzyme concentrate into a scutellaria radix; Fermenting the gold; And aging the fermented gold for a predetermined period of time and drying the saccharomyces cerevisiae, wherein the yeast is Saccharomyces cerevisiae.

The present invention provides health functional foods by mixing the composition with pharmaceutically acceptable additives. The health functional foods may be provided in various forms such as functional beverages, health supplements, tea, confectionery, and the like.

The fermented golden Gunma complex extract powder according to the present invention promotes the growth of neurites and promotes mRNA expression of GAP-43. In addition, the complex extract powder of the present invention can promote protein expression of beta-III tubulin, GAP-43, and pERK1 / 2. As described above, the extract powder of fermented golden Cheongma seed complex extract of the present invention can be used as a functional food for prevention and improvement of cerebropathy without side effects and cytotoxicity by utilizing a substance derived from natural origin.

FIG. 1 is a flow chart showing a process for preparing fermented gold by fermentation aging and drying a golden herbal medicine using yeast and a complex fermented liquor according to an embodiment of the present invention.
FIG. 2 is a flow chart showing a process for preparing a fermented mixture of golden thymus extracts by mixing fermented gold, chymargia, dermis, gruel and ginseng herbal medicines, followed by extraction, concentration and spraying.
FIG. 3 is a graph showing the results of concentration of the fermented golden chromosomal complex extract solution on the cell survival rate of PC12 cells. FIG.
FIG. 4 is a photograph showing the pattern of neuronal differentiation and the length of neurite outgrowth of PC12 cells treated with NGF and fermented golden chromosome complex extract solution at different concentrations.
FIG. 5 is a bar graph showing percentages of cells showing neural differentiation over time after treatment of PC12 cells with NGF and fermented golden-chromosomal extract solution together.
FIG. 6 is a histogram of the growth of neural prolate length of PC12 cells treated with NGF and fermented golden-chromosomal extract solution at different concentrations, over time.
FIG. 7 is an immunocytochemical staining image showing the expression of GAP-43 protein by treatment with NGF and fermented golden-chromosomal extract solution during neurite outgrowth in PC12 cells.
FIG. 8 is a photograph and analysis graph of Western blot test showing the expression of beta-III tubulin and GAP-43 protein in neurite outgrowth by treatment with NGF and fermented golden chromosomal extract solution in PC12 cells.
FIG. 9 is an RT-PCR photograph and analysis graph showing the expression amount of GAP-43 mRNA by treatment with NGF and fermented golden chromosomal extract solution during neurite outgrowth in PC12 cells.
10 is an immunocytochemical staining image showing the expression pattern of pERK1 / 2 protein by treatment with NGF and fermented golden chromosomal extract solution during neurite outgrowth in PC12 cells.
FIG. 11 is a photograph and analysis graph of a Western blot test showing the expression pattern of pERK1 / 2 protein by treatment with NGF and fermented golden chromosomal extract solution during neurite outgrowth in PC12 cells.

FIG. 1 is a flowchart showing a process of manufacturing fermentation gold according to an embodiment of the present invention. FIG. 2 is a flow chart showing a process for producing a fermented golden Ginseng complex extract powder according to an embodiment of the present invention.

Referring to FIG. 1, a method for preparing fermented gold includes culturing yeast, preparing a plum concentrate, mixing yeast powder and enzyme concentrate, and a golden fermentation step.

The yeast powder may be subcultured by inoculating a yeast strain in a medium and then dried to be powdered.

In addition, the natural enzyme can be obtained by mixing white sugar with plum, filtering it for 6 months, and then concentrating it by filtration.

Enzyme-enzyme complex fermentation broth is prepared by mixing yeast powder and enzyme concentrate prepared previously.

The yeast strains usable in the present invention may be any one or more of Saccharomyces cerevisiae, Aspergillus oryzae, Saccharomyces ellipsoideus, and the like. .

In the present invention, the yeast-enzyme complex fermentation broth is fermented in gold for a predetermined period of time. The gold is used to dry and cut gold medicinal herbs.

The fermentation may be carried out in one to three different ways.

The yeast-enzyme complex fermentation broth is added so that the gold is immersed therein. The gold can be allowed to stand in the immersed state for a predetermined time, preferably at room temperature for a day.

The primary fermentation step is a fermentation step for several days in a fermentation tank. For example, the primary fermentation can be fermented for 2 to 10 days, preferably 3 days, while maintaining the temperature at 50 to 60 ° C.

The secondary fermentation may be performed by spraying the yeast-enzyme complex fermentation broth onto the gold and fermenting the fermentation for 2 to 10 days, preferably 3 days, while maintaining the temperature at 50 to 60 ° C. The third fermentation can be further fermented under the same conditions as the second fermentation.

The aging and drying step may be carried out in various ways, but preferably the convection heat circulation method at a temperature of 40 to 50 DEG C and the wet heating method using convection heat are alternately performed while maintaining a humidity of 40 to 60%. After aging, it can be air dried at 35 ~ 45 ℃.

The fermentation gold manufacturing method of the present invention is advantageous in that the entire process of fermentation, aging, and drying is continuously performed in the same fermentation vessel, so that the process is simple and the uniformity of the product can be obtained. The method of the present invention includes three stages of fermentation, aging and drying in order to make the fermentation state of the product more uniform and increase the commerciality.

Referring to FIG. 2, the fermentation method of the present invention comprises mixing, extracting, preparing a concentrated concentrate, and drying.

The mixing step is a step of pulverizing, washing and mixing the fermented golden, chestnut, dermis, pupa, and ginseng.

50 to 80 parts by weight of ginseng, 50 to 80 parts by weight of ginseng, 50 to 80 parts by weight of ginseng root, and 20 to 40 parts by weight of ginseng can be contained in 100 parts by weight of the fermented gold.

The combined extract produces a combined extract from the fermented golden, chunmei, dermis, pupa, and ginseng.

The extracting step is a step of putting the mixture into an extractor and preparing a combined extract using the extract.

As the method for producing the complex extract, various known methods, solvent extraction method, heated pressure extraction method and the like can be used. For example, the fermented golden, ginseng, dermis, pupa, and ginseng may be placed in an extraction container, alcohol solution or water may be added, and the extract may be heated and extracted at a predetermined temperature.

In the present invention, an extract is prepared in three steps. By this method, it is possible to extract by minimizing the loss of the active ingredient. Further, in the solvent extraction method of the present invention, gastrodine, which is an active ingredient of horse chestnut, is hydrolyzed to a parahydroxybenzyl alcohol in a third solvent extraction process to increase extraction efficiency of residual gastrodine, Roxy benzyl alcohol has been reported to restore memory impairment by scopolamine, apomorphine, and cycloheximide.

More specifically, in the step of preparing the complex extract, fermented gold, chymus, dermis, pupa, and ginseng are placed in each extraction container, and the mixture is warmed at 80 to 90 ° C with 50% alcohol solution. The extract is filtered through a filter The tea extract and the 30% alcohol solution were added and the mixture was warmed at 80 to 90 ° C. The extract was filtered through a filter, and purified water was added thereto. The extract was warmed at 80 to 90 ° C and the extract was filtered through a filter And the extract can be combined.

The content of each component can be appropriately controlled in the step of preparing the extract.

In the step of preparing the complex concentrate, the complex extract may be placed in a concentrator and concentrated under reduced pressure by heating. For example, the extract is put in a concentrator and concentrated under reduced pressure of 500 to 700 mmHg to obtain a concentrate in the range of 60 to 100 brix.

The combined extract powder can be obtained by sterilizing and drying the complex concentrate.

The present invention can provide a health functional food by mixing the compound extract powder with pharmaceutically acceptable additives. The health functional foods may be provided in various forms such as functional beverages, health supplements, tea, confectionery, and the like.

The combined extract powder may be mixed with the health functional food at a concentration of less than 200 μg / ml.

The objects of the embodiments of the present invention are not limited to the above-mentioned objects, and other objects not mentioned can be clearly understood by those skilled in the art from the following description .

[Example 1]

1. Manufacture of fermented gold

36 kg of dried gold was taken and 2 L was added to the yeast culture (Saccharomyses sereviciae) and the culture of the mash enzyme, and the mixture was immersed in the fermentation container for 3 hours at 50 to 60 ° C. Then, 1 L of the yeast and enzyme culture was sprayed, Day, followed by the third fermentation by spraying 1 L of yeast and enzyme culture. In the fermentation vessel, wet heat and convective heat circulation method using convection heat were alternately performed for 3 days (aging step) while maintaining a humidity of 50-60% at 40-50 ° C. After aging, the mixture was dried at 35 to 45 ° C for 3 days to prepare fermented gold (yield: 80 to 90%).

Yeast and plum enzyme cultures were performed as follows.

First, Saccharomyses sereviciae MG111 strain was inoculated into a liquid medium, followed by subculturing, and then dispersed in a high-medium medium and dried at 30 to 40 ° C for 24 to 48 hours to obtain a powder (1 x 10 6 cfu / g) 40 g of each of the fermented mussel enzyme cultures was added to 4 L of purified water and homogeneously mixed to prepare yeast and enzyme cultures.

2. Fermented Golden Chunmean Extract Powder Production

A) Selection: Golden, Chunma, Dermis, Pungkin and Ginseng were selected as fresh and dried according to the standard of herbal medicine standard.

B) Crushing: Each herbal medicine was cut using a cutting machine.

C) Washing: The fermented gold (30 kg), the chewing gum (20 kg), the dermis (20 kg), the gruel (20 kg) and the ginseng (10 kg) prepared in the above were put into a tank and washed with purified water.

Extraction: After washing, the crude drug complex was placed in an extractor, and 50% ethanol was added thereto. The mixture was firstly extracted at 80 to 90 ° C for 12 hours, further extracted with 30% ethanol at the same temperature for 12 hours, Purified water was used for the third extraction at the same temperature for 12 hours.

E) Filtration: The extract was put into a filter stepwise and filtered under reduced pressure at 70 to 80 ° C.

F) Concentration: The filtered extract was put into a concentrator and concentrated at 60 DEG C while maintaining a reduced pressure of 500 to 700 mmHg to obtain a concentrate having a solid content of 65% or more.

G) Sterilization: The crude herbal medicine concentrate was sterilized at 100 ° C for 2 hours.

A) Drying: Each of the concentrated liquids was diluted to 30 Brix and spray dried at 185 to 195 ° C.

Charging Charge: The dried concentrated complex powder of herbal medicine was charged under a sterilized condition according to the packing unit.

Tea) Inspection: After completion of charging, the raw materials of herbal medicine were tested for quality according to the standard test method of fermented golden chromosome complex extract powder.

K) Packing: Packed raw materials passed quality inspection and stored at room temperature.

Experiment 1: PC12 cells  Effect of Fermented Golden Chamaemia Extract Powder on Survival Rate

1) Cell culture

The PC12 cell line used in this experiment was derived from the chromatin cell line of the adrenal gland of the rat and received a sale from the Korean Cell Line Bank (KCLB, Korea). PC12 cells were cultured in RPMI 1640 (Gibco BRL, USA) medium containing 10% horse serum (HS) and 5% fetal bovine serum (FBS) in a cell incubator maintained at 37 ° C and 5% For 48 hours.

2) Treatment of Fermented Golden Chunmean Extract Solution

10 llg / ml laminin solution was added to a 24 well plate and stored at 3-5 ° C. After 24 hours, the laminin solution was removed and washed with sterilized third distilled water. PC12 cells were cultured in RPMI 1640 medium containing 1% HS and 0.5% FBS at a rate of 5 × 10 3 per well. After incubation for 24 hours, PC12 cells were treated with 5, 10, 50, 100, and 200 ㎍ / ml of fermented golden chromosomal complex extract solution at different concentrations. After 3 days, the medium was exchanged and cultured for 5 days. Respectively.

3) MTT quantitative experiment

MTT assays were performed to determine cell viability of PC12 cells.

MTT solution was added to the culture solution at a ratio of 1: 8-12 at 4, 6, and 5 days after 1 day, 3 days, and 5 days after the fermentation, The supernatant was removed and 200 μl dimethylsulfoxide (DMSO) solution was added to each well to dissolve the non-aqueous purple formazan. The amount of formazan was measured with a spectrophotometer (ELISA reader; Molecular Devices, Inc.) at a wavelength of 540 nm. The measured values were calculated as percentage (%) compared with the values of normal cells.

4) Statistical processing

Statistical analysis was performed using mean standard deviation (SD). Statistical significance was verified by ANOVA test using SPSS (version 10.0, SPSS Inc., USA).

5) Experimental results

FIG. 3 is a graph showing the results of concentration of the fermented golden chromosomal complex extract solution on the cell survival rate of PC12 cells. FIG. As a result of treatment of fermented golden chromosome complex extract solution at different concentrations, there was no significant difference in cell viability after 1 day of solution treatment compared with control (control). Treatment with 50 ㎍ / ml fermented golden chromosomal extracts resulted in higher cell survival than the other treatment groups.

After 3 days of fermentation, the cell survival rate was higher at 5, 10 ㎍ / ml than at the control, especially at 10 ㎍ / ml, the cell survival rate was significantly increased to 118.3 ± 2.9% (p < 0.05). However, the cell viability was decreased at 200, 500 ㎍ / ml rather than the control, and the cell viability was decreased to 88.6 ± 2.9% at 500 ㎍ / (p < 0.05). After 5 days, cell viability was similar to that of the control group when 5, 10 ㎍ / ml of extract solution of fermented Ganoderma lucidum was treated. However, cell survival rate was 83.6 ± 2.4% (p <0.01) ± 2.8 (p <0.01), indicating that the extract of fermented golden chromosomal complex was slightly cytotoxic at high concentration.

Experiment 2: Neural differentiation and neurite length change of PC12 cells treated with fermented golden chromatin complex extract solution

1) Experimental method

    PC12 cells were treated with fermented golden chromosome complex extraction solution and NGF, and the growth of neurite was observed in differentiation of PC12 cells using a phase contrast microscope (Olympus IX71, Japan) at 1, 3, and 5 days Respectively.

Three sections were randomly selected from each culture dish and images of PC12 cells differentiated by a digital camera mounted on a microscope were taken and stored in a computer.

Image J (Image J, HIH, USA) Image analysis program was used to calculate the proportion of PC12 cells with neurites in all PC12 cells.

In addition, when multiple neurites were observed in one PC12 cell, the length of the neurite was selected by selecting the largest neurite, and then the mean value was calculated and compared according to the concentration of fermented golden chromosome complex extraction solution and NGF.

2) Experimental results

FIG. 4 is a photograph showing the growth of neurites observed in the differentiation of cells after 5 days treatment with 5 ng / ml of NGF alone or with fermented golden-chromosomal combination extract solution with NGF.

No neurites were observed in vehicle cells not treated with NGF, but neurites were observed when treated with 5 ng / ml of NGF alone.

On the other hand, the treatment of NGF with fermented golden chromosomal complex extract solution increased the size of neurites more than NGF alone. Especially, the growth of neurites at 10, 50, and 200 ㎍ / Respectively.

FIG. 5 is a graph showing percentages of cells showing neural differentiation of PC12 cells according to the passage of time after treatment of NGF and fermented golden-chromosome complex extract solution with PC12 cells at different concentrations.

PC12 cells were treated with either 5 ng / ml of NGF alone or NGF combined fermentation solution and 1, 3, and 5 days of culture, and the percentage of cells bearing neurites in PC12 cells was analyzed.

The proportion of neurite-positive PC12 cells increased with the passage of time following NGF-only treatment. In addition, the ratio of neurite - positive PC12 cells treated with NGF alone increased when the fermented golden - chroma complex extract solution was treated together with NGF. Especially, the ratio of neurite - positive cells was significantly increased at 10, 50 and 100 ㎍ / ml of fermented golden chromosomal extract solution from the first day of treatment. Significant increases in the percentage of PC12 cells with these neurites were also observed at 3 and 5 days, especially at 10 and 50 μg / ml of fermented golden chromosomal extract solution.

FIG. 6 is a graph showing the growth of neurite length according to the neural differentiation of PC12 cells according to the passage of time when the extract solution of NGF and fermented golden chromosomes was treated with PC12 cells at different concentrations.

From day 1 of drug administration, neurite outgrowth of PC12 cells was significantly increased at 50, 100 ㎍ / ml concentration of fermented golden - chroma complex extract solution. Significant growth of these neurites was also observed at 3 and 5 days, especially at 10 and 50 ㎍ / ml of fermented golden chromosomal extract solution.

The group with the longest neurites were group treated with 50 ㎍ / ml of fermented golden - chromosome extract solution and 5 ng / ml of NGF. The average length of neurites was 64.2 ± 4.8 ㎛.

Experiment 3; Neuron  Beta-III by differentiation Tubulin and  Protein expression of GAP-43

1) Experimental method

(1) Immunocytochemical staining test

In order to perform immunocytochemistry, 10 μg / ml of laminin solution was added to a 24-well plate and stored at 3-5 ° C. After 24 hours, the laminin solution was removed and washed with sterilized tertiary distilled water. PC12 cells were cultured in RPMI 1640 medium containing 1% HS and 0.5% FBS at 5 × 10 3 cells / well.

PC12 cells were cultured for 24 hours. Then, 5 ng of NGF and a fermented golden chromosome extract solution were treated at a concentration of 100 μg / ml for 5 days. Cells were washed with phosphate buffered saline (PBS), and incubated in phosphate buffered saline (PBS) supplemented with 4% paraformaldehyde and 4% sucrose for 40 min at room temperature Respectively.

To prevent nonspecific binding, the cells were immersed in a blocking buffer containing 2.5% BSA and 0.5% horse serum and reacted at 4 ° C for 6 hours. The polyclonal anti-GAP rabbit antibody (Abcam, USA), a primary antibody against GAP-43 protein, was mixed in blocking buffer at a ratio of 1: 1000 and then incubated at room temperature for 6 hours Respectively.

After the reaction with the primary antibody, the cells were washed with PBS for 3 rd hours. The secondary antibody, biotinylated anti-rabbit immunoglobulin (Dako Co., U.S.A.) was diluted at a ratio of 1: 1000 and reacted at room temperature for 30 minutes. The cells were washed with PBS, treated with streptavidin peroxidase (Vector ABC kit), and developed with 0.05% diaminobenzidine as a chromogen.

GAP-43 immunoreactive cells of dark brown color were observed under an optical microscope and images were stored in a computer using an image analysis program.

 (2) Western blot test

To analyze the expression pattern of beta-III tubulin and GAP-43 protein, western blotting was performed using beta-III tubulin and GAP-43 antibody, respectively.

Protein separation was carried out with PRO-PREP protein extraction solution (iNtRON, USA). Protein was separated and quantified with nanodrop.

The electrophoresis was performed by SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) method.

First, the protein was mixed with 0.125 M sample buffer (pH 6-7 Tris buffer solution, 1% SDA, 20% glycerol, bromophenol blue and 2-mercaptoethanol) and boiled in boiling water. The mixture was applied to 8% polyacrylamide gel And electrophoresed. The electrophoresis was carried out using a mini-gel electrophoresis apparatus (SE 600, Hofer, USA) at a voltage of 110 V for 3 hours. After electrophoresis, the protein of the gel was transferred to PVDF (polyvinylidene difluoride) membrane (Millipore Co., USA) at 130 mA for 1 hour using a protein conversion device (Semiphor, Phamacia Bio., USA).

To prevent nonspecific binding of the primary antibody of the gel protein transferred to the PVDF membrane, a PVDF membrane was added to a blocking buffer containing 5% skim milk dissolved in 0.1% Tween-20-TBS (TBS-T) Lt; / RTI &gt;

The primary antibodies, beta-III tubulin (1: 1000) and GAP-43 (1: 500, abcam, USA) were each incubated at 4 ° C for at least 24 hours in the blocking buffer.

After the primary antibody reaction, the cells were washed three times with 0.1% TBS-T and the secondary antibody anti-rabbit conjugated HRP (Santa Cruze, USA) was diluted 1: 1,000 in TBS- And reacted for 2 hours.

The reacted anti-rabbit-binding HRP was washed three times with TBS-T and specific protein was observed by ECL (enhanced chemiluminescent) method.

In order to analyze the degree of expression of the observed protein, it was stored in a computer with FCED system (FluorChem E Digital Darkroom system, SelectScience, UK).

2) Experimental results

In order to measure neuronal differentiation and neurite production at the molecular level, we examined the neurospecific marker beta-III tubulin and the protein expression of GAP-43 involved in neurite outgrowth and neuroplasticity Respectively.

FIG. 7 is a photograph of immunocytochemical staining showing GAP-43 protein expression by neurite differentiation in PC12 cells. FIG.

GAP-43 protein was confined to the cell body and showed almost no expression in the cells of the control group not treated with NGF or FSG-200 solution.

In the 5 ng / ml NGF group, GAP-43 protein was increased in the cell body compared to the control group, but the expression was weak in the neurite.

However, treatment with 5 ng / ml of NGF and 50 ㎍ / ml of FSG-200 solution resulted in an increase in GAP-43 protein in not only the cell body but also neurites.

8 is a Western blot photograph and analysis graph showing the expression pattern of beta-III tubulin and GAP-43 protein by neurite differentiation in PC12 cells.

Western blot analysis was performed using beta-III tubulin antibody to determine the expression pattern of beta-III tubulin in PC12 cells. As a result, the amount of protein was 50 kDa.

The amount of actin in the control group was set at a reference value of 1, and the expression patterns of the control and the expression levels of the respective groups were compared with each other.

The NGF 5 ng / ml group and the FSG-200 group treated with 50 ㎍ / ml of NGF and FSG-treated groups showed a significant increase of 3.3 ± 0.4 times (p <0.05) ± 0.5 fold higher than that of NGF alone (p <0.01).

Western blot analysis was performed using GAP-43 antibody to determine the expression pattern of GAP-43 protein. As a result, the amount of protein was 43 kDa.

The amount of protein in the control group was set at a reference value of 1, and the expression pattern was observed by comparing the expression values of the control group and the respective groups.

The NGF 5 ng / ml treatment group showed a significant increase of 2.4 ± 0.3 fold (p <0.05) compared to the control group (p <0.05) and treated with 5 ng / ml of NGF and 50 ㎍ / (P <0.01) than the control group.

Experiment 4 ; Neuron  GAP-43 by differentiation mRNA  Expression

1) Experimental method

The expression of GAP-43 mRNA, which is involved in neurite outgrowth and neuroplasticity during neural differentiation of PC12 cells, was measured by RT-PCR (Reverse Transcription-Polymerase Chain Reaction).

PC12 cells were washed with PBS, disrupted with Trizol (Invitrogen Life Technology Co., USA), and the RNA layer was separated using a centrifuge. Chloroform was added to the separated RNA layer, mixed and centrifuged to remove the protein. Total RNA (1 ㎍) was isolated by adding isopropanol to the protein-free cells and centrifuged, and quantified by ND-1000 spectrophotometer (NanoDrop-1000 spectrophotometer, NanoDrop, Netherland).

To perform RT-PCR, oligonucleotide dT primer (Promega, USA) and RNA premix (RNA premix, Bioneer, Korea) were added to total RNA and reacted at 40 to 45 ° C for 1 to 2 hours. The cDNA was inactivated by inactivation at ~ 75 ° C for 10 to 20 minutes. The reverse transcribed cDNA was injected with 0.1-0.3 mol / L fry and the gene was amplified using PCR Master Mix (Promega, USA). Primers were prepared according to the composition shown in [Table 1] below. GADPH was set as a control group.

GADPH is a representative house keeping gene, and the housekeeping gene refers to a gene whose expression level is constant under any conditions.

The polymerase reaction of each primer was carried out under the conditions shown in Table 2 below. The amplified gene, that is, the polymerization reaction product, was electrophoresed using a 1-3% agarose gel at 90-110 V for 15-25 minutes, and then electrophoresed using a FCED system (FluorChem E Digital Darkroom system, SelectScience, UK) Lt; / RTI &gt;

primer Nucleotide sequence (5'-3 ') Temperature (℃) Product size (bp) Rat GAPDH F  ATG CCA TCA CTG CCA CTC AG 55.5 21.5 Rat GAPDH R GCA TGT CAG ATC CAC AAC GG 55.8 GAP-43 F AAG ATG GTG TCA AAC CGG AG 53.6 18.2 GAP-43 R CTT CTC CAC ACC ATC AGC AA 53.1

Here, F means forward (forward) and R means reverse (reverse).

Number of cycles Step 1 condition 2-step condition Three-step condition Temperature time Temperature time Temperature time GAP-43 94 ° C 30 seconds 55 ° C 30 seconds 72 ℃ 30 seconds 32 GAPDH 94 ° C 30 seconds 55 ° C 30 seconds 72 ℃ 30 seconds 28

Table 2 is a step-by-step condition of the primer.

2) Experimental results

9 is an RT-PCR photograph and analysis graph of the expression pattern of GAP-43 mRNA by neurite outgrowth in PC12 cells. The expression level of GAP-43 mRNA was measured and compared with the expression level of RT-PCR of control cells not treated with both NGF and fermented golden chromosomal extracts.

As a result, the NGF 5 ng / ml-treated group showed a significant increase of 2.2 ± 0.4 times (p <0.05) compared with the control group, and the NGF 5 ng / ml and fermented golden- (P <0.01), which was significantly higher than that of the control group by 3.2 ± 0.5 times.

Experiment 5: By neurite differentiation pERK1 / 2 protein expression

1) Experimental process

GAP-43 mRNA expression by neurite differentiation (Experiment 4).

2) Experimental results

10 is a photograph showing the results of immunocyte staining for pERK1 / 2 protein expression by neurite outgrowth in PC12 cells. The expression of pERK1 / 2 protein was weak in the control cells but the expression of pERK1 / 2 protein was increased in the cell body compared to the control group in the NGF 5 ng / ml treated group, but not in the neurite.

On the other hand, Fig. 10 shows that pERK1 / 2 protein was increased not only in the cell body but also in the neurite as well as in the treated group of 50 ng / ml of NGF 5 ng / ml and fermented golden chromosomal extract solution.

11 is a photograph and a graph showing the appearance of pERK1 / 2 protein expression by western blot test by neurite outgrowth in PC12 cells. Western blot analysis was performed to examine the expression pattern of pERK1 / 2 protein in PC12 cells. As a result, the pERK1 and pERK2 proteins were isolated at a molecular weight of 44 kDa and a molecular weight of 42 kDa, respectively, and compared with the control pERK1 / 2 protein expression Expression values for each experimental group were obtained.

NGF 5 ng / ml and NGF 5 ng / ml and 10 ㎍ / ml of fermented golden chromosomal extract solution were significantly higher than the control group (2.3 ± 0.5 times, p <0.05) (P <0.01), which was 3.9 ± 0.7 times higher than that of the control group.

The expression of pERK1 / 2 protein was significantly increased in the treated group with 5 ng / ml of NGF and 10 ㎍ / ml of fermented golden chromosomal extract solution than the 5 ng / ml group treated with NGF. On the other hand, GAPDH protein expression of housekeeping gene showed similar expression level in each experimental group. Referring to FIG. 11, pERK1 / 2 protein expression was significantly increased when NGF was treated together with the fermented golden chromosomal complex extract of the present invention and treated with NGF alone.

Referring to the results of the experiment, the fermented golden Gunma complex extract powder according to the present invention promotes growth of neurite and promotes mRNA expression of GAP-43. In addition, the complex extract powder of the present invention can promote protein expression of beta-III tubulin, GAP-43, and pERK1 / 2.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, It will be readily apparent that such substitutions, modifications, and alterations are possible.

Claims (4)

Crushing, washing and mixing fermented golden, rhubarb, dermis, squid, and ginseng;
Adding the mixture to an extractor and preparing a combined extract using an alcohol solution or water;
A step of preparing a complex concentrate which is obtained by concentrating the complex extract in a concentrator and heating under reduced pressure;
Sterilizing and drying the complex concentrate to prepare a powder,
The fermentation gold is fermented and aged with gold by a yeast-enzyme complex fermentation broth. The yeast-enzyme complex fermentation broth is a mixture of yeast powder and enzyme concentrate. The yeast powder is Aspergillus oryzae, Or Saccharomyces ellipsoideus is inoculated on a medium, followed by lyophilization and lyophilization followed by pulverization.
Wherein the mixing step comprises mixing 50 to 80 parts by weight of ginseng, 50 to 80 parts by weight of ginseng, 50 to 80 parts by weight of ginseng root, and 20 to 40 parts by weight of ginseng with respect to 100 parts by weight of the fermented gold,
In the step of preparing the complex extract, fermented gold, chymus, dermis, purple ginseng, and ginseng are put into each extraction container, and a 50% alcohol solution is added thereto and the mixture is warmed at 80 to 90 ° C. The extract is filtered through a filter, % Alcohol solution, heating the mixture at 80 to 90 ° C, extracting the extract with a secondary filtrate through a filter, adding purified water, heating at 80 to 90 ° C, and filtering the extract through a filter to obtain a tertiary extract Wherein the fermented golden ginseng extract powder is prepared by a method comprising the steps of:


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