KR20130128677A - Composition made of the compounds extracted from morus alba for prevention and treatment of cerebropathia - Google Patents

Abstract

The present invention relates to a novel morusinon having the following characteristics by separating from mulberry and / or mountain mulberry and a method for separating the morusinon.
The present invention also relates to a composition for the prevention and treatment of degenerative brain diseases comprising components isolated from the bark and / or mulberry trees. More particularly, the present invention relates to a composition for preventing and treating degenerative brain diseases, Characterized in that it comprises at least one selected from Morusinon, Licoflavone C, Moracin P, Moracin O, Mulberrofuran Q, Moracenin D, Methyl 2,4-dihydroxybenzoate, Trans-oxyresveratrol, Mulberrofuran G and Chalcomoracin isolated from mulberry. And to compositions for preventing and treating diseases.
Morusinon: yellow powder, [[alpha]] _D -1.1 [deg.] ( C 0.11 MeOH); UV (MeOH)? _Max nm (log?) 228 (4.15), 287 (4.03), 339 (SH, 3.71); ESIMS m / z 459 [M - H] -, 919 [2M - H] - HRESIMS m / z 459.2024 (calcd for C 25 H 31 O 8, 459.2019).
_{^{1 H NMR (DMSO- d 6,}} 500 MHz): δ 12.2 (1H, s, OH-5), 8.31 (1H, s, OH-2 '), 8.29 (1H, s, OH-4'), 6.98 (1H, s, H-6 '), 5.88 (each 1H, s, H-6, H-8), 5.68 (1H, dd, H-2), 3.26 (1H, dd, H-3), 2.64 _{(2H, m, H 2 -1} "), 2.57 (1H, dd, H-3), 2.55 (2H, m, H 2 -1 '''), 1.58 (2H, m, H 2 -2'''), 1.53 (2H, m , H 2 -2 "), 1.14 (6H, s, H 3 -4", H 3 -5 "), 1.13 (6H, s, H 3 -4''', H ₃ -5 ''').
_{^{13 C NMR (DMSO- d 6,}} 125 MHz): δ 196.9 (s, C-4), 166.6 (s, C-7), 163.5 (each s, C-5, C-8a), 153.3 (s, C-4 '), 150.5 ( s, C-2'), 124.5 (d, C-6 '), 121.7 (s, C-5'), 118.5 (s, C-3 '), 117.2 (s, C-1 '), 101.7 ( s, C-4a), 95.7 (d, C-6), 95.0 (d, C-8), 74.2 (s, C-2), 69.4 (s, C-3 " ), 69.0 (s, C- 3 '''), 44.0 (t, C-2'''), 42.5 (t, C-2 "), 41.6 (s, C-3), 29.3 (each q, C-4 ", C-5 ", C-4 '''), 29.2 (q, C-5'''), 24.6 (t, C-1 '''), 18.4 (t, C-1 " ).

Description

TECHNICAL FIELD The present invention relates to a composition for preventing and treating cerebrospinal fluid including compounds isolated from mulberry trees. BACKGROUND OF THE INVENTION 1. Field of the Invention [0001]

The present invention relates to a composition for preventing and treating cerebral diseases including compounds isolated from mulberry. More particularly, the present invention relates to a compound isolated from mulberry, such as licoflavone C, moracin P, morasin O moracin O, morusinon, mulberrofuran Q, moracenin D, methyl-2,4-dihydroxybenzoate, Wherein the composition comprises at least one selected from trans-oxyresveratrol, mulberrofuran G, and chalcomoracin.

The present invention also relates to a novel compound, morusinon, which is represented by the following structural formula (1) among the compounds isolated from the above mulberry, and a method for its separation.

... 구조식(1)

... Structural formula (1)

With the exception of some countries since 2000, the aging of human beings has been progressing rapidly over the life expectancy of the world over the world.

This aging of humanity is particularly prominent in most advanced countries such as the United States and Europe. The aging of this population is an extension of human life due to the development of medicine, a decline in war, one of the causes of the decline of old natural population, There is a variety of reasons, including increased food production.

The aging of human beings shows various problems. Typically, there is an increasing disease according to the aging of a person. Representative examples of such diseases include ischemic stroke (cerebral ischemia), cognitive dysfunction, Alzheimer's disease (dementia), Parkinson's disease The number of patients with cranial nervous system diseases, a kind of geriatric diseases, has increased rapidly, and the social costs of treating geriatric brain diseases due to the aging of human beings have also increased astronomically.

However, most of the brain diseases of the elderly are known as degenerative senile cerebral diseases. Parkinson's disease, except for Alzheimer's disease, is not found in the elderly brain disease. , Stroke is the number one cause of death in Korea due to a single disease, but no clear treatment has been developed.

In the human body, all proteins produced from DNA to RNA are degraded at different times with different lifetimes, most of which is controlled by the ubiquitin-proteasome. If the degradation ability of the ubiquitin-proteasome against a specific protein is decreased, the amount of the protein relative to the specific protein is increased, and as a result, the function is promoted and the disease can be induced. Conversely, an increase in the proteolytic capacity of the ubiquitin-proteasome for a specific protein leads to a decrease in the amount of the protein, a decrease in function, and a decrease or disappearance of the protein.

Dysfunction of these ubiquitin-proteasome pathways has been known for many years in chronic degenerative neurological diseases such as idiopathic Alzheimer's disease (sporadic AD) and Parkinson's disease, and the fact that the metabolism of various proteins is not smooth due to these abnormalities is a major cause of degenerative changes .

It is suggested that ubiquitin-proteasome abnormality is closely related to Alzheimer's disease induced by accumulation of ubiquitin with phosphorylated Tau in the neurofibrillary tangle or senile plaque of Alzheimer's disease. Ubiquitin can not be activated by E1, so it can not be linked to proteins, but other ubiquitin is linked to ubiquitin and accumulates in the cell because there is no enzyme capable of cleaving ubiquitin effectively. The accumulated polyubiquitin ultimately blocks proteasome activity Leading to apoptosis.

The presence of ubiquitin was found not only in Alzheimer's disease but also in Down's syndrome, supra nuclear palsy, and Parkinson's disease. In addition, recently, abnormalities of genes involved in the ubiquitin-proteasome pathway have been found to be important in various diseases, and a new interpretation has been made about the pathological significance of these pathways.

There is a growing interest in the role of these pathways in chronic neurological diseases with the discovery of specific genes involved in the ubiquitin-proteasome pathway in Parkinson's disease or Alzheimer's disease. In particular, α-synuclein abnormalities inducing Parkinson's disease cause an abnormal α-synuclein that is not eliminated by the ubiquitin-proteasome pathway, resulting in an increase in intracellular α-synuclein levels compared with normal, resulting in dopaminergic Resulting in apoptosis of neuronal cells.

It has been shown that α-synuclein, a misfolded / unfolded protein in nature, accumulates in cells and degrades proteasome function. E6-associated protein (E6-AP), which is involved in the degradation of p53 by E6 oncoprotein (E3) ligase, is recently known to be a factor controlling ubiquitin ligase and promoting proteolysis in relation to proteasome. E6-AP plays a crucial role in the degradation of agglutinated a-synuclein, which has been shown to be an important factor in Parkinson's disease, as it has been shown that overexpressed E6-AP promotes the degradation of a-synuclein, which is toxic. In addition, Nurr1 has been shown to be an upper-level factor that suppresses and regulates the overexpression of α-synuclein, which plays a role in the maintenance of the homeostasis of dopamine in neurons. Dysfunctions of the ubiquitin-proteasome pathway in degenerative nervous system diseases have been known for a long time, but the molecular biology of the elements that make up this pathway has deepened, and the pathological significance has been re-emphasized again. It is possible to develop a novel approach to control the progression of degenerative nervous system diseases by regulating the function of these ubiquitin-proteasome pathways as a common mechanism for degenerative changes in neuronal cells in degenerative nervous system diseases .

Recent studies have identified and identified new E3 ligases that determine the specificity of the ubiquitin-proteasome pathway and identified mechanisms that control their function, ultimately controlling the intracellular levels of targeted proteins and their function We are concentrating on adjusting to the direction we want, which is important for the treatment of neurodegenerative diseases.

Many studies have already been made that abnormal protein expression and aggregation and accumulation of α-synuclein lead to degeneration of dopamine neurons by interfering with the function of basic cells. In this pathology, α-synuclein abnormality damages dopaminergic neurons in Parkinson's disease and induces the death of dopaminergic neurons.

The death of cells leading to the loss and damage of these neurons is the ultimate cause of degenerative nervous system brain diseases such as Parkinson's disease as well as senile dementia and stroke. For example, in the case of senile dementia, neurons in the cerebral cortex and hippocampus, which are involved in memory, cognition, etc., are thought to be lost, leading to major symptoms including memory loss.

Similarly, in the case of Parkinson's disease, dopaminergic neurons in the midbrain are selectively lost, resulting in dysfunction of the basal ganglia involved in motor function, resulting in motor dysfunction.

Cerebral ischemia is one of the types of strokes that occurs when the cerebral blood vessels become stuck or clogged and the blood supply to the brain is blocked and oxygen and nutrient deficiencies occur. Among human organs, the brain is an organ that can not produce or store energy on its own, and is fully dependent on blood supply to receive energy. Therefore, if the blood supply is not smooth, it will be immediately damaged. In general, 70-80% of cerebral ischemia has been reported to cause partial or total damage of brain function due to temporary interruption of blood supply due to clogging of the thrombus and rapid blood supply due to instantaneous thrombectomy. Such direct brain cell damage induces apoptosis, and behavioral disorders, movement disorders, and cognitive disorders also occur depending on the site and degree of brain damage. In other words, such blood supply interruption may cause various disorders due to general brain tissue damage as well as ischemic stroke.

The model of induction of apoptosis through oxygen-glucose deprivation (OGD) induces an ischemic state similar to that of blood supply interruption, and induces brain nerve cell death by ischemic conditions.

In the present invention, human neuroblastoma SH-SY5Y cells were artificially stopped from supplying glucose and serum, and an oxygen-deficient environment was created to induce apoptosis. OGD-induced apoptosis is a method of inducing oxidative stress in cells by temporarily interrupting the supply of nutrients and oxygen, and then inducing cell death by re-supplying nutrients and oxygen. In the environment that reflects actual clinical cerebral ischemia . Active oxygen species can directly induce cellular damage, or indirectly cause cell damage through a signaling system. These reactive oxygen species play a pivotal role in many central nervous system disorders, including stroke, and in a variety of degenerative brain diseases. Especially, superoxide anion plays an important role in pathophysiological mechanism of cerebral ischemia by being involved in the activation of apoptosis signal transduction system. Therefore, we sought to find a substance that blocks ultimate apoptosis in the brain ischemic cell model and blocks reactive oxygen species directly or indirectly affecting it.

Morus alba is a deciduous broad-leaved arboreous tree belonging to Moraceae. Mulberry is used as a treatment for neuralgia, antipyretic, tonic, diuretic and hypertensive treatment in one room, and it is known that recent studies promote recovery due to physical stress. However, the substances isolated and extracted from mulberry trees such as alfalfa and mountain mulberry have not been studied in cerebral ischemia or Parkinson's disease, nor have they been related to the neuroprotective effect or to the proteolytic mechanism.

In the present invention, the compounds isolated from mulberry have a protective effect against neurons and overexpression of α-synuclein, and confirms E6-AP, Nurr1 protein, protein aggregation inhibitory effect, inhibition of ubiquitin accumulation and proteasome activity And thus it is considered to be effective as a preventive and / or therapeutic agent for brain diseases such as Parkinson's disease. In addition, Dementia with Lewy bodies, α-synuclein-related disease, Multiple-system atrophy, Alzheimer's disease with common pathology with Parkinson's disease related to ubiquitin-proteasome-related protein degradation dysfunction It is expected to have effects on diseases such as amyotrophic lateral sclerosis, Down's syndrome and supranuclear palsy as well as on brain ischemia and senile dementia, stroke and memory loss caused by the disappearance of nerve cells by inhibiting the production of reactive oxygen species can do.

As a prior art related to the present invention, Korean Patent Laid-Open Publication No. 2009-0082154 promotes nerve cell death and / or neuronal cell generation to prevent or treat neuropsychiatric diseases, particularly brain diseases, The present invention relates to a composition for preventing or treating neurological diseases, particularly brain diseases, and a composition for improving cognitive function, which comprises stanniocalcin 2 as an active ingredient.

Korean Patent Publication No. 2011-0065151 also discloses a pharmaceutical composition for treating amelioration and degenerative brain diseases, which contains an extract of Aspergillus oryzae as a mature fruit of Crataegus pinnatifida Bunge belonging to Rosaceae as an active ingredient, .

Korean Patent Publication No. 2012-0007139 discloses a composition for protecting the brain, a composition for preventing or treating degenerative brain disease or ischemic brain disease, or a composition for treating a traumatic central nerve injury, which contains carbon nanotubes as an effective ingredient.

However, the present invention differs from the technical features of the prior arts described above, and the inventions are different from each other in composition.

It is an object of the present invention to provide a composition for preventing and treating brain diseases, which comprises a compound isolated from mulberry.

Another object of the present invention is to provide a novel compound, morusinon, a method for its separation, and a novel compound, morusinone, And to provide a composition for preventing and treating diseases.

A composition for preventing and treating cerebral diseases comprising a compound isolated from mulberry of the present invention is a food composition for preventing and treating cerebral diseases and / or a pharmaceutical composition comprising a pharmaceutically acceptable excipient and / or a pharmaceutically acceptable excipient Can be provided.

... 구조식(1)

... Structural formula (1)

The present invention relates to a compound isolated from mulberry, wherein the compound is licoflavone C, moracin P, moracin O, morusinon, mulberrofuran Q, But are not limited to, moracinin D, methyl-2,4-dihydroxybenzoate, trans-oxyresveratrol, mulberrofuran G, and chalcocoracin. The present invention also provides a composition for preventing and treating brain diseases.

The present invention relates to a novel compound, morusinon, which is isolated from mulberry and is represented by the following structural formula (1), and a method for its isolation and prevention of brain diseases including morusinone, a novel compound isolated from the mulberry A therapeutic composition can be provided.

... 구조식(1)

... Structural formula (1)

According to the present invention, morusinon, which is a novel compound represented by the following structural formula (1), can be obtained by separating from mulberry, and a compound, licoflavone C ), Moracin P, moracin O, mulberrofuran Q, moracenin D, methyl 2,4-dihydroxybenzoate (methyl 2,4 the present invention can provide a composition for preventing and treating brain diseases comprising at least one selected from the group consisting of dihydroxybenzoate, trans-oxyresveratrol, mulberrofuran G, and chalcomoracin.

The composition for preventing and treating brain diseases according to the present invention may include a food-acceptable excipient in addition to a compound isolated from mulberry, thereby providing a food composition for preventing or treating brain diseases.

The composition for preventing and treating brain diseases according to the present invention may further comprise a pharmaceutically acceptable excipient in addition to the compound isolated from mulberry, thereby providing a pharmaceutical composition for preventing or treating brain diseases.

... 구조식(1)

... Structural formula (1)

FIG. 1 shows the results of a single component (a) Moracinin D (b) Moracin P and dopamine treatment for 24 hrs, followed by staining with Propidium Iodide (PI) to determine apoptosis will be.
FIG. 2 shows the protective effect of OGD-induced cell death in SH-SY5Y cells treated with mulberrofuran G, a single component of mulberry, and carnosine as a positive control, through MTT assay (** p <0.001 versus non- treated control, ## p <0.001, #p <0.05 OGD-treated control).
FIG. 3 shows the results of OGD and reoxygenation of SH-SY5Y cells treated with mulberrofuran G, a positive control substance of mulberry, and carnosine as a positive control, and the incidence of intracellular ROS (%) using DCF-DA ** p <0.001 versus non-treated control, ## p <0.001, #p <0.05 OGD-treated control).
FIG. 4 shows the results of simultaneous treatment of the SH-SY5Y cells with concentrations of 600 μM dopamine, 0.16 μM, 0.8 μM and 4 μM of (a) Moracinin D (b) .
FIG. 5 shows the results of a Western blot analysis of α-synuclein, E6-AP, and ubiquitin after extracting proteins from SH-SY5Y cells treated with 600 μM of dopamine, 0.16 μM, 0.8 μM, 4 μM of Moracinin D or Moracin P for 15 hours will be.
FIG. 6 shows RT-PCR of α-synuclein and Nurr1 expression of mRNA after treatment with 600 μM, 0.16 μM, 0.8 μM and 4 μM of Moracenin D for SH-SY5Y cells for 9 hours.
FIG. 7 shows the results of western blot analysis of α-synuclein and Nurr1 expression by extracting proteins after treating dopamine 600 μM, 0.16 μM, 0.8 μM and 4 μM of Moracenin D for 15 hours in SH-SY5Y cells.
FIG. 8 shows the results of simultaneous treatment of 600 μM of dopamine, 0.16 μM, 0.8 μM and 4 μM of Moracenin D in the SH-SY5Y cells for 24 hours, and the degree of proteasome activity was measured and quantified by fluorescence staining.

The present invention represents a composition for preventing and treating brain diseases.

The present invention relates to a composition for preventing and treating brain diseases, which comprises a compound isolated from mulberry.

The present invention relates to a composition for preventing and treating cerebral diseases, which comprises a compound isolated from mulberry (licoflavone C, moracin P, moracin O) represented by the following structural formula (1) Morusinon, mulberrofuran Q, moracenin D, methyl-2,4-dihydroxybenzoate, trans-oxygen resveratrol trans-oxyresveratrol, mulberrofuran G, and chalcomoracin. The present invention also relates to a composition for preventing and treating brain diseases.

... 구조식(1)

... Structural formula (1)

Of the compounds isolated from the above mulberry, moruscinone has the following characteristics.

Morusinon: yellow powder, [[alpha]] _D -1.1 [deg.] ( C 0.11 MeOH); UV (MeOH)? _Max nm (log?) 228 (4.15), 287 (4.03), 339 (SH, 3.71); ESIMS m / z 459 [M - H] -, 919 [2M - H] - HRESIMS m / z 459.2024 (calcd for C 25 H 31 O 8, 459.2019).

_{^{1 H NMR (DMSO- d 6,}} 500 MHz): δ 12.2 (1H, s, OH-5), 8.31 (1H, s, OH-2 '), 8.29 (1H, s, OH-4'), 6.98 (1H, s, H-6 '), 5.88 (each 1H, s, H-6, H-8), 5.68 (1H, dd, H-2), 3.26 (1H, dd, H-3), 2.64 _{(2H, m, H 2 -1} "), 2.57 (1H, dd, H-3), 2.55 (2H, m, H 2 -1 '''), 1.58 (2H, m, H 2 -2'''), 1.53 (2H, m , H 2 -2 "), 1.14 (6H, s, H 3 -4", H 3 -5 "), 1.13 (6H, s, H 3 -4''', H ₃ -5 ''').

_{^{13 C NMR (DMSO- d 6,}} 125 MHz): δ 196.9 (s, C-4), 166.6 (s, C-7), 163.5 (each s, C-5, C-8a), 153.3 (s, C-4 '), 150.5 ( s, C-2'), 124.5 (d, C-6 '), 121.7 (s, C-5'), 118.5 (s, C-3 '), 117.2 (s, C-1 '), 101.7 ( s, C-4a), 95.7 (d, C-6), 95.0 (d, C-8), 74.2 (s, C-2), 69.4 (s, C-3 " ), 69.0 (s, C- 3 '''), 44.0 (t, C-2'''), 42.5 (t, C-2 "), 41.6 (s, C-3), 29.3 (each q, C-4 ", C-5 ", C-4 '''), 29.2 (q, C-5'''), 24.6 (t, C-1 '''), 18.4 (t, C-1 " ).

Morusachenone represented by the above structural formula (1) can be isolated from the mulberry bark.

Among the compounds isolated from the above mulberry, licoflavone C (see the following structural formula (2)) has the following characteristics.

licoflavone C: Pale yellow needles; UV (MeOH)? _Max (log?) 274 (4.24), 305 (4.12), 326 (4.15); ESIMS m / z 337 [M - H] -.

_{^{1 H NMR (acetone- d 6 400}} MHz): δ 13.0 (1H, s, OH-5), 9.40 (2H, br s, OH-7, OH-4 '), 7.96 (2H, d, H-2 ', H-6'), 7.05 (2H, d, H-3 ', H-5'), 6.63 (1H, s, H-3), 6.34 (1H, s, H-6), 5.30 (1H , br t, H-2 " ), 3.57 (2H, d, CH 2 -1"), 1.82 (3H, s, Me-4 "), 1.67 (3H, s, Me-5").

_{^{13 C NMR (acetone- d 6,}} 100 MHz): δ 183.5 (s, C-4), 165.0 (d, C-2), 162.2 (s, C-7), 161.9 (s, C-4 ') , 161.0 (s, C-5 ), 156.0 (s, C-9), 132.1 (s, C-3 "), 129.3 (d, C-2 'C-6'), 123.7 (d, C-1 '), 123.6 (d, C -2 "), 116.9 (d, C-3', C-5 '), 107.4 (s, C-10), 105.4 (s, C-8), 103.9 (d, C-3), 99.3 (d , C-6), 25.8 (q, C-4 "), 22.4 (t, C-1"), 18.1 (q, C-5 ")

...구조식(2)

... Structural formula (2)

Among the compounds isolated from the above mulberry, moracin P (see the following structural formula (3)) has the following characteristics.

moracin P: brown powder; UV (MeOH)? _Max (log?) 219 (3.88), 321 (3.92), 338 (3.89); ESIMS m / z 325 [M - H] -.

_{^{1 H NMR (acetone- d 6,}} 500 MHz): δ 7.26 (1H, s, H-4), 7.00 (1H, s, H-3), 6.87 (1H, s, H-7), 6.86 (each 1H, d, H-2 ' , H-6'), 6.37 (1H, br s, H-4 '), 3.82 (1H, dd, H-2 "), 3.10 (1H, dd, H-1" ), 2.83 (1H, dd , H-1 "), 1.37 (1H, s , H-5"), 1.26 (1H, s , H-4 ").

_{^{13 C NMR (acetone- d 6,}} 125 MHz): δ 159.9 (each s, C-3 ', C-5'), 156.0 (s, C-7a), 155.5 (s, C-2), 152.5 ( s, C-6), 133.1 (s, C-1 '), 123.6 (s, C-3a), 121.8 (d, C-4), 118.0 (s, C-5), 103.9 (each d, C -2 ', C-6') , 103.6 (d, C-4 '), 101.9 (d, C-3), 99.5 (d, C-7), 78.1 (s, C-3 "), 70.0 ( d , C-2 "), 32.5 ( t , C-1"), 26.3 ( q , C-4 "), 20.6 ( q , C-5").

...구조식(3)

... Structural formula (3)

Among the compounds isolated from the above mulberry, moracin O (see the following structural formula (4)) has the following characteristics.

moracin O: brown powder; UV (MeOH)? _Max (log?) 219 (3.54), 320 (3.59), 335 (3.55); ESIMS m / z 325 [M - H] -.

_{^{1 H NMR (acetone- d 6,}} 500 MHz): δ 7.33 (1H, s, H-4), 7.01 (1H, s, H-7), 6.84 (1H, s, H-3), 6.84 (each 1H, d, H-2 ' , H-6'), 6.37 (1H, br s, H-4 '), 4.68 (1H, t, H-2 "), 3.31 (1H, dd, H-1" ), 3.20 (1H, dd , H-1 "), 1.26 (1H, s , H-4"), 1.23 (1H, s , H-5 ").

_{^{13 C NMR (acetone- d 6,}} 125 MHz): δ 159.9 (each s, C-3 ', C-5'), 159.8 (s, C-2), 155.9 (s, C-6), 155.7 ( s, C-7a), 133.5 (s, C-1 '), 125.3 (s, C-3a), 123.4 (s, C-5), 117.0 (d, C-4), 103.7 (each d, C -2 ', C-6') , 103.4 (d, C-4 '), 102.5 (d, C-3), 93.1 (d, C-7), 91.3 (d, C-2 "), 71.5 ( s , C-3 "), 30.8 ( t , C-1"), 26.1 ( q , C-4 "), 25.6 ( q , C-5").

...구조식(4)

... Structural formula (4)

Among the compounds isolated from the above mulberry, mulberrofuran Q (see the following structural formula (5)) has the following characteristics.

mulberrofuran Q: Reddish violet powder; UV (MeOH)? _Max (log?) 222 (4.41), 280 (4.15), 285 (SH, 4.08), 321 (4.43), 335 (SH 4.29); ESIMS m / z 591 [M - H] -.

_{^{1 H NMR (acetone- d 6,}} 300 MHz): δ 7.43 (1H, d, H-4), 7.37 (1H, d, H-14 "), 7.00 (1H, br s, H-3), 6.92 (1H, br s, H- 7), 6.86 (1H, br s, H-6 '), 6.79 (1H, dd, H-5), 6.67 (1H, d, H-20 "), 6.63 (1H , br s, H-2 ' ), 6.54 (1H, dd, H-13 "), 6.36 (1H, d, H-17"), 6.35 (1H, dd, H-19 "), 6.13 (1H, d, H-11 "), 3.50 (1H, s, H-3"), 2.81 (1H, br s, H-5 "), 2.73 (1H, dd, H-6"), 1.88 (1H, dd , H-6 "), 1.73 (3H, s , H-7").

_{^{13 C NMR (acetone- d 6,}} 100 MHz): δ 195.8 (s, C-2 "), 172.9 (s, C-12"), 166.8 (s, C-10 "), 160.9 (s, C- 3 '), 158.8 (s, C-2), 156.6 (s, C-18 ", C-7a), 155.8 (s, C-16"), 155.4 (s, C-5'), 155.3 (s , C-6), 134.4 ( d, C-14 "), 132.6 (s, C-1 '), 126.5 (d, C-20"), 122.5 (s, C-3a), 121.9 (d, C -4), 114.8 (s, C -9 "), 113.4 (s, C-15"), 113.2 (d, C-5), 112.2 (d, C-19 "), 110.4 (s, C-4 '), 109.6 (s, C -8 "), 107.7 (d, C-6'), 105.2 (d, C-2 '), 103.1 (d, C-13"), 102.2 (d, C-3 ), 988.9 (d, C- 7), 98.6 (d, C-17 "), 98.3 (d, C-11"), 92.5 (s, C-4 "), 75.5 (s, C-1") , 49.1 ( d , C-3), 37.9 ( d , C-5 "), 31.0 ( t , C-6"), 22.5 ( q , C-7 ").

...구조식(5)

... Structural formula (5)

Among the compounds isolated from the above mulberry, moracenin D (see the following structural formula (6)) has the following characteristics.

moracenin D: Brown powder; UV (MeOH)? _Max (log?) 209 (4.79), 230 (sh 4.52), 265 (4.51), 280 (sh 4.27), 316 (4.13); ESIMS m / z 709 [M - H] -.

_{^{1 H NMR (acetone- d 6,}} 500 MHz): δ 12.82 (1H, s, OH-5 or OH-2 "), 12.80 (1H, s, OH-5 or OH-2"), 7.50 ~ 9.60 ( 6H, OH × 6), 7.40 (1H, d, H-6 "), 7.30 (1H, d, H-6 '), 6.78 (1H, d, H-20"), 6.64 (1H, d, H -3 '), 6.56 (1H, dd, H-5'), 6.20 (1H, d, H-17 "), 6.06 (1H, dd, H-19"), 6.00 (1H, s, H-6 ), 5.98 (1H, d, H-3 "), 5.94 (1H, dd, H-5"), 5.16 (1H, br s, H-10 "), 4.65 (1H, t, H-8") , 4.37 (1H, br d, H-9 "), 3.40 ~ 3.90 (1H, m, H-14"), 2.54 (2H, t, H-11), 1.80 ~ 2.20 (2H, m, H-13 ), 1.72 (2H, t , H-12), 1.50 (3H, br s , H-12 "), 1.14 (6H, s , H-15, H-14).

_{^{13 C NMR (acetonitrile- d 3,}} 125 MHz): δ 208.8 (s, C-7 "), 182.5 (s, C-4), 164.7 (s, C-4"), 164.0 (s, C-2 "), 161.0 (s, C -4 '), 160.2 (s, C-2'), 160.0 (s, C-7), 160.0 (s, C-9), 156.3 (s, C-2), 155.8 (s, C-16 " ), 155.8 (s, C-18"), 154.9 (s, C-5), 133.2 (s, C-11 "), 132.7 (d, C-6"), 131.2 (d, C-6 ') , 128.8 (d, C-20 "), 122.9 (d, C-10"), 121.5 (s, C-15 "), 121.5 (s, C-3), 114.5 ( s, C-1 "), 112.4 (s, C-1 '), 107.3 (d, C-5"), 107.1 (d, C-5'), 107.0 (s, C-8), 107.0 (d , C-19 "), 104.7 (s, C-10), 102.8 (d, C-3"), 102.6 (d, C-3 '), 102.0 (d, C-17 "), 97.5 (d, C-6), 70.2 (s , C-13), 47.0 (d, C-8 "), 41.9 (t, C-12), 37.6 (d, C-9"), 37.6 (d, C-14 "), 37.1 (t, C -13"), 28.2 (q, C-14), 28.1 (q, C-15), 22.0 (q, C-12 "), 20.0 (t, C-11).

...구조식(6)

... Structural formula (6)

Among the compounds isolated from the above mulberry, methyl 2,4-dihydroxybenzoate (see the following structural formula (7)) has the following characteristics.

methyl 2,4-dihydroxybenzoate: White powder. TOF-MS m / z: 167 [M + H] - = 168.

_{^{1 H-NMR (methanol- d 4}} , 500 MHz): δ 3.92 (3H, s, OMe), 6.33 (1H, d, J = 2.0 ㎐, H-3), 6.37 (1H, dd, H-5) , 7.71 (1H, d, H-6).

_{^{13 C-NMR (methanol- d 4}} , 125 MHz): δ 5.89 (s, OMe), 102.03 (s, C-3), 104.12 (s, C-1), 107.68 (s, C-5), 131.25 ( s , C-6), 163.51 ( s , C-2), 164.28 ( s , C-4), 170.35 ( s , C-7).

...구조식(7)

... Structural formula (7)

Among the compounds isolated from the above mulberry, trans-oxyresveratrol (see the following structural formula (8)) has the following characteristics.

trans-oxyresveratrol: Blue amorphous powder. TOF-MS m / z: 561 [M + H] - = 562.

_{^{1 H-NMR (methanol- d 4}} , 500 MHz): δ 1.82 (3H, s, H-7 "), 2.05 (1H, t, H-6"), 2.69 (1H, d, H-6 ") , 2.98 (1H, m, H -4 "), 3.36 (2H, m, H-5", H-3 "), 6.17 (1H, dd, H-13"), 6.34 (1H, d, H- 17 "), 6.37 (1H, d, H-11"), 6.44 (1H, br s, H-2 "), 6.48 (1H, dd, H-19"), 6.76 (1H, dd, H-5 ), 6.85 (1H, br s , H-6 '), 6.94 (3H, m, H-2', H-3, H-7), 7.12 (1H, d, H-20 "), 7.16 (1H , dd, H-14 "), 7.37 (1H, d, H-14").

_{^{13 C-NMR (methanol- d 4}} , 125 MHz): δ 24.05 (s, C-7 "), 28.99 (s, C-4"), 35.57 (s, C-5 "), 36.88 (s, C -6 "), 37.80 (s, C-3"), 98.67 (s, C-7), 102.29 (s, C-3), 103.24 (s, C-8 "), 104.32 (s, C-11 "), 104.70 (s, C -17"), 105.17 (s, C-2 '), 105.64 (s, C-6'), 107.16 (s, C-13 "), 110.19 (s, C-19 "), 113.39 (s, C -9"), 114.08 (s, C-5), 117.52 (s, C-15 "), 118.41 (s, C-4 '), 122.11 (s, C-4) , 123.26 (s, C-3a ), 123.51 (s, C-2 "), 128.14 (s, C-20"), 130.72 (s, C-14 "), 131.69 (s, C-1 '), 134.07 (s, C-1 " ), 153.75 (s, C-16"), 155.07 (s, C-18 "), 155.89 (s, C-6), 156.94 (s, C-2), 157.40 ( s , C-5 '), 157.90 ( s , C-3'), 158.05 ( s , C-7a), 158.54 ( s , C-10 "), 160.20 ( s , C-12").

...구조식(8)

... Structural formula (8)

Among the compounds isolated from the above mulberry, mulberrofuran G (see the following structural formula (9)) has the following characteristics.

mulberrofuran G: Yellowish amorphous powder. TOF-MS m / z: 647 [M + H] - = 648.

^{1 H-NMR (500 ㎒,} MeOD): δ 1.64 (3H, s, H-24 "), 1.75 (3H, s, H-25"), 1.95 (3H, s, H-7 "), 2.20 ( 1H, br d, H-6 "), 2.49 (1H, br d, H-6"), 3.26 (2H, t, H-21 "), 3.75 (1H, m, H-5"), 4.10 ( 1H, s, H-3 " ), 4.60 (1H, t, H-4"), 5.20 (1H, m, H-22 "), 5.76 (1H, s, H-2"), 6.26 (1H, dd, H-19 "), 6.38 (2H, m, H-3, 13"), 6.74 (3H, m, H-5, H-2 ', H-6'), 6.85 (1H, d, H -17 "), 6.88 (1H, d, H-7), 6.96 (1H, d, H-20"), 7.33 (1H, d, H-4), 8.37 (1H, d, H-14 ") .

^{13 C-NMR (500 ㎒,} MeOD): δ 18.02 (s, C-25 "), 22.58 (s, C-21"), 23.95 (s, C-7 "), 26.04 (s, C-24" ), 33.07 (s, C- 6 "), 33.87 (s, C-3"), 37.22 (s, C-5 "), 48.46 (s, C-4"), 98.58 (s, C-7) , 101.90 (s, C-3 ), 103.64 (s, C-17 "), 105.06 (s, C-2 ', C-6'), 107.38 (s, C-19"), 108.17 (s, C -13 "), 113.25 (s, C-5), 113.90 (s, C-9"), 116.32 (s, C-11 "), 117.25 (s, C-4 '), 121.97 (s, C- 4), 122.92 (s, C -15 "), 123.29 (s, C-3a), 123.75 (s, C-22"), 125.08 (s, C-2 "), 129.11 (s, C-20" ), 131.53 (s, C- 1 '), 131.98 (s, C-23 "), 132.66 (s, C-14"), 134.29 (s, C-1 "), 156.23 (s, C-6) , 156.78 (s, C-16 ", 18"), 157.21 (s, C-7a), 157.31 (s, C-2), 157.93 (s, C-3 '), 158.11 (s, C-5' ), 163.96 ( s , C-12 "), 164.74 ( s , C-10"), 210.33 ( s , C-8 ").

...구조식(9)

... (9)

Among the compounds isolated from the above mulberry, chalcomoracin (see the following structural formula (10)) has the following characteristics.

chalcoloracin: Yellowish needles. TOF-MS m / z : 579 [M + H] ^- , 581 [M + H] &lt; ⁺ &gt; = 580.

^{1 H-NMR (500 ㎒,} MeOD): δ 1.82 (3H, s, H-7 "), 2.23 (1H, d, H-6"), 2.46 (1H, d, H-6 "), 3.75 ( 1H, s, H-3 " ), 4.11 (1H, s, H-5"), 4.59 (1H, t, H-4 "), 5.75 (1H, s, H-2"), 6.21 (1H, d, H-11 "), 6.26 (1H, dd, H-19"), 6.35 (1H, dd, H-13 "), 6.38 (1H, d, H-7"), 6.74 (3H, m, H-5, H-2 ' , H-6'), 6.85 (1H, s, H-3), 6.88 (1H, d, H-7), 6.96 (1H, d, H-20 "), 7.33 (1H, d, H-4), 8.46 (1H, d, H-14 ").

^{13 C-NMR (500 ㎒,} MeOD): δ 23.98 (s, C-7 "), 33.24 (s, C-6"), 33.93 (s, C-3 "), 37.04 (s, C-5" ), 48.57 (s, C- 4 "), 98.61 (s, C-7), 101.93 (s, C-3), 103.68 (s, C-11"), 103.72 (s, C-17 "), 105.04 (br s, C-2 ', C-6'), 107.43 (s, C-19 "), 109.09 (s, C-13"), 113.30 (s, C-5), 114.21 (s, C -4 '), 117.11 (s, C-9 "), 122.01 (s, C-4), 122.91 (s, C-3a), 123.31 (s, C-15"), 125.03 (s, C-2 "), 129.19 (s, C -14"), 131.59 (s, C-1 '), 134.48 (s, C-1 "), 135.60 (s, C-20"), 156.26 (s, C-6 ), 156.84 (s, C- 18 "), 157.23 (s, C-2), 157.36 (br s, C-3 ', C-5'), 158.00 (s, C-16"), 158.18 (s , C-7a), 166.71 ( s , C-12 "), 167.14 ( s , C-10"), 210.20 ( s , C-8 ").

...구조식(10)

... Structural formula (10)

In the above, the compound isolated from mulberry comprises 0.1 to 99.9% by weight based on the total weight of the composition.

In the above, the compound isolated from mulberry comprises 10 to 50% by weight based on the total weight of the composition.

In the above, the composition for preventing and treating cerebral diseases may be a food composition containing a food-acceptable ingredient in addition to the compound isolated from mulberry.

The composition for the prevention and treatment of cerebral diseases includes a powder, a round, a granule, a tablet, a capsule (hereinafter, referred to as &quot; capsule, beverage, and the like.

The composition for preventing and treating brain diseases may be a pharmaceutical composition containing pharmaceutically acceptable excipients in addition to the compound isolated from mulberry.

The composition for preventing and treating cerebral diseases may be a powder, a pill, a granule, a tablet, a capsule, or the like so as to contain a pharmaceutically acceptable excipient in addition to the compound isolated from mulberry capsule, liquid, and the like.

The composition for preventing and treating cerebral diseases may be a pharmaceutical composition formulated with liquid injections so as to include pharmaceutically acceptable excipients in addition to compounds isolated from mulberry.

The pharmaceutically acceptable excipient and / or pharmaceutically acceptable excipient used in the composition for the prevention and treatment of brain diseases as described above may be prepared by mixing the excipients and / or excipients conventionally used in foods and / It is sufficient for the person having the knowledge of the present invention to be able to select and use it, so that details of such food acceptable ingredients and / or pharmaceutically acceptable excipients will be omitted.

The present invention includes morusinon, a novel compound separated from mulberry and represented by the following structural formula (1).

The present invention includes morusinon which is a novel compound which is separated from the mulberry white mulberry and is represented by the following structural formula (1).

The present invention includes a morusinon which is a novel compound represented by the following structural formula (1), which is isolated from the mulberry white mulberry and can be used as a component of a composition for preventing and treating brain diseases .

... 구조식(1)

... Structural formula (1)

Morusinon, which is a novel compound, which is separated from the mallow's morus alba and is represented by the structural formula (1), may have the following characteristics.

Morusinon: yellow powder, [[alpha]] _D -1.1 [deg.] ( C 0.11 MeOH); UV (MeOH)? _Max nm (log?) 228 (4.15), 287 (4.03), 339 (SH, 3.71); ESIMS m / z 459 [M - H] -, 919 [2M - H] - HRESIMS m / z 459.2024 (calcd for C 25 H 31 O 8, 459.2019).

_{^{1 H NMR (DMSO- d 6,}} 500 MHz): δ 12.2 (1H, s, OH-5), 8.31 (1H, s, OH-2 '), 8.29 (1H, s, OH-4'), 6.98 (1H, s, H-6 '), 5.88 (each 1H, s, H-6, H-8), 5.68 (1H, dd, H-2), 3.26 (1H, dd, H-3), 2.64 _{(2H, m, H 2 -1} "), 2.57 (1H, dd, H-3), 2.55 (2H, m, H 2 -1 '''), 1.58 (2H, m, H 2 -2'''), 1.53 (2H, m , H 2 -2 "), 1.14 (6H, s, H 3 -4", H 3 -5 "), 1.13 (6H, s, H 3 -4''', H ₃ -5 ''').

_{^{13 C NMR (DMSO- d 6,}} 125 MHz): δ 196.9 (s, C-4), 166.6 (s, C-7), 163.5 (each s, C-5, C-8a), 153.3 (s, C-4 '), 150.5 (s, C-2'), 124.5 (d, C-6 '), 121.7 C-1 '), 101.7 (s, C-4a), 95.7 (d, C-6), 95.0 ), 69.0 (s, C-3 '''), 44.0 (t, C-2' C-4 ", C-5", C-4 '"), 29.2 (q, C-5' ).

The present invention relates to a method for separating morusinon from mulberry, which is represented by the following structural formula (1) and which is a novel compound.

The present invention relates to a method for separating morusinon, which is a novel compound, represented by the following structural formula (1) from the morus alba of Mulberry.

The present invention relates to a method for separating morusinon, which is a novel compound, represented by the following structural formula (1) from the morus alba of Mulberry.

The present invention relates to a method for producing a methanol extract, which comprises extracting a mulberry bark from methanol at room temperature (20 to 25 ° C) to obtain a methanol extract; Separating the methanol extract from which the solvent was removed from the methanol extract of Corynebacterium with the distilled water, a mixed solvent of hexane and ethyl acetate (distilled water, hexane, ethyl acetate: 1: 1: 1, v / v); An aliquot of the ethyl acetate layer was gradually added dropwise to the methanol mixture (chloroform: methanol ratio 100: 0 to 0: 100, v: 1: 1 v / v) while gradually decreasing the amount of chloroform in the mixed solvent of chloroform: methanol / v) finally obtaining 12 fractions by collecting with increasing polarity, such as using only methanol; The seventh fraction obtained from the above 12 fractions was fractionated by reverse phase chromatography using acetonitrile and distilled water (acetonitrile: distilled water = 20 to 100: 80 to 0, v / v) to obtain nine fractions ; The third fraction obtained from the above nine fractions was characterized by comprising the step of obtaining morusinon using a reversed-phase HPLC on a mixed solvent of methanol and distilled water (55 vol% methanol and 45 vol% distilled water) And a method for separating morusinon, which is a novel compound, from mulberry.

In the above, the methanol extract of Mulberry can be obtained by adding 15 to 50 liters of methanol to 10 kg of the mulberry macaque and then performing the extraction for 2 to 5 times at room temperature of 20 to 25 ° C for 12 to 24 hours.

In the above, the methanol extract of Cowpea can be obtained by adding 30 liters of methanol to 10 kg of mulberry macaque and extracting it for 24 hours at 25 ° C for 3 times.

An aliquot of the ethyl acetate layer was gradually added to a mixture of chloroform: methanol (1: 0 v / v) (chloroform: methanol: v: 100: 0 to v: / v) 12 fractions can be obtained by using the following mixed solvents in the step of obtaining 12 fractions by increasing the polarity such as methanol only.

(1) A mixed solvent of chloroform: methanol (100: 0, v / v)

(2) A mixed solvent of chloroform: methanol (95: 5, v / v)

(3) A mixed solvent of chloroform: methanol (90:10, v / v)

(4) A mixed solvent of chloroform: methanol (80:20, v / v)

(5) A mixed solvent of chloroform: methanol (70:30, v / v)

(6) A mixed solvent of chloroform: methanol (60:40, v / v)

(7) A mixed solvent of chloroform: methanol (50:50, v / v)

(8) A mixed solvent of chloroform: methanol (40: 60, v / v)

(9) A mixed solvent of chloroform: methanol (30:70, v / v)

(10) A mixed solvent of chloroform: methanol (20:80, v / v)

(11) A mixed solvent of chloroform: methanol (10: 90, v / v)

(12) A mixed solvent of chloroform: methanol (0: 100, v / v)

The seventh fraction obtained from the twelve fractions obtained by increasing the polarity using a mixed solvent of chloroform: methanol (100: 0 to 0: 100, v / v) in the above ethyl acetate layer was chloroform: methanol (50: 50, v / v).

The seventh fraction obtained from the twelve fractions was fractionated by reverse phase chromatography using a mixture of acetonitrile and distilled water (acetonitrile: distilled water = 20 to 80: 90 to 10, v / v) The mixed solvent of acetonitrile and distilled water may be a mixed solvent of the following (a) to (e).

(A) Acetonitrile: a mixed solvent in which distilled water was mixed at 20:80 (v / v)

(B) Acetonitrile: A mixed solvent of distilled water 30:70 (v / v)

(C) Acetonitrile: A mixed solvent in which distilled water was mixed at 40:60 (v / v)

(D) Acetonitrile: A mixed solvent in which distilled water was mixed at 50:50 (v / v)

(E) Acetonitrile: Mixed solvent mixed with distilled water at 60:40 (v / v)

(F) Acetonitrile: Mixed solvent mixed with distilled water 70:30 (v / v)

(G) Acetonitrile: Mixed solvent mixed with distilled water 80:20 (v / v)

(H) Acetonitrile: Mixed solvent mixed with distilled water 90:10 (v / v)

(G) Acetonitrile: a mixed solvent of 100: 0 (v / v) of distilled water

The third fraction obtained from the above nine fractions was obtained by dividing the seventh fraction obtained from the seventh fraction among the twelve fractions into a mixture of acetonitrile and distilled water (acetonitrile: distilled water = 40: 60, v / v). The results are shown in Table 1. &lt; tb &gt;&lt; TABLE &gt;

The present invention relates to a composition for preventing and treating brain diseases, which comprises a compound isolated from mulberry, a novel compound morusinon from mulberry, and a method for separating the compound from mulberry, under various conditions to achieve the object of the present invention It is desirable to provide a composition for preventing and treating brain diseases, which comprises a compound isolated from mulberry by the above-mentioned conditions.

Hereinafter, the present invention will be described in detail with reference to examples, experimental examples and application examples. However, these are for the purpose of illustrating the present invention in more detail, and the scope of the present invention is not limited thereto.

&Lt; Example 1 >

10 kg of dried corn syrup was added to 30 liters of methanol and extracted at 25 캜 for 24 hours to obtain a methanol extract of MBE and the solvent was removed.

1.5 kg of the methanol extract of C. pertussis from which the solvent was removed was collected (610 g) in a mixed solvent of distilled water, hexane and ethyl acetate at a volume ratio (v / v) of 1: 1: 1.

The neuroprotective activity of the ethyl acetate layer (75 g) fractionated on a separatory funnel was measured. The chloroform-methanol mixed solvent (1: 1, v / v) Of 100: 0 to 0: 100, v / v), and 12 fractions were obtained. That is, a fraction obtained by fractionating with a mixed solvent of chloroform: methanol 100: 0 (v / v) was fractionated with a mixed solvent of chloroform: methanol at a ratio of 95: 5 (v / v) The fractions were collected with a mixed solvent of 90:10 (v / v) of chloroform and methanol, and the fractions were divided into three fractions and were collected with a mixed solvent of chloroform and methanol mixed at 80:20 (v / v) And fractionated with a mixed solvent in which chloroform: methanol was mixed at 70:30 (v / v) was fractionated into 5 fractions, and the fraction was collected with a mixed solvent of chloroform and methanol mixed at 60:40 (v / v) The fractions were collected by a mixed solvent in which 50:50 (v / v) of chloroform and methanol were mixed, and the fractions were fractioned into 7 fractions and were collected with a mixed solvent of chloroform and methanol mixed at 40:60 (v / v) The fraction was collected with a mixed solvent of 30:70 (v / v) of chloroform and methanol, and the fraction was collected by fraction 9, and the fraction was collected with a mixed solvent of chloroform and methanol at 20:80 (v / v) minute The fraction obtained by fractionation with a mixed solvent of 10:90 (v / v) of methanol and chloroform: methanol was collected by fraction 11 and mixed solvent of chloroform and methanol at 0: 100 (v / v) The fractions were divided into 12 fractions.

Of the 12 fractions, fraction 5 was separated by increasing the mixed solvent of methanol and distilled water at 40:60 (v / v) to 90:10 (v / v) to obtain pure licoflavone C (11 mg).

Nine fractions were obtained by increasing the concentration of acetonitrile and distilled water mixed solvent at 20:80 (v / v) to 100: 0 (v / v) using reverse phase chromatography. That is, fraction 1 obtained using a mixed solvent of acetonitrile: distilled water at a ratio of 20:80 (v / v), acetonitrile: distilled water at a ratio of 30:70 (v / v) Fraction 2, acetonitrile: fraction 3 obtained using a mixed solvent of 40:60 (v / v) mixed with distilled water, acetonitrile: distilled water 50:50 (v / v) (4), acetonitrile: distilled water 60:40 (v / v), acetonitrile: distilled water 70:30 (v / v) , Fraction 7 obtained using a mixed solvent of acetonitrile: distilled water 80:20 (v / v), acetonitrile: distilled water 90:10 (v / v) Nine fractions were obtained from the 9th fraction obtained from the mixed solvent, the 8th fraction obtained from the mixed solvent, and the acetonitrile: distilled water 100: 0 (v / v) mixed solvent. 3 fractions were obtained from pure moracin P (5 mg), moracin O (5 mg) and morusinon (10 mg) in reversed phase HPLC in a mixed solvent of 55 vol% methanol and 45 vol% distilled water.

Ten fractions out of the above 12 fractions were subjected to reversed phase chromatography to obtain five fractions while using a mixed solvent of acetonitrile / distilled water (1: 1, v / v), and the second fraction was diluted with a mixed solvent of 62% methanol / distilled water , Pure mulberrofuran Q (21 mg) was obtained by reversed-phase HPLC, and the fourth fraction was obtained moracenin D (15 mg) on silica gel by increasing the polarity of chloroform and acetone mixed solvent

Among the compounds separated from the above-mentioned bark, licoflavone C is represented by the following structural formula (2) and has the following characteristics.

licoflavone C: Pale yellow needles; UV (MeOH)? _Max (log?) 274 (4.24), 305 (4.12), 326 (4.15); ESIMS m / z 337 [M - H] -.

_{^{1 H NMR (acetone- d 6 400}} MHz): δ 13.0 (1H, s, OH-5), 9.40 (2H, br s, OH-7, OH-4 '), 7.96 (2H, d, H-2 ', H-6'), 7.05 (2H, d, H-3 ', H-5'), 6.63 (1H, s, H-3), 6.34 (1H, s, H-6), 5.30 (1H , br t, H-2 " ), 3.57 (2H, d, CH 2 -1"), 1.82 (3H, s, Me-4 "), 1.67 (3H, s, Me-5").

_{^{13 C NMR (acetone- d 6,}} 100 MHz): δ 183.5 (s, C-4), 165.0 (d, C-2), 162.2 (s, C-7), 161.9 (s, C-4 ') , 161.0 (s, C-5 ), 156.0 (s, C-9), 132.1 (s, C-3 "), 129.3 (d, C-2 'C-6'), 123.7 (d, C-1 '), 123.6 (d, C -2 "), 116.9 (d, C-3', C-5 '), 107.4 (s, C-10), 105.4 (s, C-8), 103.9 (d, C-3), 99.3 ( d , C-6), 25.8 ( q , C-4 "), 22.4 ( t , C-1"), 18.1 ( q , C-5 ").

...구조식(2)

... Structural formula (2)

Moracin P (moracin P) is represented by the following structural formula (3) and has the following characteristics.

moracin P: brown powder; UV (MeOH)? _Max (log?) 219 (3.88), 321 (3.92), 338 (3.89); ESIMS m / z 325 [M - H] -.

_{^{1 H NMR (acetone- d 6,}} 500 MHz): δ 7.26 (1H, s, H-4), 7.00 (1H, s, H-3), 6.87 (1H, s, H-7), 6.86 (each 1H, d, H-2 ' , H-6'), 6.37 (1H, br s, H-4 '), 3.82 (1H, dd, H-2 "), 3.10 (1H, dd, H-1" ), 2.83 (1H, dd , H-1 "), 1.37 (1H, s , H-5"), 1.26 (1H, s , H-4 ").

_{^{13 C NMR (acetone- d 6,}} 125 MHz): δ 159.9 (each s, C-3 ', C-5'), 156.0 (s, C-7a), 155.5 (s, C-2), 152.5 ( s, C-6), 133.1 (s, C-1 '), 123.6 (s, C-3a), 121.8 (d, C-4), 118.0 (s, C-5), 103.9 (each d, C -2 ', C-6') , 103.6 (d, C-4 '), 101.9 (d, C-3), 99.5 (d, C-7), 78.1 (s, C-3 "), 70.0 ( d , C-2 "), 32.5 ( t , C-1"), 26.3 ( q , C-4 "), 20.6 ( q , C-5").

...구조식(3)

... Structural formula (3)

Moracine O (moracin O) among the compounds isolated from the above-mentioned bovine periwinkle is represented by the following structural formula (4) and has the following characteristics.

moracin O: brown powder; UV (MeOH)? _Max (log?) 219 (3.54), 320 (3.59), 335 (3.55); ESIMS m / z 325 [M - H] -.

_{^{1 H NMR (acetone- d 6,}} 500 MHz): δ 7.33 (1H, s, H-4), 7.01 (1H, s, H-7), 6.84 (1H, s, H-3), 6.84 (each 1H, d, H-2 ' , H-6'), 6.37 (1H, br s, H-4 '), 4.68 (1H, t, H-2 "), 3.31 (1H, dd, H-1" ), 3.20 (1H, dd , H-1 "), 1.26 (1H, s , H-4"), 1.23 (1H, s , H-5 ").

_{^{13 C NMR (acetone- d 6,}} 125 MHz): δ 159.9 (each s, C-3 ', C-5'), 159.8 (s, C-2), 155.9 (s, C-6), 155.7 ( s, C-7a), 133.5 (s, C-1 '), 125.3 (s, C-3a), 123.4 (s, C-5), 117.0 (d, C-4), 103.7 (each d, C -2 ', C-6') , 103.4 (d, C-4 '), 102.5 (d, C-3), 93.1 (d, C-7), 91.3 (d, C-2 "), 71.5 ( s , C-3 "), 30.8 ( t , C-1"), 26.1 ( q , C-4 "), 25.6 ( q , C-5").

...구조식(4)

... Structural formula (4)

Among the compounds separated from the above-mentioned monobasma, moruscinone is represented by the following structural formula (1) and has the following characteristics.

Morusinon: yellow powder, [[alpha]] _D -1.1 [deg.] ( C 0.11 MeOH); UV (MeOH)? _Max nm (log?) 228 (4.15), 287 (4.03), 339 (SH, 3.71); ESIMS m / z 459 [M - H] -, 919 [2M - H] - HRESIMS m / z 459.2024 (calcd for C 25 H 31 O 8, 459.2019).

_{^{1 H NMR (DMSO- d 6,}} 500 MHz): δ 12.2 (1H, s, OH-5), 8.31 (1H, s, OH-2 '), 8.29 (1H, s, OH-4'), 6.98 (1H, s, H-6 '), 5.88 (each 1H, s, H-6, H-8), 5.68 (1H, dd, H-2), 3.26 (1H, dd, H-3), 2.64 _{(2H, m, H 2 -1} "), 2.57 (1H, dd, H-3), 2.55 (2H, m, H 2 -1 '''), 1.58 (2H, m, H 2 -2'''), 1.53 (2H, m , H 2 -2 "), 1.14 (6H, s, H 3 -4", H 3 -5 "), 1.13 (6H, s, H 3 -4''', H ₃ -5 ''').

_{^{13 C NMR (DMSO- d 6,}} 125 MHz): δ 196.9 (s, C-4), 166.6 (s, C-7), 163.5 (each s, C-5, C-8a), 153.3 (s, C-4 '), 150.5 ( s, C-2'), 124.5 (d, C-6 '), 121.7 (s, C-5'), 118.5 (s, C-3 '), 117.2 (s, C-1 '), 101.7 ( s, C-4a), 95.7 (d, C-6), 95.0 (d, C-8), 74.2 (s, C-2), 69.4 (s, C-3 " ), 69.0 (s, C- 3 '''), 44.0 (t, C-2'''), 42.5 (t, C-2 "), 41.6 (s, C-3), 29.3 (each q, C-4 ", C-5 ", C-4 '''), 29.2 (q, C-5'''), 24.6 (t, C-1 '''), 18.4 (t, C-1 " ).

...구조식(1)

... Structural formula (1)

Among the compounds separated from the above-mentioned bark, mulberrofuran Q is represented by the following structural formula (5) and has the following characteristics.

mulberrofuran Q: Reddish violet powder; UV (MeOH)? _Max (log?) 222 (4.41), 280 (4.15), 285 (SH, 4.08), 321 (4.43), 335 (SH 4.29); ESIMS m / z 591 [M - H] -.

_{^{1 H NMR (acetone- d 6,}} 300 MHz): δ 7.43 (1H, d, H-4), 7.37 (1H, d, H-14 "), 7.00 (1H, br s, H-3), 6.92 (1H, br s, H- 7), 6.86 (1H, br s, H-6 '), 6.79 (1H, dd, H-5), 6.67 (1H, d, H-20 "), 6.63 (1H , br s, H-2 ' ), 6.54 (1H, dd, H-13 "), 6.36 (1H, d, H-17"), 6.35 (1H, dd, H-19 "), 6.13 (1H, d, H-11 "), 3.50 (1H, s, H-3"), 2.81 (1H, br s, H-5 "), 2.73 (1H, dd, H-6"), 1.88 (1H, dd , H-6 "), 1.73 (3H, s , H-7").

_{^{13 C NMR (acetone- d 6,}} 100 MHz): δ 195.8 (s, C-2 "), 172.9 (s, C-12"), 166.8 (s, C-10 "), 160.9 (s, C- 3 '), 158.8 (s, C-2), 156.6 (s, C-18 ", C-7a), 155.8 (s, C-16"), 155.4 (s, C-5'), 155.3 (s , C-6), 134.4 ( d, C-14 "), 132.6 (s, C-1 '), 126.5 (d, C-20"), 122.5 (s, C-3a), 121.9 (d, C -4), 114.8 (s, C -9 "), 113.4 (s, C-15"), 113.2 (d, C-5), 112.2 (d, C-19 "), 110.4 (s, C-4 '), 109.6 (s, C -8 "), 107.7 (d, C-6'), 105.2 (d, C-2 '), 103.1 (d, C-13"), 102.2 (d, C-3 ), 988.9 (d, C- 7), 98.6 (d, C-17 "), 98.3 (d, C-11"), 92.5 (s, C-4 "), 75.5 (s, C-1") , 49.1 ( d , C-3), 37.9 ( d , C-5 "), 31.0 ( t , C-6"), 22.5 ( q , C-7 ").

...구조식(5)

... Structural formula (5)

Moracenin D is a compound represented by the following structural formula (6) and has the following characteristics.

moracenin D: Brown powder; UV (MeOH)? _Max (log?) 209 (4.79), 230 (sh 4.52), 265 (4.51), 280 (sh 4.27), 316 (4.13); ESIMS m / z 709 [M - H] -.

_{^{1 H NMR (acetone- d 6,}} 500 MHz): δ 12.82 (1H, s, OH-5 or OH-2 "), 12.80 (1H, s, OH-5 or OH-2"), 7.50 ~ 9.60 ( 6H, OH × 6), 7.40 (1H, d, H-6 "), 7.30 (1H, d, H-6 '), 6.78 (1H, d, H-20"), 6.64 (1H, d, H -3 '), 6.56 (1H, dd, H-5'), 6.20 (1H, d, H-17 "), 6.06 (1H, dd, H-19"), 6.00 (1H, s, H-6 ), 5.98 (1H, d, H-3 "), 5.94 (1H, dd, H-5"), 5.16 (1H, br s, H-10 "), 4.65 (1H, t, H-8") , 4.37 (1H, br d, H-9 "), 3.40 ~ 3.90 (1H, m, H-14"), 2.54 (2H, t, H-11), 1.80 ~ 2.20 (2H, m, H-13 ), 1.72 (2H, t , H-12), 1.50 (3H, br s , H-12 "), 1.14 (6H, s , H-15, H-14).

_{^{13 C NMR (acetonitrile- d 3,}} 125 MHz): δ 208.8 (s, C-7 "), 182.5 (s, C-4), 164.7 (s, C-4"), 164.0 (s, C-2 "), 161.0 (s, C -4 '), 160.2 (s, C-2'), 160.0 (s, C-7), 160.0 (s, C-9), 156.3 (s, C-2), 155.8 (s, C-16 " ), 155.8 (s, C-18"), 154.9 (s, C-5), 133.2 (s, C-11 "), 132.7 (d, C-6"), 131.2 (d, C-6 ') , 128.8 (d, C-20 "), 122.9 (d, C-10"), 121.5 (s, C-15 "), 121.5 (s, C-3), 114.5 ( s, C-1 "), 112.4 (s, C-1 '), 107.3 (d, C-5"), 107.1 (d, C-5'), 107.0 (s, C-8), 107.0 (d , C-19 "), 104.7 (s, C-10), 102.8 (d, C-3"), 102.6 (d, C-3 '), 102.0 (d, C-17 "), 97.5 (d, C-6), 70.2 (s , C-13), 47.0 (d, C-8 "), 41.9 (t, C-12), 37.6 (d, C-9"), 37.6 (d, C-14 "), 37.1 (t, C -13"), 28.2 (q, C-14), 28.1 (q, C-15), 22.0 (q, C-12 "), 20.0 (t, C-11).

...구조식(6)

... Structural formula (6)

<Example 2>

The mulberry root was shredded at room temperature for one week and then powdered in a mill. The mulberry root powder was immersed in methanol (MeOH) for 72 hours at room temperature and extracted three times to obtain MeOH crude extract.

The extracted MeOH crude extract was fractionated by sequentially using n- hexane, dichrolomethane (CH ₂ Cl ₂ ), and ethyl acetate (EtOAc). The material was separated using a double EtOAc soluble portion (MRE).

4 fractions of MRE-4 were obtained by collecting 500 ml of each of 23.86 g of the soluble portion of EtOAc using a column packed with Sephadex LH-20 (8 x 40 cm, MeOH) and collecting the MRE-4 fractions by thin layer chromatography (TLC ) And finally divided into seven fractions (MRE-4-1 to MRE-4-7).

(1) Methyl 2,4-dihydroxybenzoate

The purified MRE-4 fractions (6.522 g) were collected in a volume of 100 ml with a silica gel column (6 x 20 cm, Hexane: Acetone = 2: 1, v / v) to obtain 64 fractions. 4 fractions (MRE-4-1 to MRE-4-7) were observed under ultraviolet light (UV) and developed on TLC (Toluene: Ethyl formate: Formic acid (5: The 5th fraction (MRE-4-5) of the seven fractions was confirmed by TLC (silica gel 60, toluene-ethyl formate-formic acid - 5: 4: 1, v / v / v) To isolate Methyl 2,4-dihydroxybenzoate (10 mg).

The methyl 2,4-dihydroxybenzoate thus obtained is represented by the following structural formula (7) and has the following characteristics.

methyl 2,4-dihydroxybenzoate: White powder. TOF-MS m / z: 167 [M + H] - = 168.

_{^{1 H-NMR (methanol- d 4}} , 500 MHz): δ 3.92 (3H, s, OMe), 6.33 (1H, d, J = 2.0 ㎐, H-3), 6.37 (1H, dd, H-5) , 7.71 (1H, d, H-6).

_{^{13 C-NMR (methanol- d 4}} , 125 MHz): δ 5.89 (s, OMe), 102.03 (s, C-3), 104.12 (s, C-1), 107.68 (s, C-5), 131.25 ( s , C-6), 163.51 ( s , C-2), 164.28 ( s , C-4), 170.35 ( s , C-7).

...구조식(7)

... Structural formula (7)

(2) Trans-oxyresveratrol

(1) 7 fractions (MRE-4-1~MRE-4-7) in the fourth fraction (MRE-4-4) 1.31g of Sephadex LH-20 column (3.5 × 132㎝, CHCl ₃ obtained in (TLC) (developing solvent: ethyl formate: formic acid (5: 4; 1: 1, v / v) , v / v) and divided into eight fractions (MRE-4-4-1 to MRE-4-4-8) identified under ultraviolet light (UV). 18.0 mg of Trans-oxyresveratrol compound was isolated using HPLC (MeOH 40-80%, 20 min).

The trans-oxyresveratrol obtained above is represented by the following structural formula (8) and has the following characteristics.

trans-oxyresveratrol: Blue amorphous powder. TOF-MS m / z: 561 [M + H] - = 562.

_{^{1 H-NMR (methanol- d 4}} , 500 MHz): δ 1.82 (3H, s, H-7 "), 2.05 (1H, t, H-6"), 2.69 (1H, d, H-6 ") , 2.98 (1H, m, H -4 "), 3.36 (2H, m, H-5", H-3 "), 6.17 (1H, dd, H-13"), 6.34 (1H, d, H- 17 "), 6.37 (1H, d, H-11"), 6.44 (1H, br s, H-2 "), 6.48 (1H, dd, H-19"), 6.76 (1H, dd, H-5 ), 6.85 (1H, br s , H-6 '), 6.94 (3H, m, H-2', H-3, H-7), 7.12 (1H, d, H-20 "), 7.16 (1H , dd, H-14 "), 7.37 (1H, d, H-14").

_{^{13 C-NMR (methanol- d 4}} , 125 MHz): δ 24.05 (s, C-7 "), 28.99 (s, C-4"), 35.57 (s, C-5 "), 36.88 (s, C -6 "), 37.80 (s, C-3"), 98.67 (s, C-7), 102.29 (s, C-3), 103.24 (s, C-8 "), 104.32 (s, C-11 "), 104.70 (s, C -17"), 105.17 (s, C-2 '), 105.64 (s, C-6'), 107.16 (s, C-13 "), 110.19 (s, C-19 "), 113.39 (s, C -9"), 114.08 (s, C-5), 117.52 (s, C-15 "), 118.41 (s, C-4 '), 122.11 (s, C-4) , 123.26 (s, C-3a ), 123.51 (s, C-2 "), 128.14 (s, C-20"), 130.72 (s, C-14 "), 131.69 (s, C-1 '), 134.07 (s, C-1 " ), 153.75 (s, C-16"), 155.07 (s, C-18 "), 155.89 (s, C-6), 156.94 (s, C-2), 157.40 ( s , C-5 '), 157.90 ( s , C-3'), 158.05 ( s , C-7a), 158.54 ( s , C-10 "), 160.20 ( s , C-12").

...구조식(8)

... Structural formula (8)

(3) Mulberrofuran G

50.0 mg of the seventh fraction (MRE-4-4-7) out of the eight fractions (MRE-4-4-1 to MRE-4-4-8) obtained in the above (1) 18.0 mg of Mulberrofuran G compound corresponding to MRE-4-4-7-1 was isolated using HPLC (MeOH 40-80%, 20 min).

The mulberrofuran G thus obtained is represented by the following structural formula (9) and has the following characteristics.

mulberrofuran G: Yellowish amorphous powder. TOF-MS m / z: 647 [M + H] - = 648.

^{1 H-NMR (500 ㎒,} MeOD): δ 1.64 (3H, s, H-24 "), 1.75 (3H, s, H-25"), 1.95 (3H, s, H-7 "), 2.20 ( 1H, br d, H-6 "), 2.49 (1H, br d, H-6"), 3.26 (2H, t, H-21 "), 3.75 (1H, m, H-5"), 4.10 ( 1H, s, H-3 " ), 4.60 (1H, t, H-4"), 5.20 (1H, m, H-22 "), 5.76 (1H, s, H-2"), 6.26 (1H, dd, H-19 "), 6.38 (2H, m, H-3, 13"), 6.74 (3H, m, H-5, H-2 ', H-6'), 6.85 (1H, d, H -17 "), 6.88 (1H, d, H-7), 6.96 (1H, d, H-20"), 7.33 (1H, d, H-4), 8.37 (1H, d, H-14 ") .

^{13 C-NMR (500 ㎒,} MeOD): δ 18.02 (s, C-25 "), 22.58 (s, C-21"), 23.95 (s, C-7 "), 26.04 (s, C-24" ), 33.07 (s, C- 6 "), 33.87 (s, C-3"), 37.22 (s, C-5 "), 48.46 (s, C-4"), 98.58 (s, C-7) , 101.90 (s, C-3 ), 103.64 (s, C-17 "), 105.06 (s, C-2 ', C-6'), 107.38 (s, C-19"), 108.17 (s, C -13 "), 113.25 (s, C-5), 113.90 (s, C-9"), 116.32 (s, C-11 "), 117.25 (s, C-4 '), 121.97 (s, C- 4), 122.92 (s, C -15 "), 123.29 (s, C-3a), 123.75 (s, C-22"), 125.08 (s, C-2 "), 129.11 (s, C-20" ), 131.53 (s, C- 1 '), 131.98 (s, C-23 "), 132.66 (s, C-14"), 134.29 (s, C-1 "), 156.23 (s, C-6) , 156.78 (s, C-16 ", 18"), 157.21 (s, C-7a), 157.31 (s, C-2), 157.93 (s, C-3 '), 158.11 (s, C-5' ), 163.96 ( s , C-12 "), 164.74 ( s , C-10"), 210.33 ( s , C-8 ").

...구조식(9)

... (9)

(4) Chalcoloracin

50.0 mg of MRE-4-4-7 was prepared. 18.0 mg of Chalcomoracin compound corresponding to MRE-4-4-7-2 was isolated by HPLC (MeOH 40-80%, 20 min).

The chalcomoracin obtained above is represented by the following structural formula (10) and has the following characteristics.

chalcoloracin: Yellowish needles. TOF-MS m / z : 579 [M + H] ^- , 581 [M + H] &lt; ⁺ &gt; = 580.

^{1 H-NMR (500 ㎒,} MeOD): δ 1.82 (3H, s, H-7 "), 2.23 (1H, d, H-6"), 2.46 (1H, d, H-6 "), 3.75 ( 1H, s, H-3 " ), 4.11 (1H, s, H-5"), 4.59 (1H, t, H-4 "), 5.75 (1H, s, H-2"), 6.21 (1H, d, H-11 "), 6.26 (1H, dd, H-19"), 6.35 (1H, dd, H-13 "), 6.38 (1H, d, H-7"), 6.74 (3H, m, H-5, H-2 ' , H-6'), 6.85 (1H, s, H-3), 6.88 (1H, d, H-7), 6.96 (1H, d, H-20 "), 7.33 (1H, d, H-4), 8.46 (1H, d, H-14 ").

^{13 C-NMR (500 ㎒,} MeOD): δ 23.98 (s, C-7 "), 33.24 (s, C-6"), 33.93 (s, C-3 "), 37.04 (s, C-5" ), 48.57 (s, C- 4 "), 98.61 (s, C-7), 101.93 (s, C-3), 103.68 (s, C-11"), 103.72 (s, C-17 "), 105.04 (br s, C-2 ', C-6'), 107.43 (s, C-19 "), 109.09 (s, C-13"), 113.30 (s, C-5), 114.21 (s, C -4 '), 117.11 (s, C-9 "), 122.01 (s, C-4), 122.91 (s, C-3a), 123.31 (s, C-15"), 125.03 (s, C-2 "), 129.19 (s, C -14"), 131.59 (s, C-1 '), 134.48 (s, C-1 "), 135.60 (s, C-20"), 156.26 (s, C-6 ), 156.84 (s, C- 18 "), 157.23 (s, C-2), 157.36 (br s, C-3 ', C-5'), 158.00 (s, C-16"), 158.18 (s , C-7a), 166.71 ( s , C-12 "), 167.14 ( s , C-10"), 210.20 ( s , C-8 ").

...구조식(10)

... Structural formula (10)

<Experimental Example>

[A] Experimental method

(1) Cell culture and treatment

Human neuroblastoma SH-SY5Y cells (ATCC no. CRL-2266) were cultured in Dulbecco's modified eagle's medium (Sigma-Aldrich, USA) containing 10% fetal bovine serum (Gibco-BRL, USA) and antibiotics ) Culture medium. The incubator maintained a temperature of 37 캜, and a mixed gas of 95% by volume of air and 5% by volume of CO ₂ was continuously supplied to obtain appropriate conditions for cell culture. Cells were cultured in 6-well plates at 3 × 10 ⁵ cells / well. Dopamine concentration was determined to be 600 μM after cell viability. Moracenin D and moracin P (dissolved in dimethyl sulfoxide, final concentration 0.25%) were simultaneously treated with dopamine (Sigma-Aldrich, USA) Respectively.

(2) flow cytometry for the measurement of dopaminergic neuronal death

Flow cytometry analysis was performed to determine the effect on DNA content. The collected cells were washed three times with PBS, fixed with cold 70% ethanol at -20 ° C for 1 day, centrifuged at 1000 rpm for 5 minutes, and washed with PBS. The cells were then stained with 40 μg / ml Propidium Iodide (PI, Sigma-Aldrich, USA) and analyzed by flow cytometry (FACS, Coulter EPICS XL, USA). Apoptosis cells were decreased in PI staining intensity and compared with normal cells. The half-value effect concentration (EC ₅₀ ) was calculated from the survival rate using the table curve program.

(3) Measurement of nerve cell death induced by oxygen-glucose deficiency

96-well plate to ^{18x10 4 cells / well SH-SY5Y} cell one after After 24 hours incubation in is washed with DMEM medium without glucose that the medium is removed, enough to contain a conventional glucose, gives replace it with a non-glucose medium . After replacing the medium, the cells were allowed to adapt for 1 hour at 37 ° C in an atmosphere of 95% by volume of air and 5% by volume of CO ₂ gas continuously, and then each sample and the positive control group were treated, . The plate was then transferred to an anaerobic container (MIC-101, Billups-Rothernberg Inc., USA), filled with a mixture of 95 vol.% Nitrogen and 5 vol.% CO ₂ , Time. After 18 hours, glucose was reprocessed to a final concentration of 4.5 mg / L in each well and reoxygenated for 24 hours by transfer to an incubator feeding 95% by volume of air and 5% by volume of CO ₂ (Tabakman et al., 2005). The OGD-treated group was cultured in glucose-containing DMEM. To induce reoxygenation of the OGD-treated group, medium was replaced with non-glucose-containing DMEM, and a concentration of 4.5 mg / L Glucose. After 24 hours, the cell viability (%) of each cell was determined by MTT assay.

(4) Intracellular ROS measurement

The amount of ROS in SH-SY5Y cells was measured using a fluorescent probe, DCFDA (2'7'-dichlorofluorescin diacetate, Sigma-Aldrich, USA). Cells were cultured in a concentration-dependent manner (0.08 μM, 0.4 μM, and 2 μM) and mulberrofuran G or carnosine, which has been proven to be effective against cerebral ischemia, as a positive control to induce oxygen-glucose deficiency. After 4 hours, DCFDA was added to the well Treated at a final concentration of 40 μM and cultured for 30 minutes. The peroxide in the cells formed after the reaction was measured for fluorescence at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.

(5) Experiment to measure protein aggregation inhibition efficacy

Fluorescence analysis was used to measure the degree of protein aggregation. Thioflavin S (5 μM) used as a fluorescent substance was dissolved in PBS solution. SH-SY5Y cells were plated in a 96-well plate for 48 hours and incubated in a 37 ° C incubator for 48 hours. Then, the cells were treated with moracinin D or moracin P or 600 μM dopamine at concentrations of 0.16 μM, 0.8 μM and 4 μM, The cells were incubated at 37 ° C for 24 hours to coagulate the proteins, and the cells were incubated with 5 μM thioflavin S (200 μl) for 30 minutes. The supernatant was transferred to a black plate and analyzed by fluorescence spectrophotometer. The wavelength of the fluorescence spectrometer used was measured at Excitation 444 nm and Emission 4985 nm.

(6) RT-PCR

One mL of Easy Blue solution (iNtRON Biotech, Korea) was added to the cells collected by treating with a single component substance and dopamine and shaken. chloroform (SIGMA, USA) was added and the mixture was shaken again for 30 seconds. The mixture was centrifuged at 15,000 rpm for 15 minutes at 4 ° C and the same amount of isopropanol (SIGMA, USA) was added to the supernatant. The mixture was shaken for 30 seconds and centrifuged at room temperature and 15,000 rpm. At this time, the obtained pellet was washed with EtOH, and 100 μl of the third distilled water in which the RNase was removed by drying was added and the mixture was kept at 55 ° C for 15 minutes. The extracted RNA was subjected to RT-PCR using One step RT-PCR premix (iNtRON Biotech, Korea).

The RT-PCR primers used were Nurr1 (forward: GAACTGCACTTGGGCAGAGTTG; reverse: GGTGGACAGTGTCGTAATTC) and α-synuclein (forward: AGGAATTCATTAGCCATGGATGTATTC; reverse: AGATATTTCTTAGGCTTCAGGTTCGTAGT) and β-actin (forward: GACCCAGATCATGTTTGAGA; reverse: GCTTGCTGATCCACATCTGC).

PCR conditions were: 35 cycles of 95 ° C for 45 s (second, s), 58 ° C for 45 s, and 72 ° C for 45 s. α-synuclein has 38 cycles of 94 ° C for 30s, 53 ° C for 30s, and 72 ° C for 60s. β-actin was performed at 32 cycles of 94 ° C for 30s, 58 ° C for 23s, and 72 ° C for 35s.

(7) Western blot analysis

SH-SY5Y cells were cultured, washed with PBS, and total cellular proteins were extracted with PRO-PREP protein solution (Intron biotechnology, Korea). Protein concentration was measured using a protein assay solution (Intron biotechnology, Korea) . The membrane was transferred to a PVDF membrane (Amersham Pharmacia Biotech, UK) by electrophoresis on SDS-polyacrylamide gel and the membrane was washed with 5% skim milk in tris buffered-0.1% tween 20 (TBST) For 2 hours. After incubation at 4 ° C for 24 h with a primary antibody (α-synuclein; IBL Co., JAPAN, -actin, E6-AP, ubiquitin; Santa Cruz Biotechnology, CA) anti-mouse HRP-conjugated IgG; Chemicon, USA) for 1 hour at room temperature. After washing, the image was analyzed with an image analyzer LAS1000 (Fuji photo film, Japan). All western blot analyzes were repeated three or more times.

(8) Proteasome activity measurement experiment

To measure proteasome activity, Cells were treated with 0.4 μM, 2 μM, and 10 μM of a single component of Moracinin D and Moracin P, and 600 μM of dopamine, and incubated for 24 hours. Proteasome buffer [10 mM Tris-HCl, 1 mM EDTA, 2 mM ATP, 4 mM dithiothreitol (Sigma, St Louis, MO). Thereafter, centrifugation was carried out at 4 DEG C for 10 minutes at a speed of 13,000 rpm. 20 μg of the upper layer was incubated at 37 ° C for 1 hour in proteasome activity buffer [0.05M Tris-HCl (pH 8.0), 0.5 mM EDTA, 20 μM succinyl-leucine-leucine-valine-tyrosine-amidomethylcoumarin (Sigma, St Louis, do. Fluorescence density of each solution was measured using a fluorescence spectrometer. All readings were normalized using the fluorescence density of the same amount of AMC solution (50 [mu] M).

(9) Statistical processing

All measurements were carried out by the t -test were expressed as mean value ± standard error, and statistical analysis of each experimental group using the Sigma Plot program 10.0. P <0.05 was considered statistically significant.

[B] Experimental results

(1) Effects of monoclonal antibodies including moracinin D and moracin P on dopamine-induced neuronal apoptosis

In order to examine the effect of monocomponents such as Moracinin D and Moracin P, which are single components separated from mulberry in SH-SY5Y cells, on neurotoxicity of dopamine, SH-SY5Y cells were treated with 0.16 μM Moracenin D and Moracin P increased the cell survival rate in a dose-dependent manner, especially Moracenin D, EC ₅₀ Values (see Table 1, below) were better with 5.1 +/- 0.1 [mu] M (see Figure 1).

(2) Effects of monomers including Mulberrofuran G on neuronal death induced by oxygen-glucose deficiency

The SH-SY5Y cells were used to examine the protective effect of mulberrofuran G (0.08 μM, 0.4 μM, and 2 μM) on cell death by oxygen-glucose deprivation (see FIG. 2). In addition, carnosine, which has been proven to be effective against cerebral ischemia, was treated with a positive control and compared with the protective effect of mulberrofuran G on the neuronal cell death. The cell viability (%) of the group induced by oxygen - glucose deficiency decreased to about 60.7 ± 7.3 compared to the cell viability (%) 101.9 ± 4.0 of the control group without any treatment. The cell viability (%) of mulberrofuran G 0.08 μM treated group was 75.9 ± 1.6, the cell survival rate (%) of treated group was 79.9 ± 3.1 and the cell survival rate (%) of treated group was 81.0 ± 3.6 Showed excellent protection against apoptosis. In addition, it was confirmed that the cell survival rate (%) of carnosine 10 μM treatment group was superior to that of 75.7 ± 7.1 (see Table 1 below).

Table showing the effect concentration (EC ₅₀ ) of a substance that survived 50% of the cell survival rate after treatment with three concentrations of single component materials and treated with dopamine or OGD Number of species Used part Minute Active on DA
(EC ₅₀ , uM) Active on OGD
(EC ₅₀ , uM) mulberry tree Root extract and fraction > 100 23.1 ([mu] g / ml) mulberry tree Root extract and fraction > 100 0.6 ([mu] g / ml) mulberry tree Root extract and fraction > 100 16.2 ([mu] g / ml) mulberry tree Mallow 모루 민 hydroperoxide 5.2 μM > 100 mulberry tree Mallow moracenin D 5.1 μM > 100 mulberry tree Mallow moracin P 9.0 μM 10.4 μM mulberry tree Mallow moracin O > 100 12.6 μM mulberry tree Mallow mulberrofuran Q > 100 15.9 μM mulberry tree Mallow morusinon (new) > 100 25.5 μM mulberry tree Mallow licoflavone C > 100 16.4 μM Mountain mulberry fell total extract > 100 21.9 ([mu] g / ml) Mountain mulberry xylem total extract > 100 12.1 ([mu] g / ml) Mountain mulberry Muscle total extract > 100 27.6 (占 퐂 / ml) Mountain mulberry Muscle methyl 2,4-dihydroxybenzoate 122 μM > 100 Mountain mulberry Muscle trans-oxyresveratral > 100 28.4 (占 퐂 / ml) Mountain mulberry Muscle mulberrofuran G > 100 1.7 [mu] M Mountain mulberry Muscle chalcoloracin > 100 1.8 ([mu] g / ml)

(3) Inhibitory effect of Mulberrofuran G on intracellular ROS generation

(0.08 μM, 0.4 μM, 2 μM and 10 μM) on the excessive production of ROS induced by oxygen-glucose deficiency using SH-SY5Y cells. As a positive control group, carnosine (2 μM, 10 μM and 20 μM), which has been proven to be effective against cerebral ischemia, was treated with mulberrofuran G to inhibit intracellular ROS. The percentage of intracellular ROS in the group treated with OGD followed by reoxygenation was as high as 50.2 ± 4.4, compared to the intracellular ROS incidence (11.7 ± 2.1%) in the control group without any treatment, Was generated excessively. As a result of treatment with mulberrofuran G by concentration, intracellular ROS incidence (%) by concentration was 43.2 ± 1.0 at 0.08 μM, 39.8 ± 3.6 at 0.4 μM, 31.4 ± 2.4 at 2 μM and 23.1 ± 1.2 at 10 μM Showed an excellent ROS inhibitory effect on the concentration, and showed a much better inhibitory effect than the ROS incidence (41.8 ± 1.1) when 20 μM carnosine was treated (see FIG. 3).

(4) Effect of Moracinin D and Moracin P on protein aggregation inhibition

As a result of treatment with 600 μM of dopamine for 15 hours, the degree of protein aggregation increased about 2.7 ± 0.1 times as compared with the control without any treatment. However, Moracinin D at concentrations of 0.16 μM, 0.8 μM, and 4 μM was 2.6 ± 0.1, 2.1 ± 0.2, and 1.1 ± 0.1 times the highest concentration-dependent effect inhibiting protein aggregation, and Moracin P was 2.6 ± 0.2 , 2.1 ± 0.2, and 1.3 ± 0.1, respectively (FIG. 4).

(5) Effects of Moracinin D, Moracin P on protein accumulation and expression

After 15 hours of treatment with 600 μM of Dopamine, the expression level of -synuclein was increased. The levels of E6-AP and Nurr1 proteins were decreased, and the ubiquitin conjugates were increased. In order to evaluate the effect on these proteins, 0.16 μM, 0.8 μM, 4 μM of Moracinin D or Moracin P was treated with 600 μM of dopamine for 15 hours and then the degree of protein expression was determined by western blot analysis. Moracenin D and Moracin P were found to be more effective in other mechanisms. First, Moracenin D was found to reduce overexpression of α-synuclein by 4 μM, and overexpression of E6-AP And it was confirmed that the ubiquitin conjugates of the polymer were significantly reduced in a concentration dependent manner. On the other hand, it was confirmed that the overexpression of Moracin P and α-synuclein was significantly reduced in a concentration-dependent manner, and that E6-AP was prevented from decreasing and the ubiquitin conjugates of the polymer were reduced (see FIG. 5) .

(6) Influence of Moracenin D -synuclein and its higher protein Nurr1

In order to investigate the effect of α-synuclein overexpression of Moracenin D on Nurr1, which is known to be a high-level protein, we measured both mRNA and protein expression levels. As a result, it was confirmed that the expression level of α-synuclein increased and that Nurr1, which regulates it, decreased after 9 hours of treatment with 600 μM of Dopamine at the mRNA stage (see FIG. 6) -synuclein increased and Nurr1 regulates the expression of N-acetyltransferase. Decreased Nurr1 expression was observed in cells treated with 0.16 μM, 0.8 μM, and 4 μM of Moracenin D, indicating that α-synuclein overexpression was decreased and Nurr1 was also increased (See FIG. 7).

(7) Effect of Moracenin D, Moracin P on proteasome activity

After 24 hours of dopamine treatment at 600 μM, the activity of proteasome was 54.5 ± 5.3%, which was significantly lower than that of control. As shown in FIG. 7, when concurrent treatment of 0.16 μM, 0.8 μM and 4 μM of Moracenin D and dopamine was carried out, the proteasome activity was significantly increased in a concentration-dependent manner by 57.5 ± 3.1, 79.2 ± 3.7, and 87.2 ± 5.1, respectively (See FIG. 8).

&Lt; Application Example 1-1 >

10 mg of licoflavone C obtained in Example 1, 2 g of corn starch and 2 g of lactose were mixed and filled in an airtight container to prepare a composition for preventing and treating cerebral vascular disease.

&Lt; Application Example 1-2 > Preparation of tablets

A composition for preventing and treating cerebrovascular disease was prepared by mixing 10 mg of licoflavone C obtained in Example 1, 150 mg of corn starch, 150 mg of lactose, and 3 mg of magnesium stearate, followed by tabletting.

&Lt; Application Example 1-3 > Preparation of capsules

A mixture of 10 mg of licoflavone C, 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate obtained by the method of Example 1 was filled in a gelatin capsule to prepare a composition for the prevention and treatment of cerebrovascular disease in a capsule preparation.

&Lt; Application 1-4 >

A mixture of 10 mg of licoflavone C obtained in Example 1, 2 g of corn starch, 3 g of lactose, 1 g of glycerin and 1 g of xylitol was poured into a ventilator to prepare a composition for prevention and treatment of cerebrovascular disease .

<Application Example 1-5> Preparation of granules

After mixing 10 mg of licoflavone C, 50 mg of soybean extract, 200 mg of glucose and 600 mg of corn starch obtained by the method of Example 1, 100 mg of 30% ethanol was added and dried at 60 ° C to form granules to prevent and treat cerebrovascular diseases Lt; / RTI &gt;

&Lt; Application example 2-1 >

10 g of moracin P obtained by the method of Example 1, 2 g of corn starch and 1 g of lactose were mixed and filled in an airtight container to prepare a composition for preventing and treating cerebrovascular diseases.

&Lt; Application example 2-2 > Preparation of tablet

A composition for preventing and treating cerebrovascular disease was prepared by mixing 10 mg of moracin P obtained by the method of Example 1, 150 mg of corn starch, 150 mg of lactose and 3 mg of magnesium stearate and then tableting.

&Lt; Application Example 2-3 > Preparation of capsules

A mixture of 10 mg of moracin P obtained by the method of Example 1, 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate was filled in a gelatin capsule to prepare a composition for the prevention and treatment of cerebrovascular disease in a capsule formulation.

&Lt; Application Example 2-4 >

A mixture of 10 g of moracin P obtained in the method of Example 1, 2 g of corn starch, 3 g of lactose, 1 g of glycerin and 1 g of xylitol was poured into a ventilator to prepare a composition for prevention and treatment of cerebrovascular disease .

&Lt; Application example 2-5 > Preparation of granules

After mixing 10 mg of moracin P, 50 mg of soybean extract, 200 mg of glucose and 600 mg of corn starch obtained by the method of Example 1, 100 mg of 30% ethanol was added and dried at 60 ° C to form granules to prevent and treat cerebrovascular diseases Lt; / RTI &gt;

&Lt; Application example 3-1 >

10 g of moracin O obtained by the method of Example 1, 2 g of corn starch and 1 g of lactose were mixed and filled in an airtight container to prepare a composition for preventing and treating cerebrovascular diseases.

&Lt; Application example 3-2 > Preparation of tablet

10 mg of moracin O, 150 mg of corn starch, 150 mg of lactose and 3 mg of magnesium stearate obtained by the method of Example 1 were mixed and then tableted to prepare a composition for the prevention and treatment of cerebrovascular disease.

&Lt; Application example 3-3 > Preparation of capsules

A mixture of 10 mg of moracin O obtained by the method of Example 1, 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate was filled in a gelatin capsule to prepare a composition for the prevention and treatment of cerebrovascular disease in a capsule formulation.

&Lt; Application example 3-4 >

A mixture of 10 mg of moracin 0 obtained in the method of Example 1, 2 g of corn starch, 3 g of lactose, 1 g of glycerin and 1 g of xylitol was poured into a ventilator to prepare a composition for preventing and treating cerebrovascular disease .

&Lt; Application example 3-5 > Preparation of granules

After mixing 10 mg of moracin O, 50 mg of soybean extract, 200 mg of glucose, and 600 mg of corn starch obtained by the method of Example 1, 100 mg of 30% ethanol was added and dried at 60 ° C to form granules to prevent and treat cerebrovascular diseases Lt; / RTI &gt;

&Lt; Application example 4-1 >

10 mg of mulberrofuran Q obtained in Example 1, 2 g of corn starch and 1 g of lactose were mixed and filled in an airtight container to prepare a composition for preventing and treating cerebral vascular disease.

&Lt; Application example 4-2 > Preparation of tablet

A composition for preventing and treating cerebrovascular disease was prepared by mixing 10 mg of mulberrofuran Q, 150 mg of corn starch, 150 mg of lactose, and 3 mg of magnesium stearate obtained by the method of Example 1, followed by tabletting.

&Lt; Application example 4-3 > Preparation of capsules

A mixture of 10 mg of mulberrofuran Q, 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate obtained by the method of Example 1 was filled in a gelatin capsule to prepare a composition for the prevention and treatment of cerebrovascular disease in a capsule formulation.

&Lt; Application Example 4-4 >

A mixture of 10 mg of mulberrofuran Q obtained in Example 1, 2 g of corn starch, 3 g of lactose, 1 g of glycerin and 1 g of xylitol was poured into a ventilator to prepare a composition for preventing and treating cerebrovascular disease .

&Lt; Application example 4-5 > Preparation of granules

After mixing 10 mg of mulberrofuran Q obtained by the method of Example 1, 50 mg of soybean extract, 200 mg of glucose and 600 mg of corn starch, 100 mg of 30% ethanol was added and dried at 60 ° C to form granules to prevent and treat cerebrovascular diseases Lt; / RTI &gt;

&Lt; Application Example 5-1 >

10 g of moracenin D obtained by the method of Example 1, 2 g of corn starch and 1 g of lactose were mixed and filled in an airtight container to prepare a composition for preventing and treating cerebrovascular diseases.

&Lt; Application example 5-2 > Preparation of tablet

10 g of moracenin D obtained in Example 1, 150 mg of corn starch, 150 mg of lactose and 3 mg of magnesium stearate were mixed and then tableted to prepare a composition for preventing and treating cerebrovascular disease.

&Lt; Application example 5-3 > Preparation of capsules

A mixture of 10 mg of moracenin D obtained in Example 1, 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate was filled in a gelatin capsule to prepare a composition for preventing and treating cerebrovascular disease in a capsule preparation.

&Lt; Application 5-4 >

A mixture of 10 g of moracenin D obtained by the method of Example 1, 2 g of corn starch, 3 g of lactose, 1 g of glycerin and 1 g of xylitol was poured into a ventilator to prepare a composition for prevention and treatment of cerebrovascular disease .

&Lt; Application example 5-5 > Preparation of granules

After mixing 10 mg of moracenin D obtained in Example 1, 50 mg of soybean extract, 200 mg of glucose, and 600 mg of corn starch, 100 mg of 30% ethanol was added and dried at 60 ° C to form granules to prevent and treat cerebrovascular diseases Lt; / RTI &gt;

&Lt; Application Example 6-1 >

10 mg of morusinon, a novel compound of the following structural formula (1) obtained by the method of Example 1, 2 g of corn starch and 1 g of lactose were mixed and filled in an airtight container to prepare a composition for preventing and treating cerebrovascular diseases.

... 구조식(1)

... Structural formula (1)

&Lt; Application example 6-2 > Preparation of tablet

10 mg of morusinon, 150 mg of corn starch, 150 mg of lactose and 3 mg of magnesium stearate, which is a novel compound of the following structural formula (1) obtained by the method of Example 1, were mixed and then tableted to prepare a composition for the prevention and treatment of cerebrovascular diseases.

... 구조식(1)

... Structural formula (1)

&Lt; Application example 6-3 > Preparation of capsules

A mixture of 10 mg of morusinon, 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate, which is a novel compound of the following structural formula (1) obtained by the method of Example 1, was filled into a gelatin capsule to prevent and treat cerebrovascular diseases Lt; / RTI &gt;

... 구조식(1)

... Structural formula (1)

&Lt; Application Example 6-4 >

A mixture of 10 mg of morusinon, 2 g of corn starch, 3 g of lactose, 1 g of glycerin, and 1 g of xylitol, obtained by the method of Example 1, which is a novel compound of the following structural formula (1) A composition for prevention and treatment of vascular diseases was prepared.

&Lt; Application example 6-5 > Preparation of granules

10 mg of morusinon, 50 mg of soybean extract, 200 mg of glucose and 600 mg of corn starch obtained by the method of Example 1, which is a novel compound of the following structural formula (1), was added and then 100 mg of 30% ethanol was added and dried at 60 ° C to form granules Thereby preparing a composition for preventing and treating cerebral vascular diseases.

... 구조식(1)

... Structural formula (1)

&Lt; Application example 6-6 >

10 mg of morusinon, 1 g of citric acid, 50 g of oligosaccharide, 2 g of a plum concentrate, 1 g of taurine and purified water were added to a new compound of the following structural formula (1) obtained in Example 1 to obtain a total volume of 1 liter (L) The mixture was stirred at 500 rpm and then sterilized at 85 DEG C for 15 minutes to prepare a beverage composition for cerebral vascular prevention and treatment.

... 구조식(1)

... Structural formula (1)

&Lt; Application example 6-7 > Injection preparation

5 mg of anhydrous ethanol and 527 mg of Cremophor EL were added to 10 ml of morusinon, a novel compound of the following structural formula (1) obtained by the method of Example 1, and the mixture was stirred at 100 rpm for 30 minutes to prepare cerebral blood vessels A composition for prevention and treatment was prepared.

... 구조식(1)

... Structural formula (1)

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be taken by way of illustration in and of itself. It will be understood that the invention may be variously modified and changed.

According to the present invention, morusinon, which is a novel compound isolated from mulberry, can be obtained, and licoflavone C, moracin P, which is a compound isolated from mulberry, Moracin O, mulberrofuran Q, moracenin D, methyl-2,4-dihydroxybenzoate, trans-oxy It is possible to provide a composition for preventing and treating brain diseases comprising at least one selected from the group consisting of trans-oxyresveratrol, mulberrofuran G, and chalcomoracin.

Meanwhile, the composition for preventing and treating cerebral diseases according to the present invention includes food-acceptable excipients and / or pharmaceutically acceptable excipients in addition to compounds isolated from mulberry, Compositions can be provided, which can contribute to the development of food- and / or pharmaceutical-related industries, thus being industrially applicable.

<110> THE REPUBLIC OF KOREA (FORESTRY ADMINISTRATION FORESTRY RESEARCH INSTITUTE) <120> COMPOSITION MADE OF THE COMPOUNDS EXTRACTED FROM Morus alba FOR          PREVENTION AND TREATMENT OF CEREBROPATHIA <130> 8784 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Nurr1 forward primer <400> 1 gaactgcact tgggcagagt tg 22 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Nurr1 reverse primer <400> 2 ggtggacagt gtcgtaattc 20 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> a-synuclein forward primer <400> 3 aggaattcat tagccatgga tgtattc 27 <210> 4 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> a-synuclein reverse primer <400> 4 agatatttct taggcttcag gttcgtagt 29 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> B-actin forward primer <400> 5 gacccagatc atgtttgaga 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> B-actin reverse primer <400> 6 gcttgctgat ccacatctgc 20

Claims (16)

Morusinon, a novel compound represented by the following structural formula (1) separated from mulberry.

... Structural formula (1) The method of claim 1,
Wherein the morus ison is characterized by having the following characteristics.
Morusinon: yellow powder, [[alpha]] _D -1.1 [deg.] ( C 0.11 MeOH); UV (MeOH)? _Max nm (log?) 228 (4.15), 287 (4.03), 339 (SH, 3.71); ESIMS m / z 459 [M - H] -, 919 [2M - H] - HRESIMS m / z 459.2024 (calcd for C 25 H 31 O 8, 459.2019).
_{^{1 H NMR (DMSO- d 6,}} 500 MHz): δ 12.2 (1H, s, OH-5), 8.31 (1H, s, OH-2 '), 8.29 (1H, s, OH-4'), 6.98 (1H, s, H-6 '), 5.88 (each 1H, s, H-6, H-8), 5.68 (1H, dd, H-2), 3.26 (1H, dd, H-3), 2.64 _{(2H, m, H 2 -1} "), 2.57 (1H, dd, H-3), 2.55 (2H, m, H 2 -1 '''), 1.58 (2H, m, H 2 -2'''), 1.53 (2H, m , H 2 -2 "), 1.14 (6H, s, H 3 -4", H 3 -5 "), 1.13 (6H, s, H 3 -4''', H ₃ -5 ''').
_{^{13 C NMR (DMSO- d 6,}} 125 MHz): δ 196.9 (s, C-4), 166.6 (s, C-7), 163.5 (each s, C-5, C-8a), 153.3 (s, C-4 '), 150.5 ( s, C-2'), 124.5 (d, C-6 '), 121.7 (s, C-5'), 118.5 (s, C-3 '), 117.2 (s, C-1 '), 101.7 ( s, C-4a), 95.7 (d, C-6), 95.0 (d, C-8), 74.2 (s, C-2), 69.4 (s, C-3 " ), 69.0 (s, C- 3 '''), 44.0 (t, C-2'''), 42.5 (t, C-2 "), 41.6 (s, C-3), 29.3 (each q, C-4 ", C-5 ", C-4 '''), 29.2 (q, C-5'''), 24.6 (t, C-1 '''), 18.4 (t, C-1 " ). The method of claim 1,
The morus nongon is characterized by that the morus nongon is separated from the mulberry bark. Extracting the mulberry bark from methanol at room temperature to obtain a methanol extract;
Separating the methanol extract obtained by removing the solvent from the above methanol extract of Corynebacterium sp. With distilled water, a mixed solvent of hexane and ethyl acetate (distilled water, hexane, ethyl acetate: 1: 1: 1, v / v);
Obtaining an aliquot of the ethyl acetate layer by using a mixed solvent of chloroform: methanol (1: 1 v / v) to increase the polarity to 12 fractions;
Seventh fraction obtained from the above 12 fractions was fractionated by reverse phase chromatography using 9% acetonitrile and distilled water as a solvent (acetonitrile: distilled water = 20 to 100: 80 to 0, v / v) Obtaining fractions thereof;
The third fraction obtained from the above nine fractions was subjected to reverse phase HPLC using a mixed solvent of methanol and distilled water (55 vol% methanol and 45 vol% distilled water) as a solvent to obtain morusinon Wherein the morusinon is a novel compound from mulberry. 5. The method of claim 4,
The methanol extract of Corynebacterium phalaenopsis is obtained by adding 15 to 50 liters of methanol to 10 kg of the cowpea and then performing the extraction for 2 to 5 times at a temperature of 20 to 25 캜 for 12 to 24 hours. A method for separating morusinon, a compound. 5. The method of claim 4,
The methanol extract of Corynebacterium annuum was obtained by adding 30 liters (liter) of methanol to 10 kg of mulberry macaque and extracting it at room temperature for 24 hours at 25 ° C for 3 times. morusinon. 5. The method of claim 4,
A novel compound, morusinon, obtained from the mulberry tree bark, has the following characteristics: a novel compound from mulberry, morusinon.
Morusinon: yellow powder, [[alpha]] _D -1.1 [deg.] ( C 0.11 MeOH); UV (MeOH)? _Max nm (log?) 228 (4.15), 287 (4.03), 339 (SH, 3.71); ESIMS m / z 459 [M - H] -, 919 [2M - H] - HRESIMS m / z 459.2024 (calcd for C 25 H 31 O 8, 459.2019).
_{^{1 H NMR (DMSO- d 6,}} 500 MHz): δ 12.2 (1H, s, OH-5), 8.31 (1H, s, OH-2 '), 8.29 (1H, s, OH-4'), 6.98 (1H, s, H-6 '), 5.88 (each 1H, s, H-6, H-8), 5.68 (1H, dd, H-2), 3.26 (1H, dd, H-3), 2.64 _{(2H, m, H 2 -1} "), 2.57 (1H, dd, H-3), 2.55 (2H, m, H 2 -1 '''), 1.58 (2H, m, H 2 -2'''), 1.53 (2H, m , H 2 -2 "), 1.14 (6H, s, H 3 -4", H 3 -5 "), 1.13 (6H, s, H 3 -4''', H ₃ -5 ''').
_{^{13 C NMR (DMSO- d 6,}} 125 MHz): δ 196.9 (s, C-4), 166.6 (s, C-7), 163.5 (each s, C-5, C-8a), 153.3 (s, C-4 '), 150.5 ( s, C-2'), 124.5 (d, C-6 '), 121.7 (s, C-5'), 118.5 (s, C-3 '), 117.2 (s, C-1 '), 101.7 ( s, C-4a), 95.7 (d, C-6), 95.0 (d, C-8), 74.2 (s, C-2), 69.4 (s, C-3 " ), 69.0 (s, C- 3 '''), 44.0 (t, C-2'''), 42.5 (t, C-2 "), 41.6 (s, C-3), 29.3 (each q, C-4 ", C-5 ", C-4 '''), 29.2 (q, C-5'''), 24.6 (t, C-1 '''), 18.4 (t, C-1 " ). A composition for preventing and treating brain diseases,
Licoflavone C, moracin P, moracin O, a compound isolated from mulberry, morusinon represented by the following structural formula (1), mulberofuran Q (mulberrofuran) Q), Moracenin D, methyl-2,4-dihydroxybenzoate, trans-oxyresveratrol, mulberrofuran G , Chalcomoracin (chalcomoracin) comprising a composition for the prevention and treatment of brain diseases, including any one or more.

... Structural formula (1) 9. The method of claim 8,
Among the compounds isolated from mulberry, moruscinone has the following characteristics.
Morusinon: yellow powder, [[alpha]] _D -1.1 [deg.] ( C 0.11 MeOH); UV (MeOH)? _Max nm (log?) 228 (4.15), 287 (4.03), 339 (SH, 3.71); ESIMS m / z 459 [M - H] -, 919 [2M - H] - HRESIMS m / z 459.2024 (calcd for C 25 H 31 O 8, 459.2019).
_{^{1 H NMR (DMSO- d 6,}} 500 MHz): δ 12.2 (1H, s, OH-5), 8.31 (1H, s, OH-2 '), 8.29 (1H, s, OH-4'), 6.98 (1H, s, H-6 '), 5.88 (each 1H, s, H-6, H-8), 5.68 (1H, dd, H-2), 3.26 (1H, dd, H-3), 2.64 _{(2H, m, H 2 -1} "), 2.57 (1H, dd, H-3), 2.55 (2H, m, H 2 -1 '''), 1.58 (2H, m, H 2 -2'''), 1.53 (2H, m , H 2 -2 "), 1.14 (6H, s, H 3 -4", H 3 -5 "), 1.13 (6H, s, H 3 -4''', H ₃ -5 ''').
_{^{13 C NMR (DMSO- d 6,}} 125 MHz): δ 196.9 (s, C-4), 166.6 (s, C-7), 163.5 (each s, C-5, C-8a), 153.3 (s, C-4 '), 150.5 ( s, C-2'), 124.5 (d, C-6 '), 121.7 (s, C-5'), 118.5 (s, C-3 '), 117.2 (s, C-1 '), 101.7 ( s, C-4a), 95.7 (d, C-6), 95.0 (d, C-8), 74.2 (s, C-2), 69.4 (s, C-3 " ), 69.0 (s, C- 3 '''), 44.0 (t, C-2'''), 42.5 (t, C-2 "), 41.6 (s, C-3), 29.3 (each q, C-4 ", C-5 ", C-4 '''), 29.2 (q, C-5'''), 24.6 (t, C-1 '''), 18.4 (t, C-1 " ). 9. The method of claim 8,
A composition for preventing and treating cerebral diseases, characterized in that the moruscinone represented by the structural formula (1) is isolated from the mallow's mantle. 9. The method of claim 8,
Wherein the compound isolated from mulberry comprises 0.1 to 99.9% by weight based on the total weight of the composition. 9. The method of claim 8,
Wherein the compound isolated from mulberry comprises 10 to 50 wt% of the total weight of the composition. 9. The method of claim 8,
The composition is a composition for preventing and treating brain diseases, characterized in that the food composition comprising a food acceptable material. 9. The method of claim 8,
The composition may be formulated in one or more forms selected from powders, pills, granules, granules, tablets, capsules and beverages, including food acceptable ingredients. A composition for preventing and treating brain diseases, characterized in that the food composition. 9. The method of claim 8,
Wherein the composition is a pharmaceutical composition comprising a pharmaceutically acceptable excipient. 9. The method of claim 8,
The composition may be in the form of any one or more oral dosage forms selected from powders, pills, granules, tablets, capsules and liquids, including pharmaceutically acceptable excipients, Or a pharmaceutically acceptable excipient. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is formulated into liquid injections.

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