KR20170140647A - Herbal composition containing of fermented scutellarae radix and gastrodia rhizoma for prevention and improvement of oxidative neurologic damage by hidrogen peroxide - Google Patents

Abstract

The present invention relates to an extract comprising a complex extract of fermented Scadelaria, Gastrodia elata Blume, driven orange peel, arrowroot roots, and ginseng for preventing and alleviating hydrogen peroxide oxidation damage of neurocytes, to powder, and to a method for preparing a pharmaceutical composition comprising the same. A Scutellaria and Gastrodia elata Blume complex extract fermented liquid and powder of the present invention show effects of preventing, alleviating, and treating neurological diseases by inhibiting oxidative nerve apoptosis by hydrogen peroxide. The Scutellaria and Gastrodia elata Blume complex extract fermented liquid and powder of the present invention show the effect of increasing the formation of field excitatory postsynaptic potentials (fEPPs) in the CA1 region of hippocampus. In addition, the Scutellaria and Gastrodia elata Blume complex extract fermented liquid and powder of the present invention show an alleviation and treatment effect on the formation disorder of longer-term potentiation (LTP) caused by nerve cell oxidative damage induced by hydrogen peroxide in the CA1 region of a hippocampal segment. The Scutellaria and Gastrodia elata Blume complex extract fermented liquid and powder of the present invention can minimize side effects and cytotoxicity by using a naturally derived material. The Scutellaria and Gastrodia elata Blume complex extract fermented liquid and powder of the present invention show an effect of inhibiting ischemic brain damage in a stroke experiment model by occlusion of middle cerebral artery.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for preparing a fermented golden-germanium complex extract powder for preventing and improving damage of hydrogen peroxide oxidative damage of neurons,

The present invention relates to an extract powder containing as an active ingredient a complex extract of fermented golden, ginseng, dermis, gruel and ginseng for preventing and improving damage of hydrogen peroxide oxidative damage of nerve cells, and a method of producing a herbal composition containing the extract powder.

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, the rapid death of many neurons in the aging process causes degenerative brain diseases such as cerebral infarction, stroke, 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 they are supplied with about 30% of the blood, which is the total amount of 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 flow supply or decline, is a terrible disease that causes a significant loss of 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, structural and functional changes of the neural network formed by the surviving neurons can be substituted for 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, it is urgently necessary to develop drugs capable of promoting the plasticity of the neural network by promoting the production of the dendrites and the dendritic spines of nerve cells.

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 loops of neurons as a nerve growth factor in PC12 cells as an experimental model for neuronal cell growth and neuroplasticity at the cellular level ( in vitro ).

While many mammalian species meet the criteria for stroke models, rodent rats are currently the most commonly used model. The reason for this is that the brain tissue of the mouse is similar to the human brain and anatomical structure and physiological functions.

If the staple is maintained by inserting the monofilament suture into the middle cerebral artery through the internal carotid artery to prevent blood flow to the middle cerebral artery, it is a permanent closure. Removal may be transient closure.

This model induces damage to the striatum, parietal lobule, and temporal lobe, and can be reproduced well in repeated experiments, making it suitable for the study of neuroprotective agents. Since the development of the ischemic paralytic animal model, numerous materials have been applied to these models and studies are under way to clarify the effects.

In cerebral infarcts where cerebral blood vessels are closed, nerve cells are damaged due to a decrease in blood supply. Specific mechanisms are glutamate toxicity, active oxygen, inflammation and pneumocyte activation, and many studies It has been progressed.

In the meantime, existing therapies for the treatment of neurological diseases are aimed at maintaining the damage no longer proceeding, rather than regenerating damaged nerves in most cases.

For example, local treatment such as a thrombolytic agent is performed in some neurological diseases such as stroke and cerebral infarction. In addition, techniques for applying ceria, which is a rare earth element and has the effect of preventing oxidation and apoptosis, to 3 nm nanoparticles and applying it to the brain are being developed.

 The thrombolytic agent is effective only within 3 to 4 hours after onset, and the treatment success rate is only about 12%.

There are also risks associated with the technology of making ceria as nanoparticles and applying it in vivo. That is, nanoparticles have a high surface reaction force and can permeate the cell membrane, which can easily enter the human body from the outside through the respiratory tract or skin. Thus, nanoparticles can travel across the body in the blood, causing brain or cardiovascular disease.

Unlike previous studies, neurodevelopment studies and attempts have been made in various ways. Neurodevelopment studies and stem cell-based neurological diseases are actively under way. In other words, neurons generate neurites with various pathways and various factors, and studies are under way to induce neural differentiation by using various substances, so as to prevent, improve and cure neurological diseases.

Experimental studies on the treatment of various brain diseases and neuron regeneration have been actively conducted in recent years. 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.

 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, the epidural administration of the gold methanol extract in transient preconditioning animals showed a significant decrease in 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.

The major flavonoid-based chemicals extracted from gold are baicalein, baicalin, wogonin and oroxylin. 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 Baikal lane than in bicalkine.

Orcoxin improved cognitive function and memory in elderly rats and experimental animals with degenerative brain disease.

UGONIN inhibited neuronal apoptosis in the cerebral cortex in acute regional cerebral ischemic injury model and improved behavioral symptoms.

Gastrodiae Rhizoma is a dried herbaceous root of perennial parasitic herbaceous plant belonging to the Orchidaceae. It is an herbal medicine used for the treatment of antiepileptic, analgesic, dizziness suppression and paralysis. Gastrodin, a phenolic derivative of chymase, inhibits apoptosis induced by ischemia and glutamate toxicity in various types of cultured neurons and inhibits intracellular calcium and NO production as well as intracellular PI3K It is known to activate secondary signal transduction systems.

In addition, it inhibits cerebral cortical neuronal cell death due to ischemic injury by intermittently blocking the blood flow of the middle cerebral artery in the rat, inhibits the increase of glutamate during ischemia in the hippocampus, and increases the inhibitory neurotransmitter GABA concentration .

Vanillyl alcohol and 4-hydroxybenzyl alcohol in rats inhibited ferric chloride-induced seizures or induced ischemic brain damage in free radicals Production inhibition.

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

The dermis (Aurantii Nobilis Pericarpium) is a fruit-shell of a tangerine tree, which is a black-and-white fruit tree that belongs to an excess of the mountain. It is a sun-dried herbal medicine. According to Dong-bo-gyung, its efficacy is known to promote digestion, suppress coughing, .

In addition, the pharmacological action of dermis has been reported as the actinic action, the central inhibitory action, the antiallergic action, the anti-inflammatory action, the capillary lowering action and the blood lipid lowering action.

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.

Hesperidin is the most abundant ingredient in the dermis, and it has been known to have various pharmacological actions such as blood pressure lowering, capillary lowering action, antiinflammation, blood cholesterol lowering, and antitumor action.

Experimental studies also showed that hesperidin reduced the degradation of intracellular glucose utilization by beta amyloid in cultured neurons, suppressing autophagy and inhibiting oxidative cytotoxicity by hydrogen peroxide (H ₂ O _{2 )} .

It is known that CCl4 poisonous substances and transient cerebral ischemia cause neuronal cell damage by lipid and protein oxidation in brain tissue at the experimental animal level, and hesperidin suppresses this phenomenon.

In addition, Nobiletin inhibits the loss of memory by amyloid beta protein in rats, as well as promotes the stimulation of cAMP / PKA / ERK / CREB signaling pathways and neural cell growth in cultured hippocampal neurons It inhibits cell degeneration.

Puerariae Radix is a dried root of the vine of the leguminous bean. It treats the fever, the fever, the stomach, the poison, and the cold body by the medicine of the persimmon in the herbal medicine. Diabetes), fatigue, and other detoxification.

In recent studies, it has been reported that PGE has an effect of hypotensive action, increase of cerebral blood flow, and memory enhancement, dilating the coronary artery to mitigate myocardial infarction and improve the ischemic response on ECG.

The main components of the brow root include flavonoid compounds such as puerarin, daizein and daidzin and saponin such as soyasaponin and starch .

In the case of Puerariae Radix extract, it has been shown to increase the concentrations of norepinephrine and dopamine in the hippocampus in mice that occlude the bilateral carotid arteries and cause cerebral ischemia, and play an important role in restoring motor function .

 Puerarin, an active ingredient in purine roots, has been shown to have a neuroprotective effect through various mechanisms, and is widely used in China for treating acute stroke.

Puerto rarin the test and in vitro test inhibit glutamate ^{Ca 2 + (glutamate Ca 2 +} ) cells into and NO produced in (in vitro), acid-reactive ions in the channel to block the (acid-sensing ion channel) and protecting the nerve cells , It has been reported that neuronal cells are protected by various effects such as inhibition of NMDA receptor expression, inhibition of intracellular Ca ^{2 +} entry, antioxidation, anti-inflammation, and anti-apoptosis in a cerebral ischemia model, a model of local cerebral ischemia.

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.

The present invention provides a substance capable of inhibiting the growth of nerve cells and the death of nerve cells by oxidation.

The present invention relates to a method for inhibiting oxidative neuronal cell death by hydrogen peroxide, (FEPSPs, field excitatory postsynaptic potentials) and long-term potentiation (LTP) formation disorders due to oxidative damage.

 It is an object of the present invention to provide a fermented powder of fermented ginseng root which prevents, alleviates and treats neurological diseases by reducing damage caused by a decrease in blood supply of nerve cells in a cerebral infarction in which cerebral blood vessels are closed.

Fermenting gold with yeast and enzyme complex fermentation broth; Crushing, washing and mixing the fermented golden, chestnut, dermis, peel, and ginseng; Adding the mixture to an extractor and preparing a complex extract using the extract; Fermenting lactic acid bacteria and an enzyme complex fermentation broth with the complex extract to perform low temperature fermentation , ≪ / RTI & Wherein the yeast is any one or more of Aspergillus oryzae, Saccharomyces ellipsoideus and Saccharomyces cerevisiae, and the lactic acid bacterium is selected from the group consisting of Lactobacillus acidophilus The present invention relates to a method for preparing a fermented fermented product of golden thymus for the prevention and improvement of damage to nerve cells which is at least one of Lactobacillus acidophilus, Streptococcus thermophilus and Bifidobacterium longum.

The present invention relates to a method for preparing a fermented milk, And then spray-drying the concentrated fermentation broth to produce a powder.

The present invention relates to a complexed fermented golden ginseng powder, wherein the powder is associated with a fermented golden ginseng fermentation powder which inhibits oxidative nerve cell death by hydrogen peroxide.

The golden ginseng complex extract powder of the present invention exhibits an effect of preventing, ameliorating and treating neuropathy by inhibiting oxidative neuronal cell death by hydrogen peroxide.

The golden-gamma complex extract powder of the present invention has an effect of enhancing the formation of excitatory post-synaptic postsynaptic potentials (fEPSPs) in the CA1 region of the hippocampal slice, (LTP, long-term potentiation) due to oxidative damage.

The golden ginseng composite extract powder of the present invention can minimize adverse effects and cytotoxicity by using a substance derived from a natural origin.

The Golden Gene Complex Extract Powder of the present invention exhibits an effect of inhibiting ischemic brain injury in a stroke experimental model of middle cerebral artery occlusion.

The golden ginseng complex extract powder of the present invention may be formulated into powders, granules, tablets and capsules by mixing with carriers, excipients, diluents, etc., and may be administered orally or parenterally.

FIG. 1 is a flowchart showing a process of preparing fermented gold by fermentation and aging of golden herb medicine using yeast and physiological enzyme complex fermentation broth according to an embodiment of the present invention.
FIG. 2 is a view illustrating a process of preparing fermented golden fermented liquor, fermented ginseng, ginseng root, ginseng and ginseng herbal medicines by mixing and extracting them, followed by liquid fermentation and aging using a lactic acid bacterium and a physiological enzyme complex fermentation broth according to an embodiment of the present invention FIG.
FIG. 3 is a flow chart showing a process of producing a complexed fermented golden germanium powder by depressurizing and concentrating a complex herbal extract fermentation broth according to an embodiment of the present invention, followed by a high-temperature spray drying process.
Figure 4 is a device for measuring excitatory post-synaptic potentials (fEPSPs) in the CA1 region of hippocampal slices.
FIG. 5 is a graph showing the excitatory post-synaptic evoked potentials recorded in four channels after electrical stimulation in the radiating layer of the hippocampal CA1 region.
FIG. 6 is a graph showing long term synergy (LTP) by analyzing the percentage of the amplitude and the slope of the excitatory post-synaptic evoked potential.
7 is a view showing a setter burst stimulation pattern for inducing a long-term synergistic effect.
FIG. 8 is a photograph showing an example of generation of excitatory post-synaptic dystrophies (fEPSPs) by electrical stimulation in the radiating layer of hippocampal CA1 region using a 64-channel electrophysiological recording system.
Fig. 9 is a graph showing the effect of hydrogen peroxide By concentration This study shows the change of excitatory post-synaptic evoked potential by administration.
FIG. 10 is a graph showing changes in hydrogen peroxide concentration-dependent effects on long-term synergism in the CA1 region of a hippocampal slice.
FIG. 11 is a photograph showing changes in slope and amplitude of excitatory post-synaptic evoked potential over time after treatment of golden-rhombus complex fermented powder in CA1 region of a hippocampal slice.
FIG. 12 is a graph showing the rate of change of the slope and amplitude of the excitonic post-synaptic evoked potential during the 50-60 min administration of the mixed fermented golden powder.
FIG. 13 shows the results of the pretreatment of the golden chromosomal complex fermentation powder in the CA1 region of the hippocampal slice for 15 minutes, and the combination of 200μ hydrogen peroxide and the golden chromosome complex fermentation powder And the change of long-term synergism with the passage of time after administration.
Fig. 14 shows the results of a combination of 200 [mu] m hydrogen peroxide and a golden chromatin composite fermentation powder in the CA1 region of the hippocampal slice And the rate of change of long-term synergism by administration.
Fig. 15 shows the results of the pretreatment of 200 과 of hydrogen peroxide and 15 minutes of pre-treatment of the golden chromosomal complex fermentation powder in the CA1 region of the hippocampal slice, And the change of long-term synergism with the passage of time after administration.
16 is a graph showing the results of the measurement of the concentration of CaCO3 in the CA1 region of the hippocampal slice by pretreating 200 袖 g of hydrogen peroxide and 15 과 of hydrogen peroxide And the rate of change of long-term synergism with the passage of time after administration.
FIG. 17 is a graph showing the cell survival rate of PC12 cells treated with various concentrations of the mixed solution of the golden chromosomal complex fermentation powders in oxidative cell damage caused by hydrogen peroxide.
FIG. 18 is a photograph and analysis graph showing the effect of FSG-100 solution on DNA damage in cell death by hydrogen peroxide in PC12 cells.
FIG. 19 is a TTC staining showing the dose-dependent effect of the fermented golden-chromosomal combination solution on ischemic brain injury in a stroke model experiment with a cerebral aortic occlusion.
20 is an analysis graph showing dose-dependent effects of the fermented golden-chromosomal complex extract solution in the ischemic injury of a stroke model model of middle cerebral artery occlusion.

FIG. 1 shows a process for preparing fermented gold, and FIG. 2 shows the preparation of the fermented fermented product of the present invention. Referring to FIGS. 1 and 2, the fermented golden ginseng extract fermentation broth of the present invention includes preparation of fermented gold, mixing, preparation of an extract, and fermentation.

 As shown in FIG. 1, the golden fermentation is a step of fermenting gold with yeast and enzyme complex fermentation broth.

The preparation of fermented gold includes a step of immersing three types of yeast and physiological enzyme complex fermentation broth in a golden section, a fermentation step and an aging step.

  The microorganisms used in the present invention are three yeasts.

Yeast powder is obtained by inoculating a yeast strain in a medium and subculturing it, followed by drying and pulverizing.

In addition, the (natural fruit) enzyme fermentation broth can be obtained by mixing white sugar with immature physiology, fermenting it for 6 months, and then concentrating it by filtration.

Yeast (yeast) and enzyme complex fermentation broth are prepared by mixing the previously prepared yeast powder and (natural fruit) enzyme concentrate.

The yeast strains usable in the present invention may be any one of Aspergillus oryzae, Saccharomyces ellipsoideus and Saccharomyces cerevisiae, In addition, it is preferable to use all three strains.

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 microorganism-fruit enzyme complex fermentation broth is added so that 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 5 to 10 days, preferably 7 days, while maintaining the temperature at 50 to 60 ° C.

The secondary fermentation may be performed by spraying the microorganism-fruit enzyme complex fermentation broth onto the gold and fermenting the fermented product for 5 to 10 days, preferably 7 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 step may be carried out in various ways, but alternatively, 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 step 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.

As shown in FIG. 2, the mixing step is a step of pulverizing, washing, and mixing the fermented gold, gum, dermis, pupa, and ginseng prepared in FIG. The mixing step may include 50 to 100 parts by weight of ginseng, 50 to 100 parts by weight of ginseng, 50 to 100 parts by weight of ginseng, and 20 to 50 parts by weight of ginseng, based on 100 parts by weight of the fermented gold.

The step of preparing the complex extract is a step of preparing the complex extract by adding the mixture to an extractor and 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.

For example, the complex extract can be obtained by cutting fermented gold, chymus, dermis, pupa, and ginseng, adding it into purified water, heating at 90 to 100 ° C for 6 hours, and then filtering.

The low-temperature fermentation step is a step of low-temperature fermentation by mixing lactic acid bacteria and an enzyme-based fermentation broth with the combined extract.

The lactic acid bacteria may be any one or more of Lactobacillus acidophilus, Streptococcus thermophilus, and Bifidobacterium longum. In addition, .

Lactic acid bacteria can be used in powder form. The lactic acid bacteria powder is inoculated with a lactic acid bacterium strain in a culture medium, followed by subculture, followed by drying and pulverizing.

(Natural fruit) enzyme can be referred to the above description.

Lactic acid bacteria and enzyme complex fermentation broth can be prepared by mixing lactic acid bacterial powder and (natural fruit) enzyme.

In the low temperature fermentation step, lactic acid bacteria and an enzyme fermentation broth may be added to the combined extract, fermented at a low temperature of 35 to 45 ° C for 3 days, and then aged at 45 to 55 ° C again.

Referring to FIG. 3, the preparation method of the complexed fermented golden ginseng comprises the steps of concentrating the fermented solution of the extracted fermented ginseng root mixture at reduced pressure, and spray-drying the concentrated fermented solution to prepare a powder.

 The reduced-pressure concentrate can be concentrated at 80 to 90 ° C under a reduced pressure of 180 to 200 mmHg.

In the powder preparation step, the concentrated fermentation broth is subjected to high-temperature spray drying and sterilization at 170 to 190 ° C.

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

The present invention can be used as a herb medicine composition by adding a herbal medicine component known in the present invention.

The golden-gamma complex fermentation powder of the present invention can inhibit the oxidative nerve cell death by hydrogen peroxide.

The fermentation broth of Golden Gamma extract of the present invention and the powder exhibited the effect of enhancing the formation of excitatory post excitatory postsynaptic potentials (CAE1) in the CA1 region of the hippocampal slice, and also the effect of the hydrogen peroxide- cell (LTP, long-term potentiation) due to oxidative damage.

The fermentation broth of Golden Chamaese Extract and powder of the present invention can minimize side effects and cytotoxicity by using a substance derived from natural origin.

The fermentation broth of Golden Gamma Extract of the present invention and the powder exhibit the effect of suppressing ischemic brain injury in a stroke experimental model of middle cerebral artery occlusion.

The fermentation broth of Golden Gamma Combined Extract of the present invention and the powder may be formulated into powders, granules, tablets and capsules by mixing with carriers, excipients, diluents and the like, and they can be administered orally or parenterally.

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: Preparation of fermented gold

First, 3 kg of the golden slice was taken and 10 g of Aspergillus oryzae, Saccharomyces ellipsoideus and Saccharomyces cerevisiae yeast powder, 30 g of natural fruit enzyme culture, Was dispersed in 10 L of purified water, and 3 L of the solution was added so as to immerse the golden slice, and then left to stand at 50 to 60 캜 for 24 hours. After fermentation at 50 to 60 DEG C for 3 days, 1 L of yeast and enzyme culture was injected, followed by secondary fermentation for 3 days, and further 1 L of yeast and enzyme culture was injected for third fermentation.

Yeast powder and enzyme culture were prepared as follows.

First, Saccharomyses sereviciae strains were inoculated in a liquid medium, followed by subculturing. The cells were then dispersed in a high-temperature 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 enzyme cultures was added to 4 L of purified water and homogeneously mixed to prepare yeast and enzyme culture broth.

2. Manufacture of fermented fermented milk

3kg of fermented golden, 2kg of Chunmae slice, 2kg of dendritic slices, 2kg of slices of gangrove and 1kg of ginseng slices were added to 100L of purified water, heated at 90 ~ 100 ℃ for 6 hours and filtered.

The filtrate was cooled to room temperature and 10 g of each of Lactobacillus acidophilus, Streptococcus thermophilus, Bifidobacterium longum and 30 g of the natural fruit enzyme culture was dispersed in 10 L of purified water , Fermented at 35 ~ 45 ℃ for 3 days and then fermented at 45 ~ 55 ℃ for fermentation.

3. Preparation of Powder

The combined fermented extracts were prepared by high temperature spray drying and sterilization at 170 ~ 190 ℃.

Experimental Example 1 Recording and analysis of excitatory post-synaptic evoked potentials (fEPSPs) in CA1 region of hippocampal slices

Figure 4 is a device for measuring excitatory post-synaptic potentials (fEPSPs) in the CA1 region of hippocampal slices.

A 64-channel neural interface system (NIS), ETRI, and a 64-channel extracellular recording system (ETRI) developed by ETRI for induction and recording of synaptic excitatory postsynaptic potentials (fEPSPs) Korea) was used.

The 64-channel extracellular recording system controls an electrode probe composed of an 8 x 8 array (electrode spacing 200 μm), a probe head stage capable of mounting an electrode probe, a 64-channel signal amplification and electrical stimulation system, and a signal amplification system, And a computer capable of analyzing the data.

Under a digital microscope, the hippocampal slices were placed on 64 electrodes of the electrode probes and fixed using a silver anchor. At this time, the central part of the radiating layer of the CA1 region was positioned at the center of 64 electrodes. The artificial cerebrospinal fluid at 30 ° C containing 95% O 2 and 5% CO 2 is perfused at a rate of 2 ml per minute until the end of the experiment.

FEPSPs were generated and recorded in the hippocampal CA1 region using an electric stimulation program (Trigger ^® ) created by the Labview program and a signal analysis program (Signal ^® ) capable of storing and analyzing biological signals.

FIG. 5 is a graph showing the excitatory post-synaptic evoked potentials recorded in four channels after electrical stimulation in the radiating layer of the hippocampal CA1 region. For the generation of excitatory post-synaptic evoked potentials, we used the Trigger ^® program In the 64 channel signal amplification and electric stimulation device, a two-phase square wave (0.2ms, 1-5V) was generated, and electrical stimulation was applied to the central part of the radiation layer in the CA1 region via the probe head stage.

Determine the excitatory postsynaptic evoked potential signal is detected through the previous stage amplifies the signal from the 64 channels of signal amplification system, removing the japsinho following new Signal ^® program excitatory after occurrence distribution of synaptic induced potential generated in the 64 channels of the generated by the electrical stimulation Respectively. On the other hand, the slope and amplitude of the excitatory post-synaptic evoked potentials were analyzed by analyzing the representative 4 channels which recorded the signals of the excitatory post-synaptic evoked potential among the 64 channels.

FIG. 6 is a graph showing long term synergy (LTP) by analyzing the percentage of the amplitude and the slope of the excitatory post-synaptic evoked potential. The stimulation to induce long-term synergy after basal recording stimulated long-term synergy by stimulation four times at intervals of 20 seconds with stimulation of theta rupture (20 Hz, 4 pulses, 10 busts, interbust interval: 200 ms). The stimulation intensity was fixed throughout the observation of long-term synergy, and stimulation was performed once every 20 seconds for 1 hour after inducing long-term synergy, resulting in excited post-synaptic evoked potentials.

7 is a view showing a setter burst stimulation pattern for inducing a long-term synergistic effect. The slope and amplitude values of the excitatory post-synaptic evoked potentials for each hippocampal slice were calculated on-line and stored as a text file. The long-term synergistic induction rate was analyzed using the Excel program, ie, the amplitude of the excitatory post-synaptic evoked potential recorded during 60 minutes after theta tear stimulation on the amplitude and slope values of the excitatory post-synaptic evoked potential recorded during baseline recording, The slope value was converted into a percentage.

 (%) = [(Amplitude and slope of excitatory post-synaptic evoked potential during excitatory post-synaptic evoked potential gradient or amplitude-basal recording after theta-tear stimulation) / amplitude and slope of excitatory post-synaptic evoked potential during basal recording )] X 100

On the other hand, long-term synergy induction rate was calculated from each hippocampal slice in 4 channels, and the average value was calculated as the induction rate of long-term synergy per hippocampal slice. For the statistical analysis of the long - term synergy induction rate, mean value was calculated from the induction rate of organ synergy for 50 to 60 minutes after the eruption.

FIG. 8 is a photograph showing an example of generation of excitatory post-synaptic dystrophies (fEPSPs) by electrical stimulation in the radiating layer of hippocampal CA1 region using a 64-channel electrophysiological recording system.

64-channel extracellular electrophysiological recording system was used to electrically excite the Schaffer's collateral-comissural system (SCC) in the radial layer (SR) of the hippocampal CA1 region to generate an excitatory post-synaptic evoked potential .

The generation of excitatory post - synaptic evoked potentials in the CA1 region was clearly observed in the SR and SP layers and the amplitude of excitatory post - synaptic evoked potentials was recorded significantly in the recording channel near the electrical stimulation channel.

Fig. 9 is a graph showing the effect of hydrogen peroxide By concentration This study shows the change of excitatory post-synaptic evoked potential by administration.

FIG. 10 is a graph showing changes in hydrogen peroxide concentration-dependent effects on long-term synergism in the CA1 region of a hippocampal slice. Hydrogen peroxide is a substance that causes oxidative damage to cells by generating reactive oxygen species (ROS). In this experiment, oxidative injury of hydrogen peroxide in the CA1 region of the hippocampal slice was observed to observe changes in long-term synergism.

After baseline recording, hydrogen peroxide was diluted into cerebrospinal fluid at 200, 400, and 600 μ, respectively, and the change in excitatory post-synaptic evoked potential was recorded after cetaphritic stimulation.

When the hydrogen peroxide was treated for 15 minutes after the basal recording, the amplitude of the excitatory post-synaptic evoked potential decreased significantly in the CA1 region of the hippocampal slice exposed to hydrogen peroxide compared to the normal control without hydrogen peroxide for 15 minutes.

That is, as the concentration of hydrogen peroxide to be administered was higher, the amplitude of the excitatory post synaptic evoked potential was further decreased, thereby showing drug concentration dependency. This is due to hydrogen peroxide Oxidative damage inhibits synaptic excitability in the CA1 region.

On the other hand, in experiments inducing long-term synergy by the theta tear stimulation, Oxidative damage inhibited the long-term synergism and also showed concentration dependence.

In other words, the long-term synergism calculated by analyzing the slope of the excitatory post-synaptic evoked potential was 179.3 ± 4.5% in the normal control group, (N = 7) was 128.3 ± 5.7% (P <0.01), indicating a statistically significant decrease.

Hydrogen peroxide (N = 7) and the control group (n = 7) were 69.6 ± 13.2% (P <0.01) 600μ administration group showed 30.5 ± 5.9% (P <0.01), which was lower than the basal state. Therefore, in hippocampal slices, Oxidative injury not only inhibits synaptic excitability but also causes synaptic plasticity, such as long term synergistic developmental disorder.

FIG. 11 is a photograph showing changes in slope and amplitude of excitatory post-synaptic evoked potentials over time after treatment of golden chromosome complex fermented powder in CA1 region of hippocampal slice.

FIG. 12 is a graph showing the rate of change of the slope and amplitude of the excitonic post-synaptic evoked potential during the 50-60 min administration of the mixed fermented golden powder.

In order to observe the effects of the mixed fermentation of golden chromosomes on the generation of excitatory post-synaptic induction potential in the hippocampal CA1 region, stimulation of the emissive layer with stimulation intensity of 40-50% And the change of excitatory post synaptic evoked potential was observed for 60 minutes.

The amplitude and the slope of the excitatory post - excitation potential were maintained steady for 60 min without the administration of the complex fermented powder.

However, when the mixed fermented powder was mixed with cerebrospinal fluid and the hippocampal slice was administered to the hippocampal slice, the voltage and slope of the excitatory post synaptic evoked potential began to increase from 5 minutes after administration and further increased with the lapse of time.

The voltage and slope of excitatory post synaptic induction potential did not increase up to 20 min after the administration of 7.5 ug / ml of mixed fermented powder of golden chromatin, but the slope of the excitatory post synaptic induction potential increased with the passage of time.

The voltage and slope of excitatory post - synaptic evoked potentials increased more rapidly than that of 7.5 ug / ml in the case of administration of 15 ug / ml of complex fermented powder.

However, the increase of voltage and slope of excitatory post - synaptic induction potential was lower than that of 15 ug / ml of mixed - fermented golden fermented powder.

(0 ug / ml) (n = 5) in the untreated fermented powder of golden - rhinoceros were compared with those of pre - fermented golden - (N = 5) of the group containing golden gonad mixed fermented powder (n = 5) was 103.8 ± 4.1% The fermented powder (25 ug / ml) group (n = 5) showed 152.7 ± 10.6%.

Therefore, the results of this experiment, in which the fermented powders of golden chromosomes increased the amplitude and slope of the excitatory post synaptic induction potential in the steady state, indicate that the golden chromatin complex fermented powder can increase the synaptic excitability in the hippocampal CA1 region.

FIG. 13 shows the results of the pretreatment of the golden chromosomal complex fermentation powder in the CA1 region of the hippocampal slice for 15 minutes, and the combination of 200μ hydrogen peroxide and the golden chromosome complex fermentation powder And the change of long-term synergism with the passage of time after administration.

Fig. 14 shows the results of a combination of 200 [mu] m hydrogen peroxide and a golden chromatin composite fermentation powder in the CA1 region of the hippocampal slice And the rate of change of long-term synergism by administration.

Two types of experiments were carried out in order to confirm the effect of the golden chromosome complex fermentation powder on inhibition of long - term synergism by 200μ hydrogen peroxide in the CA1 region. As a first experiment, the mixed fermented golden ginseng powder was pretreated for 15 minutes at each concentration, and then 200 .mu.M hydrogen peroxide and a combination of The changes in long-term synergism were observed.

Without pretreating the complex fermented powder of golden chromatin, 200μ hydrogen peroxide The long - term synergistic effect was similar to that of the control group in all experimental groups when pretreatment and concurrent administration of the golden - gamma complex fermented powder were compared with those of the control group.

After 7.5 .mu.g / ml of complex fermented powder of golden chromatin was pretreated for 15 minutes, 200 .mu.M hydrogen peroxide And 7.5μg / ml of the combined fermented powders of golden chromosomes, the long - term synergism was similar to that of the control group.

This phenomenon was also observed in the case of 15 ug / ml of the mixed fermented powder of golden thymus. Especially, when 25 ug / ml of mixed fermented powder of golden thymus were treated, the rate of increase of the long - term synergistic effect of the control group was increased.

Based on the slope of excitatory post-synaptic induction potential during the 50-60 min after the theta tear stimulation, the induction rate of long-term synergism was 179.0 ± 4.5% in the control group (n = 7), 200μ hydrogen peroxide The treatment group (n = 14) had 129.6 ± 6.8%, hydrogen peroxide In the group of 7.5 ug / ml administration of gold - chromosomal complex fermentation powder, 162.5 ± 13.2%, 178.0 ± 8.8% of 15ug / ml and 208.6 ± 15.1 of 25ug /

Fig. 15 shows the results of the pretreatment of 200 과 of hydrogen peroxide and 15 minutes of pre-treatment of the golden chromosomal complex fermentation powder in the CA1 region of the hippocampal slice, And the change of long-term synergism with the passage of time after administration.

16 is a graph showing the results of the measurement of the concentration of CaCO3 in the CA1 region of the hippocampal slice by pretreating 200 袖 g of hydrogen peroxide and 15 과 of hydrogen peroxide And the rate of change of long-term synergism with the passage of time after administration.

As a second experiment, the mixed fermented powder of golden yeast cultivars was treated with 200μ hydrogen peroxide After 15 minutes of pretreatment, long-term synergism was observed by administering only 200μ hydrogen peroxide.

When combined with 200μ hydrogen peroxide, The increase of the rate of long - term synergism was reduced by the addition of 25 ug / ml of the mixed fermented powder.

Based on the slope of excitatory post-synaptic induction potential during the 50-60 min after the theta tear stimulation, the induction rate of long-term synergism was 179.0 ± 4.5% in the control group (n = 7), 200μ hydrogen peroxide (N = 7) and the control group (n = 7) were 129.6 ± 6.8% (n = 7), while the group treated with 7.5 ug / ml of hydrogen peroxide ) Was 184.3 ± 6.8% and that of 25 ug / ml group was 211.2 ± 6.3%

Therefore, it was confirmed that the administration of the mixed fermented powder of golden yeast gum inhibits the suppression of synaptic plasticity by hydrogen peroxide.

17 is a graph showing the cell survival rate of PC12 cell damage caused by hydrogen peroxide by concentration of fermented golden chromosome complex extraction solution.

FIG. 18 is a photograph and analysis graph showing the effect of the fermented golden chromosome complex extraction solution against DNA damage in PC12 cell death process with hydrogen peroxide.

 To determine the optimal concentration of hydrogen peroxide, PC12 cells were cultured for 24 hours, and the hydrogen peroxide solution was treated for 50 minutes, 100, 150, and 200 μM, respectively, for 30 minutes, followed by washing and remaining in the culture for 24 hours MTT assay was performed and cells treated with hydrogen peroxide were used as a control.

In order to confirm the cytoprotective effect of the fermented golden chromatin complex extract solution, fermented golden chromatin complex extract solution was treated at 5, 10, 50, 100, 200 ㎍ / ml for 2 hours and 100 .mu.M hydrogen peroxide for 30 minutes After 24 hours of treatment and washing, cell viability was measured by MTT assay.

MTT assays were performed to determine the cell viability of PC12 cells. The MTT solution containing 4 ~ 6 / / ml MTT solution was mixed with the culture solution at a ratio of 1: 8~12 at 37 째 C for 3 hours, and the supernatant containing the MTT solution was removed. A solution of methylsulfoxide (DMSO, Dimethyl sulfoxide) was added to each test tube to dissolve the water-insoluble 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.

  To observe the death of PC12 cells after treatment with 100 μM hydrogen peroxide, DAPI staining was performed after 24 h of incubation in hydrogen peroxide.

The cultured cells were washed 2 to 3 times with PBS and fixed in 4% paraformaldehyde solution for 20 minutes.

  Washed again with PBS and DAPI-methanol (1 μg / ml) and stained with DAPI-methanol solution (Sigma, USA) for 15 min at 37 ° C. The stained cells were washed with methanol and observed under a fluorescence microscope.

MTT assay showed significant reduction of MTT reduction, ie, cell survival rate, as the concentration of hydrogen peroxide solution increased. The cell viability was 67.4 ± 4.8% in 100 μM hydrogen peroxide solution.

In order to confirm the effect of the fermented golden chromatin complex extract solution, the fermented golden chromatin complex extract solution was pre-treated for 2 hours at 5, 10, 50, 100, and 200 ㎍ / ml in PC12 cells. Then, 100 μM hydrogen peroxide was added for 30 minutes Treated, washed, left for 24 hours, and then cell viability was measured by MTT staining.

At this time, the decrease of cell survival rate due to oxidative damage caused by hydrogen peroxide was remarkably suppressed as the concentration of fermented golden chromatin complex extract increased.

The cell viability was increased to 85.8 ± 4.7 and 88.5 ± 5.5% at 50, 100 ㎍ / ml concentration of fermented golden chromatin complex extract solution, respectively.

  DNA fragmentation or DNA condensation was observed in the dead cells, and apoptosis-induced cell death was observed. The DNA fragmentation phenomenon was observed by treatment with the extract solution of fermented golden chromosome at a concentration of 100 μg / ml Cell viability was increased.

FIG. 19 is a TTC staining showing the dose-dependent effect of the fermented golden-chromosomal combination solution on ischemic brain injury in a stroke model experiment with a cerebral aortic occlusion.

20 is an analysis graph showing dose-dependent effects of the fermented golden-chromosomal complex extract solution in the ischemic injury of a stroke model model of middle cerebral artery occlusion.

The experimental animals Male Sprague-Dawley (SD) rats weighing 250-300 g were used, and the experimental group was divided into three groups: a control group in which physiological saline was administered, a fermented golden-chromosome extract solution in an amount of 100 mg per kg of body weight, And 150 mg / kg, respectively. The number of each experimental group was 7.

Fermented golden chromosome complex extraction solution (Golden Chunmeal compound extract fermentation broth) was diluted to 100 mg / ml in physiological saline. The fermented golden chromosome extract solution was orally administered to the experimental animals at a dose of 100 mg / kg and 150 mg / kg for 1 hour before induction of ischemia and at 3 and 12 hours after ischemia induction. The same amount of physiological saline was administered to the control group three times at the same time as the drug administration group.

The ischemic brain damage was assessed by TTC staining, and the effect of fermented golden chromosome complex extraction solution on ischemic brain injury was assessed by permanent ischemia of the middle cerebral artery in the experimental animals.

The experimental animal was anesthetized with isofluorene and fixed to the operating table with the supine position. Under the surgical microscope, the skin at the center of the anterior cervical region was dissected and the right common carotid artery and external carotid artery (ECA) , Respectively.

  The upper thyroid artery and the laryngeal artery, which are branches of the external carotid artery, were closed with sutures and the blood vessels were closed by electrocauterizing the pelvic root canal artery, which is the branch of the internal carotid artery. An external carotid artery branching from the common carotid artery was cut and a probe made of silicone-coated 4-0 nylon filament (model 403556pk10, Doccol Coperation, USA) was inserted into the internal carotid artery via the external carotid artery. 19 mm.

24 hours after middle cerebral artery occlusion (MCAO), experimental animals were sacrificed by intraperitoneal injection of an excess of Urethane anesthetic. The brain was carefully removed and placed on ice until the brain became slightly hard. The extracted brains were placed on a rat brain matrix (Harvard Bioscience, USA), and coronal slices were prepared in 2 mm thickness using a razor blade and placed in 24 well plates in the order of cut. 2% TTC, 2,3,5-triphenyltetrazolium chloride, Sigma, USA) was poured into a test tube with brain sections and stained for 30 min in an incubator at 37 ° C, fixed with 4% neutral formalin Respectively. Brain slices and grid paper stained with TTC were placed on a scanner (Taiwan) and images were output. The area and volume of the cerebral infarct were measured in the TTC stained brain slice images using the Image J Program (USA) program as follows.

Area (area, mm ² ) = (100 × pixel value of the measured area) / pixel value of 100 mm ² (grid paper)

Edema index = area of right hemisphere / area of left hemisphere

Corrected Cerebral Infarct Area = Measured Cerebral Infarct Area / Edema Index

Volume (volume, mm3) = Sum of corrected cerebral infarct areas × 2

The central part of the TTC staining cerebral cortex was severely affected in the striatum, frontal lobes, and parietal lobes, and the cells died in necrotic form. Cerebral infarction was also observed in the thalamus and hippocampus. The amount of ischemic damage expressed in white was decreased as the volume of fermented golden chromatin complex extract increased.

The volume of cerebral infarction in the control group administered with saline was 350.5 ± 26.8 mm ³ . The concentration of fermented golden chromosomal extract solution was 310.5 ± 18.5 mm ³ in the group treated with 100 mg per kg body weight, but the difference was not significant.

However, fermented golden-chromosomal complex extract solution of 252.2 ± 20.8 mm ³ in the experimental group treated with 150 mg / kg of body weight was significantly decreased compared with the control group.

Therefore, the oral administration of the extract solution of Fermented Golden Gamma showed the effect of inhibiting brain damage due to local cerebral ischemia due to vascular occlusion in experimental animals.

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 (2)

Fermenting the gold with yeast and enzyme complex fermentation broth;
Crushing, washing and mixing the fermented golden, chestnut, dermis, peel, and ginseng;
Adding the mixture to an extractor and preparing a complex extract using the extract;
Mixing the combined extract with a lactic acid bacterium and an enzyme complex fermentation broth and fermenting the mixture at a low temperature to produce an extract fermentation broth;
Concentrating the extracted fermentation broth under reduced pressure;
And spray-drying the reduced-pressure concentrated fermentation liquid to prepare a powder,
The powder inhibits oxidative nerve cell death by hydrogen peroxide,
The yeast may be any one or more of Aspergillus oryzae, Saccharomyces ellipsoideus and Saccharomyces cerevisiae,
Wherein the lactic acid bacterium is selected from the group consisting of Lactobacillus acidophilus, Streptococcus thermophilus, and Bifidobacterium longum. (Preparation of Chun - Ma Hybrid Extraction Powder). The method according to claim 1, wherein 50 to 100 parts by weight of ginseng, 50 to 100 parts by weight of ginseng, 50 to 100 parts by weight of ginseng, and 20 to 50 parts by weight of ginseng are contained (Method for the Preparation of Golden Chamaese Composite Extract Powder for Prevention and Improvement).

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