KR102121915B1 - Composition for protecting neuronal cells comprising naphthopyrone derivatives derived from the sprout of Cassia obtusifolia L. - Google Patents

Abstract

The present invention relates to a composition comprising a naphthopyrone derivative and a modified sprout extract containing it as an active ingredient. The composition according to an aspect of the present invention has an antioxidant effect and an effect of protecting nerve cells from oxidative stress or inhibiting apoptosis, and in particular, damage or death of retinal nerve cells or hippocampal nerve cells due to glutamate toxicity. It has an inhibitory effect, and treatment or prevention of visual acuity and deterioration and eye disease due to optic nerve damage, memory loss due to brain nerve damage, learning ability decline, depressive disorder occurrence and exacerbation, and pharmacy or food for the treatment or prevention of degenerative neurological disease It can be used as a composition.

Description

Composition for protecting neuronal cells comprising naphthopyrone derivatives derived from the sprout of Cassia obtusifolia L.}

The present specification relates to a composition comprising a naphthopyrone derivative and a modified sprout extract containing it as an active ingredient.

The hippocampus (hippocampus) is a structure of the medial temporal lobe of the brain and plays an important role in the cognitive function of humans and animals. The hippocampus is vulnerable to physiological and oxidative stress stimuli and causes cognitive impairment due to stress, and is known as the central tissue. Stress can cause hippocampal structure and brain cell production, synaptic plasticity, and behavioral changes related to the hippocampus. (Kim Eun-joo, Journal of the Korean Psychological Association: Cognitive and Biological, 2012, 24, 65-88). When oxidative stress in neurons is induced in the hippocampus, pituitary gland, striatum, black matter, the whole cortex, or the hypothalamus, neuronal cell death increases and neurons and growth factors decrease, resulting in amyotrophic lateral sclerosis (ALS), Parkinson's disease, or brain It is known to cause acute or chronic neurological diseases such as ischemic nerve damage. (Rahman, T. et al. Adv. Biosci. Biotechnol. 2012, 3, 997-1019)

According to the National Health Insurance Corporation's health insurance data, the number of patients with retinal disease has increased by about 8% per year from about 830,000 in 2010 to about 12.5 million in 2015. The retina is a thin neural membrane inside the eye that is likened to a camera film. There are more than 100 million photoreceptor cells (light-sensing cells) and more than 1 million optic nerve cells in the retina, which convert light into electrical signals and transmit the images of objects seen through the nerves to the brain. If the nerves of the retina are damaged or the nerve function is abnormal, problems with vision and vision occur. Representative retinal diseases include diabetic retinopathy, age-related macular degeneration, retinal pigmentation, retinal detachment, and retinal vascular occlusion, including decreased vision, blindness and blindness. Glaucoma is a typical eye disease in which retinal optic nerve damage occurs, and the optic nerve is damaged for reasons such as increased intraocular pressure, ischemia, and oxidative stress. (Kim, NY et al. J. Korean Opthalmol. Soc. 2015, 56, 70-79; Kang, JH et al. J. Korean Ophthalmol. Soc. 2003, 44, 965-970; Lee, SM et al. J Korean Ophthalmol. Soc. 2002, 43, 2577-2584)

Glutamate is an excitatory neurotransmitter that plays an important role in the vertebrate central nervous system. Glutamate is involved in quisqualate receptors, NMDA (N-methyl-D-aspartate) receptors, and Kainite receptors, which are concentrated in the cerebellar tonsil and hippocampus, which are known to be involved in memory and learning ability among brain tissues. . On the other hand, when glutamate increases the amount of free out of the nerve cells and increases the concentration of glutamate outside the cell, it may act as an oxidative neurotoxic substance. (Kim Hyun-jung et al., J. Life Sci. 2009, 19, 963-967) Excessive glutamate is known to be associated with various acute neurological disorders, including anemia, oxygen deficiency, hypoglycemia, trauma, and several chronic degenerative neurological diseases. . Glutamate in the eye is associated with acute disorders and death of retinal ganglion cells. (Otori, Y. et al. Invest. Ophthalmol. Vis. Sci. 1998, 39, 972-981) A large amount of glutamate is generated, which activates the presynaptic glutamate receptor and activates reactive oxygen species (ROS). It is known that fruiting is stimulated. (Tarasenko A. et al. Neurochem. Int. 2012, 61, 1044-1051) Also, oxidative action exerted in response to glutamate is mediated by activation of the NMDA receptor, and is known to exhibit calcium ion dependence. It has been reported that such excessive NMDA receptor activation and uptake of calcium ions into mitochondria lead to ROS production. (Reynolds IJ et al. J. Neurosci. 1995, 15, 3318-3327) Reactive Oxygen Species (ROS) are continuously produced by the oxidation and reduction of oxygen by respiration and immune responses in mitochondria in living cells. do. Since ROS is chemically unstable and highly reactive, it can react with lipids, nucleic acids, and proteins in vivo to cause DNA damage, increase the concentration of free calcium and iron in the cell, and damage the ion transport system of the biofilm. (Kiselyov, K. et al. Cell Calcium. 2016, 60, 108-114) ROS is also known to induce light-induced damage to the optic nerve by inducing production by light. (Masuda, T. et al. Oxid.Med.Cell.Longevity. 2017; article ID 9208489, 14 pages)

It has been traditionally used for eye health and is known to have excellent antioxidant properties. The terminus is a mature seed of the herbaceous plant belonging to the legume family (Cassia obtusifolia L.) or Ginjiang Namcha (Cassia tora L.). It is grown in all parts of Korea and glosss in a pod of about 10 cm after the leaves are cut. I have a single seed. (Yen, G.-C. et al. J. Agric. Food Chem. 1998, 46, 820-824) Extracts of terminator extract have been studied and reported to improve diabetes, improve dyslipidemia, protect liver, antibacterial, and lower blood pressure. . (Dong, X. et al. Mol. Med. Rep. 2017, 16, 2331-2346)

However, the 7-hydroxymusizinyl-rubrofusarin-8'-O-glucoside (7-hydroxymusizinyl-rubrofusarin-8'-O-glucopyranoside), isotora, is a napropyrone derivative derived from the bud extract or derived therefrom. Neuroprotective action against glutamate-induced neurotoxicity of lactor (isotoralactone), toralactone (toralactone), torosachrysone (torosachrysone) and rubrofusarin (rubrofusarin) has not been reported, antioxidants and nerves using the novel sprout extract There is no known technology for protective compositions.

In addition, no research has been reported on health functional and medicinal ingredient research using the sprouted shoot extract, and the present inventors have found that the sprouted shoot extract produces a new antioxidant compared to the extract of shooter, which increases the antioxidant activity and increases significantly. The present inventors have confirmed that it has an antioxidant activity and a neuronal protective action, and a new compound 7-hydroxymushizinyl-rubbrofusarin-8'-O-glucoside component isolated from the bud extract The present invention was completed by confirming that they exhibit the efficacy of protecting retinal nerve cells and hippocampal nerve cells from glutamate-induced oxidative stress.

KR 10-1503429 B1 KR 10-2010-0082054 A KR 10-0877371 B1 KR 10-1807367 B1 KR 10-2016-0058613 A

Jung, H. A. Journal of Ethnopharmacology, 2016, vol 191, 152-160; Inhibitory activities of major anthraquinones and other constituents from Cassia obtusifolia against β-secretase and cholinesterases. Shrestha, S. et al. Archives Pharmacal Research, 2018, online publication https://doi.org/10.1007/s12272-018-1044-0; Two new naphthalenic lactone glycosides from Cassia obtusifolia L. seeds.

An object of the present specification is to provide a naphthopyrone derivative of Formula 1, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof.

[Formula 1]

Another object of the present specification is to provide a composition exhibiting an effect of protecting nerve cell damage from oxidative stress or inhibiting nerve cell death.

Another object of the present specification is to provide a composition exhibiting a prophylactic or therapeutic effect of a neuro-damaging disease caused by damage or death of retinal nerve cells or hippocampal nerve cells.

Another object of the present specification is to provide a composition having an antioxidant effect.

In order to achieve the above object, in one aspect, the present invention provides a naphthopyrone derivative of Formula 1, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof.

[Formula 1]

In another aspect, the present invention is one or more naphthopyrone derivatives selected from the group consisting of naphthopyrone derivatives of Formulas 1 to 5, stereoisomers thereof, pharmaceutically acceptable salts, hydrates thereof, or solvents thereof Provides a method for manufacturing cargo.

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

In another aspect, the present invention is one or more naphthopyrone derivatives selected from the group consisting of the naphthopyrone derivatives of Formulas 1 to 5, stereoisomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, or One or more selected from the group consisting of solvates, Cassia obtusifolia L. or Cassia tora L. bud extract, or a bud fraction containing it as an active ingredient, for protecting neurons from oxidative stress Or provides a composition for inhibiting nerve cell death.

In another aspect, the present invention is one or more naphthopyrone derivatives selected from the group consisting of the naphthopyrone derivatives of Formulas 1 to 5, stereoisomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, or One or more selected from the group consisting of solvates, Cassia obtusifolia L. or Cassia tora L. bud extract, or a fraction of the bud containing it, as an active ingredient, of retinal nerve cells or hippocampal nerve cells Provided is a composition for the prevention or treatment of a neuro-damaging disease caused by damage or death.

In another aspect, the present invention is one or more naphthopyrone derivatives selected from the group consisting of the naphthopyrone derivatives of Formulas 1 to 5, stereoisomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, or Provided is an antioxidant composition comprising at least one selected from the group consisting of solvates, Cassia obtusifolia L. or Cassia tora L. sprout extract, or a fraction of the sprout comprising it as an active ingredient.

The composition according to one aspect of the present invention has an antioxidant effect and an effect of protecting nerve cells from oxidative stress or inhibiting apoptosis, and in particular, damage or death of retinal nerve cells or hippocampal nerve cells by glutamate neurotoxicity. It has the effect of suppressing. Therefore, the composition according to one aspect of the present invention can be used for the treatment or prevention of deterioration and deterioration and ocular diseases caused by optic nerve damage by protecting the retinal nerve cells, and protect the hippocampal nerve cells to reduce memory loss due to brain nerve damage, learning It can be used for the treatment or prevention of degenerative and depressive disorder occurrence and deterioration and degenerative cranial nerve disease, and can also be used for mental health such as memory and learning ability improvement, stress relief, and eye health protection such as optic nerve protection and vision loss improvement. It can be used as a bar, pharmaceutical or food composition.

1 is a diagram showing the free radical scavenging activity of DPPH (α,α-diphenyl-β-picrylhydrazyl) of the Cassia tora extract (ST) and Cassia tora sprout extract (STS).
FIG. 2 is a diagram showing ABTS online antioxidant HPLC chromatogram of Cassia tora extract (ST) and Cassia tora sprout extract (STS). The chromatogram of the blue line indicated in the upward direction is a diagram showing the components detected at 254 nm of ultraviolet (UV), and the chromatogram of the red line indicated in the downward direction reacts with ABTS reagent to UV/VIS (ultraviolet/visible light) 734 nm It is a diagram showing the antioxidant components detected in.
FIG. 3 is a diagram showing ABTS online antioxidant HPLC chromatograms of bearer sprout extracts (STS-C, STS-385, STS-465, STS-645 and STS-780) grown under various light conditions. In each chromatogram of (A) to (F), the chromatogram indicated in the upward direction is a diagram showing the components detected at ultraviolet (UV) 254 nm, and the chromatogram indicated in the downward direction reacts with ABTS reagent to react with UV/VIS ( UV/Visible light) It is a diagram showing antioxidant components detected at 734 nm. The sample of (A) is a seedling sprout extract grown for 2 weeks under light-shielding conditions (STS of Example 1), and the sample of (B) is a seedling sprout extract grown for 2 weeks under normal light conditions (STS of Example 2) The samples of C) and (C) were cultivated for 2 weeks under 385 nm LED illumination (STS-385 of Example 2), and the samples of (D) were cultivated for 2 weeks under 465 nm LED illumination The extract (STS-465 of Example 2), the sample of (E) was cultivated for 2 weeks under 645 nm LED illumination, and the bud extract (STS-645 of Example 2), the sample of (F) was 780 nm LED illumination It was a cultivated sprout extract (STS-780 of Example 2) grown for 2 weeks under.
Figure 4 is an HPLC chromatogram showing the peaks of the main components separated from the bud extract (STS) of Example 1, which is an embodiment of the present invention.
Figure 5 is a comparative example 1 of the present invention extracts (ST), the seedling sprout extract cultivated under light-shielding conditions (STS of Example 1) and the seedling sprout extracts grown under various light conditions (STS, STS of Example 2) -C, STS-385, STS-465, STS-645 and STS-780) are graphs showing the efficacy of protecting damage to retinal progenitor cells (R28) caused by glutamate toxicity.
Figure 6 is a retinal progenitor cell induced by glutamate toxicity by concentration of each compound (Compounds 1 to 5 of Examples 4 and 5, and Compounds A to Compound X of Comparative Example 2) isolated from the seedling sprout extract (STS) (R28) It is a graph showing the efficacy of protecting damage.
Figure 7 is a comparative example 1 of the present invention extract extract (ST), seedling sprout extract grown in light-shielding conditions (STS of Example 1) and seedling sprout extracts grown under various light conditions (STS, STS of Example 2) -C, STS-385, STS-465, STS-645 and STS-780) are graphs showing the efficacy of protecting hippocampal nerve cell (HT-22) damage caused by glutamate toxicity.
8 is a hippocampal neuron induced by glutamate toxicity by concentration of each compound (Compounds 1 to 5 of Examples 4 and 5, and Compounds A to Compound X of Comparative Example 2) isolated from the seedling sprout extract (STS). (HT-22) It is a graph showing the efficacy of protecting damage.

In one aspect, the present invention may relate to a naphthopyrone derivative of Formula 1, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof.

[Formula 1]

The naphthopyrone derivative of Formula 1 may be 7-hydroxymusizinyl-rubrofusarin-8'-O-glucopyranoside.

"Pharmaceutically acceptable" herein is intended to be approved by the government or equivalent regulatory agency for use in animals, more specifically in humans, by avoiding significant toxic effects when used in conventional medicinal dosages. It means that it can be received, approved, listed in the pharmacopeia or recognized as described in other general pharmacopoeia.

“Pharmaceutically acceptable salt” as used herein refers to a salt according to an aspect of the present invention having pharmacologically acceptable and desirable pharmacological activity of a parent compound. The salt (1) is formed of an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; Or acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl) Benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-Toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo [2,2,2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert Acid addition salts formed from organic acids such as butylacetic acid, lauryl sulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, and muconic acid; Or (2) a salt formed when the acidic proton present in the parent compound is substituted.

“Isomers” as used herein, in particular optical isomers (eg, essentially pure enantiomers, essentially pure diastereomers or mixtures thereof), as well as conformational isomers ( conformation isomers (i.e., isomers differing only in the angle of one or more chemical bonds), position isomers (especially tautomers) or geometric isomers (e.g., cis-trans isomers) do.

As used herein, “essentially pure,” when used in connection with, for example, enantiomers or diastereomers, contains at least about 90%, preferably at least about 95% of specific compounds that may exemplify enantiomers or diastereomers. , More preferably at least about 97% or at least about 98%, even more preferably at least about 99%, even more preferably at least about 99.5% (w/w).

As used herein, "hydrate" refers to a compound to which water is bound, and is a broad concept including an inclusion compound having no chemical bonding force between water and the compound.

As used herein, "solvate" refers to a higher order compound formed between molecules or ions of a solute and molecules or ions of a solvent.

In one aspect, the present invention, one or more naphthopyrone derivatives selected from the group consisting of naphthopyrone derivatives of Formulas 1 to 5, stereoisomers thereof, pharmaceutically acceptable salts, hydrates thereof, or solvents thereof A method of manufacturing a cargo, comprising: the naphthopyrone derivative, its stereoisomer, its pharmaceutically acceptable salt, its hydrate, or solvate thereof from a sprout ( Cassia obtusifolia L. or Cassia tora L.) It may be related to a method for preparing a naphthopyrone derivative, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof, comprising the step of separating at least one selected from the group.

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

The compound of Formula 1 (naphthopyrone derivative) may be 7-hydroxymushizinyl-rubbrofusarin-8'-O-glucoside, and the compound of Formula 2 (naphthopyrone derivative) is isotolactone ( isotoralactone), the compound of formula 3 (naphthopyrone derivative) may be toralactone (toralactone), the compound of formula 4 (naphthopyrone derivative) may be torosacrysone (torosachrysone), the The compound of Formula 5 (naphthopyrone derivative) may be rubrofusarin.

The sprout may be grown by germinating seeds for a short period of time, or may be cultivated by germinating seeds indoors, or may be a sprout that is generally sold.

Sprout of the above-mentioned manufacturing method is a sprout or young plant grown or grown naturally for 2 to 31 days by germination of seeds of Cassia obtusifolia L. or Ginjiang Namcha ( Cassia tora L. or Senna tora ) 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, 14 days or more, 15 days or more, 16 days or more, 17 days or more, 18 days or more, 19 days or more, 20 days or more, 21 days or more, 22 days or more, 23 days or more, 24 days or more, 25 days Or more, 26 or more, 27 or more, 28 or more, 29 or more or more than 30 or more naturally grown or grown buds or young plants, 31 or less, 30 or less, 29 or less, 28 or less, 27 days or less, 26 days or less, 25 days or less, 24 days or less, 23 days or less, 22 days or less, 21 days or less, 20 days or less, 19 days or less, 18 days or less, 17 days or less, 16 days or less, 15 days 14 days or less, 13 days or less, 12 days or less, 11 days or less, 10 days or less, 9 days or less, 8 days or less, 7 days or less, 6 days or less, 5 days or less, 4 days or less, or 3 days or less It may be a sprout or a young plant that has been grown or cultivated, and more specifically, may be a sprout or a young plant that has been naturally grown or cultivated for a selected period of a period from 2 days to 21 days.

The term ( Cassia obtusifolia L. or Cassia tora L.) refers to the herbaceous genus dicotyledonous plant Rosaceae legume, and the terminus ( Cassia obtusifolia L.) or Long Gangnam ( Cassia tora L. or Senna) tora ) as a mature seed. In general, the above names ( Cassia obtusifolia L.) and Ginjiang Namcha ( Cassia tora L. or Senna tora ) are mixed and sold and used, making it difficult to distinguish between them. The extract of terminator has been known to improve diabetes, improve dyslipidemia, protect liver, antibacterial, and lower blood pressure (Dong, X. et al. Mol. Med. Rep. 2017, 16, 2331-2346), but has oxidative stress or drugs Nothing is known about the efficacy of improving damage or inhibiting damage to retinal nerve cells or hippocampal nerve cells, especially due to glutamate.

The seedling sprout or young plant may be all or part of the seedling sprout or young plant. The part may be an outpost or a ground portion or an underground portion. The above-ground portion may be a stem, a leaf, a flower, or a combination thereof. The underground portion may be a root. The plant may be naturally grown or artificially cultivated. The bud of the present invention can be easily cultivated indoors and can be cultivated for a short period of time within a few weeks without additional nutrient supply, and thus has great industrial applicability.

The above-mentioned sprout extract also includes a crude extract or a fraction in which an extract is additionally fractionated. Specifically, the extract of the sprouted bud may be a crude extract, fraction of a sprouted bud, or a combination thereof. The crude extract is obtained by contacting the sprouted bud with an extraction solvent. The fraction refers to a substance containing specific components separated from the crude extract. The extract, or a fraction thereof, may include one or more mixtures of extracts, fractions thereof, or small fractions of a named sprout plant in the composition of the present invention. The small fraction may be obtained by passing through an ultrafiltration membrane having a cut-off value, or may be obtained through column chromatography or solvent fractionation. The bud extract or fraction may be one containing at least one naphthopyrone derivative selected from the group consisting of the naphthopyrone derivatives of Formulas 1 to 5.

The separation may be by filtration, immersion, centrifugation, solvent fractionation, chromatography or a combination thereof. The chromatography is prepared for separation according to various conditions, namely, size, charge, hydrophobicity or affinity, ion exchange chromatography, affinity chromatography, size exclusion chromatography, HPLC, high-speed fluid chromatography, column chromatography, reverse phase Column chromatography or a combination thereof.

The extraction may include incubating the plant for a period of time in a solvent. The extraction may be performed with or without stirring, or may include heating. The incubation may be performed without stirring or stirring at room temperature to reflux temperature. The incubation temperature can be appropriately selected depending on the solvent selected. For example, it may be room temperature to reflux temperature, 30°C to reflux temperature, and 40°C to reflux temperature. The heating may include heating to 50°C, 60°C, 70°C, 80°C, or reflux temperature. The heating may be heating to 50°C to reflux temperature, 60°C to reflux temperature, 70°C to reflux temperature, 80°C to reflux temperature, or reflux temperature. The extraction time may vary depending on the selected temperature, for 1 hour to 2 months, for example, 1 hour to 1 month, 1 hour to 15 days, 1 hour to 10 days, 1 hour to 5 days, and 1 hour to 3 days , 1 hour to 2 days, 1 hour to 1 day, 5 hours to 1 month, 5 hours to 15 days, 5 hours to 10 days, 5 hours to 5 days, 5 hours to 3 days, 5 hours to 2 days, 5 It may be an hour to 1 day, 10 hours to 1 month, 10 hours to 15 days, 10 hours to 10 days, 10 hours to 5 days, 10 hours to 3 days, or 10 hours to 2 days. The extraction may be reflux extraction of the plant in a solvent. The solvent may have a volume of 1, 2, 5, 10, or 15 times or more with respect to the plant weight. The solvent may have a volume of 1 to 15 times, 2 to 15 times, 5 to 15 times, 10 to 15 times, or about 15 times the weight of the plant. The plant may be dried in a shade or shading facility or a warm air or drying device.

As the extraction method, a conventional method in the art may be used, such as filtration, hot water extraction, immersion extraction, cold extraction, microwave extraction, reflux cooling extraction, pressure extraction, subcritical extraction, supercritical extraction, and ultrasonic extraction. For example, it may be an immersion extraction method. The immersion extraction may be immersed at a warm temperature or a normal temperature, and may be one to five extractions. The seedling sprout plant may be in contact with the extraction solvent of 0.1 to 10 times or 1 to 6 times. Cold extraction temperature may be 20 ℃ to 40 ℃. The temperature of warming or heating extraction may be 40°C to 100°C. The cold immersion extraction time may be 24 hours to 120 hours, and the warm immersion or heating extraction temperature may be 0.5 hours to 48 hours.

The extraction may also include removing the solvent from the obtained extract by known methods, such as evaporation or concentrated under reduced pressure. The extraction may also include preparing the dried extract by drying the obtained extract such as lyophilization. The reduced pressure concentrator may be a vacuum reduced pressure concentrator or a vacuum rotary evaporator. Further, the drying may be drying under reduced pressure, vacuum drying, boiling drying, spray drying or freeze drying.

The manufacturing method may further include the step of extracting the resulting sprout as a solvent selected from the group consisting of water, C1 to C6 alcohols, and mixed solvents thereof. The alcohol may be C1 to C3 alcohol, C1 to C4, C1 to C5 or C1 to C6 alcohol. The alcohol may be a primary alcohol. The alcohol of C1 to C6 may be methanol, ethanol, propanol, isopropanol, butanol or a mixture thereof.

In addition, the manufacturing method may further include a process of fractionating one or more selected from the group consisting of water, ethyl acetate, hexane, methylene chloride, chloroform, methanol, ethanol, acetone, and mixed solvents thereof. The manufacturing method is at least one naphthopyrone derivative selected from the group consisting of the naphthopyrone derivatives of Chemical Formulas 1 to 5 based on the total weight of the extract obtained through the process of extracting the named buds, stereoisomers thereof, The pharmaceutically acceptable salt, hydrate, or solvate thereof may include a process for preparing a fraction of a sprouted bud containing 1 to 20% by weight, specifically 1% by weight or more, 2% by weight or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13 wt% or more, 14 wt% or more, 15 wt% or more, 16 wt% or more, 17 wt% or more, 18 wt% or more, or 19 wt% or more, and may include a process of preparing a sprouted fraction containing at least 19 wt%, 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% by weight, 9% by weight, 8% by weight, 7% by weight, 6% by weight, 5% by weight, 4% by weight, 3% by weight or 2% by weight It may be to include a manufacturing process.

One or more naphthopyrone derivatives selected from the group consisting of the naphthopyrone derivatives of Formulas 1 to 5, stereoisomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, or solvates thereof are defined as bud extracts or fractions It may be included in 1 to 50% by weight based on the total weight, specifically, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% % Or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% % Or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 25% or more, 26% or more, 27% or more % Or more, 28% or more, 29% or more, 30% or more, 31% or more, 32% or more, 33% or more, 34% or more, 35% or more, 36% or more, 37% or more % Or more, 38% or more, 39% or more, 40% or more, 41% or more, 42% or more, 43% or more, 44% or more, 45% or more, 46% or more, 47% or more % Or more, 48% or more, or 49% or more, 50% or less, 49% or less, 48% or less, 47% or less, 46% or less, 45% or less, 44% or less , 43% or less, 42% or less, 41% or less, 40% or less, 39% or less, 38% or less, 37% or less, 36% or less, 35% or less, 34% or less , 33% or less, 32% or less, 31% or less, 30% or less, 29% or less, 28% or less, 27% or less, 26% or less, 25% or less, 24% or less, 23% or less, 22% or less, 21% or less, 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% by weight or less, 3% by weight or less, or 2% by weight or less.

In one aspect of the present invention, at least one naphthopyrone derivative selected from the group consisting of the naphthopyrone derivatives of Formulas 1 to 5, stereoisomers thereof, pharmaceutically acceptable salts, hydrates thereof, or solvents thereof One or more selected from the group consisting of cargo, Cassia obtusifolia L. or Cassia tora L. bud extract, or a bud fraction containing it as an active ingredient, for protecting neurons from oxidative stress, or It may be related to a composition for inhibiting nerve cell death. The at least one naphthopyrone derivative selected from the group consisting of naphthopyrone derivatives of Formulas 1 to 5 is selected from the group consisting of naphthopyrone derivatives of Formula 1 and naphthopyrone derivatives of Formulas 2 to 5 It may be one or more naphthopyrone derivatives. Descriptions of the naphthopyrone derivatives of Formulas 1 to 5, pharmaceutically acceptable salts, stereoisomers, hydrates, solvates, buds, buds, extracts or fractions As described above.

The composition may be a composition for protecting nerve cells or inhibiting neuronal cell death, and specifically, may be a composition for protecting nerve cells from oxidative stress induced by metabolic toxicity, neurotoxicity, chemical causes, etc., or a composition for inhibiting neuronal cell death. The oxidative stress may be caused by glutamate, glutamate toxicity, or glutamate neurotoxicity, or calcium homeostatic disorders similar to oxidative stress caused by glutamate neurotoxicity, mitochondrial dysfunction, excitatory cytotoxicity, It may be oxidative stress caused by depletion of nutritional factors. Specifically, the composition reduces, inhibits, ameliorates, or prevents damage to retinal nerve cells or hippocampal nerve cells caused by glutamate, glutamate toxicity, or glutamate neurotoxicity, or resuscitate dead retinal nerve cells or hippocampal nerve cells, or It may be regenerating, and it protects neurons by activating antioxidants, which are defense mechanisms against oxidative stress caused by glutamate neurotoxicity, dysregulation of calcium homeostasis, dysfunction of mitochondria, excitatory cell toxicity, depletion of nutritional factors, or regulation of glutamate metabolism. Or it may be to suppress the death of nerve cells.

According to an embodiment of the present invention, the extract of the sprouted shoots has a DPPH free radical scavenging effect of 1.7 times or more superior to the extract of the shooter (Test Example 1), and the compounds of Formulas 1 to 5 corresponding to antioxidant components (naph Topirone derivative) is high in content, and exhibits a superior antioxidant effect (Test Example 2), the composition of the present invention is activated by the antioxidant mechanism, which is a defense mechanism against oxidative stress, to protect nerve cells or inhibit neuronal cell death. It was confirmed that the effect is excellent.

The composition may be to be treated before, simultaneously with, or after the occurrence of nerve cell damage or death. The composition is from the group consisting of at least one naphthopyrone derivative selected from the group consisting of the naphthopyrone derivatives of Formulas 1 to 5, stereoisomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, or solvates thereof. One or more selected, Cassia obtusifolia L. or Cassia tora L. sprout extract, or Cassia sprout fraction comprising thereof, may contain 0.001% to 80% by weight based on the total weight of the composition, specifically 0.001 Above weight%, above 0.01%, above 0.05%, above 0.1%, above 1%, above 2%, above 3%, above 4%, above 5%, above 10%, 20 It may include at least 30% by weight, at least 30% by weight, at least 40% by weight, or at least 60% by weight, 80% by weight or less, 60% by weight or less, 40% by weight or less, 30% by weight or less, 20% by weight or less, 10% by weight % Or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less.

In addition, the above-mentioned bud extract or fraction is at least one naphthopyrone derivative selected from the group consisting of naphthopyrone derivatives of Formulas 1 to 5, stereoisomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, or The solvate may include 1 to 50% by weight based on the total weight of the named bud extract or fraction, specifically, 1% by weight or more, 2% by weight or more, 3% by weight or more, 4% by weight or more, 5% by weight % Or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more % Or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 25% or more % Or more, 26% or more, 27% or more, 28% or more, 29% or more, 30% or more, 31% or more, 32% or more, 33% or more, 34% or more, 35% or more % Or more, 36% or more, 37% or more, 38% or more, 39% or more, 40% or more, 41% or more, 42% or more, 43% or more, 44% or more, 45% or more % Or more, 46% or more, 47% or more, 48% or more, or 49% or more, and 50% or less, 49% or less, 48% or less, 47% or less, 46% or more Or less, 45% or less, 44% or less, 43% or less, 42% or less, 41% or less, 40% or less, 39% or less, 38% or less, 37% or less, 36% or less Or less, 35% or less, 34% or less, 33% or less, 32% or less, 31% or less, 30% or less, 29% or less, 28% or less, 27% or less Lower, 26% or less, 25% or less, 24% or less, 23% or less, 22% or less, 21% or less, 20% or less, 19% or less, 18% or less, 17% or less Or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less Or less, 6% by weight or less, 5% by weight or less, 4% by weight or less, 3% by weight or less or 2% by weight or less.

The nerve cells may be central nerves, peripheral nerves, or optic nerves associated with the brain hippocampus or eye retina, and specifically, may be retinal nerve cells or hippocampal nerve cells.

The retinal nerve cells are rod cells, cone cells, bipolar cells, retinal amacrine cells, horizontal cells and retinal ganglion cells. ) May be one or more cells selected from the group consisting of. The rod cells and cone cells are neurons that recognize objects by sensing light, and the bipolar cells, retinal amacrine cells, and horizontal cells are neurons that transmit visual information to retinal ganglion cells, and the retinal ganglion cells are retinal cells. It is a nerve cell that transmits visual information to the brain. R28 retinal progenitor cells or R28 retinal progenitor cells of one embodiment of the present invention express the traits of various constituent cells of the retina and survive even when transplanted into the retina, and cell death occurs against hypoxia or deficiency of serum, and glaucoma It can also be useful for apoptosis and cytotoxicity studies linked to glutamate or GABA receptor expression. In addition, R28 cells can differentiate from retinal progenitor cells to retinal ganglion cells depending on the culture conditions, and can be used not only for overall cytological studies of the retina, but also for basic research of retinal ganglion cells (Jungil Lee, Jaewoo Kim, Korean Journal of Ophthalmology) , 2009, 50, 919-922).

The hippocampal nerve cells are neurons of the hippocampus, a structure of the temporal lobe of the brain, and the hippocampus is vulnerable to physiological/oxidative stress stimulation and causes cognitive impairment due to stress, and is known as the central tissue. It can cause hippocampal structure and brain cell production, synaptic plasticity, and behavioral changes related to the hippocampus (Eunjoo Kim, Journal of the Korean Psychological Association: Cognitive and Biological, 2012, 24, 65-88). The HT-22 hippocampal nerve cell of one embodiment of the present invention is sensitive to glutamate and can be used as an in vitro model system for studying neurotoxicity caused by oxidative stress (Liu, J. et al. Life Science , 2009, 84, 267-271; Jihyun Kim, Soonsil Jeon, Korean Journal of Food and Nutrition, 2017, 46, 886-890).

According to an embodiment of the present invention, when treating each of the naphthopyrone derivatives of Formula 1 to Formula 5 with the bud extract, R28 cells having oxidative stress induced by glutamate treatment, reduced cell viability by glutamate It was elevated to a degree corresponding to the control group, and this retinal cell protection efficacy was superior to that of the comparative example, the extract of the present invention. The composition of the present invention has the effect of protecting the damage of retinal nerve cells due to oxidative stress or inhibiting retinal cell death. It was confirmed that it was excellent (Test Examples 3 and 4).

In addition, according to an embodiment of the present invention, upon treatment of each of the naphthopyrone derivatives of Formula 1 to Formula 5, which are modified with glutamate treatment, each of the naphthopyrone derivatives in HT-22 cells inducing oxidative stress is reduced by glutamate. The increased cell viability was increased to a level corresponding to the control group, and the effect of protecting the hippocampal neurons was superior to that of the extract of the comparative example. The composition of the present invention protects damage to the hippocampal nerve cells by oxidative stress or kills the hippocampal nerve cells. It was confirmed that the effect of inhibiting is excellent (Test Examples 5 and 6).

In one aspect of the present invention, at least one naphthopyrone derivative selected from the group consisting of the naphthopyrone derivatives of Formulas 1 to 5, stereoisomers thereof, pharmaceutically acceptable salts, hydrates thereof, or solvents thereof Damage to retinal nerve cells or hippocampal nerve cells comprising at least one selected from the group consisting of cargo, Cassia obtusifolia L. or Cassia tora L. sprout extract, or Cassia tora sprout fraction containing it as an active ingredient. Or it may be related to a composition for the prevention or treatment of neuro-damaging diseases caused by death, or to a composition for the prevention or treatment of neuro-damaging diseases caused by glutamate neurotoxicity. One or more naphthopyrone derivatives selected from the group consisting of naphthopyrone derivatives of Formulas 1 to 5 may be selected from the group consisting of naphthopyrone derivatives of Formula 1 and naphthopyrone derivatives of Formulas 2 to 5 It may be a species of more than one naphthopyrone derivative. Descriptions of the naphthopyrone derivatives of Formulas 1 to 5, pharmaceutically acceptable salts, stereoisomers, hydrates, solvates, modified buds, modified bud extracts or fractions, and contents are as described above.

The neurodegenerative disease caused by damage or death of the retinal nerve cells or hippocampal nerve cells may be due to glutamate neurotoxicity, and the description of the retinal nerve cells and hippocampal nerve cells is as described above. The neurodegenerative disease may be one or more selected from the group consisting of diabetic retinopathy due to retinal nerve cell damage or death, macular degeneration, vision impairment due to retinal cell damage, retinal pigmentation, retinal detachment, retinal vessel occlusion and glaucoma. And may be one or more selected from the group consisting of hippocampal nerve cell damage or apoptosis due to memory loss or death, learning ability decline, depressive disorder, amyotrophic lateral sclerosis (ALS), Parkinson's disease, and cerebral ischemic neuropathy. The disease caused by damage or death of retinal nerve cells or hippocampal nerve cells caused by the disease is not limited.

The glutamate neurotoxicity refers to the toxicity that is caused by the rapid increase in the concentration of glutamate outside the neurons and the action of glutamate as an oxidative neurotoxic substance. The glutamate neurotoxicity may be caused by glutamate introduced from the outside of the subject, and the influx of glutamate may include ingestion of food containing glutamate, a therapeutic agent containing glutamate, a preventive agent containing glutamate, an antibiotic containing glutamate, and the like. Or it may be by administration, but is not limited thereto.

One or more naphthopyrone derivatives selected from the group consisting of the naphthopyrone derivatives of Formulas 1 to 5 may be isolated or purified from buds of Cassia obtusifolia L. or Cassia tora L. It may be separated or purified from the ethanol extract, after fractionating the sprouted ethanol extract with ethyl acetate, the ethyl acetate fraction is separated by chromatography or the ethyl acetate fraction is again mixed solvent of hexane, methylene chloride and methanol. It may be fractionated and separated by chromatography.

The extract of the sprouted bud may be extracted by using the sprouted bud as a solvent selected from the group consisting of water, alcohols of C1 to C6, and mixed solvents thereof. The alcohol may be C1 to C3 alcohol, C1 to C4, C1 to C5 or C1 to C6 alcohol. The alcohol may be a primary alcohol. The alcohol of C1 to C6 may be methanol, ethanol, propanol, isopropanol, butanol or a mixture thereof.

The fraction may further include a process of fractionating one or more selected from the group consisting of water, ethyl acetate, hexane, methylene chloride, chloroform, methanol, ethanol, acetone, and mixed solvents thereof.

According to one embodiment of the present invention, at least one naphthopyrone derivative selected from the group consisting of the naphthopyrone derivatives of Chemical Formulas 1 to 5 is an antioxidant component and is included in a higher content of the extract of the sprouted bud compared to the extract of Cassia tora. (Test Example 2), the naphthopyrone derivative has excellent efficacy in protecting retinal nerve cells (R28) and hippocampal nerve cells (HT-22) damaged or killed by glutamate treatment (Test Examples 4 and 6). , It was confirmed that the naphthopyrone derivatives of Chemical Formulas 1 to 5 are effective components of the buds having the efficacy of preventing or treating neuropathic diseases caused by glutamate neurotoxicity.

In one aspect of the present invention, at least one naphthopyrone derivative selected from the group consisting of the naphthopyrone derivatives of Formulas 1 to 5, stereoisomers thereof, pharmaceutically acceptable salts, hydrates thereof, or solvents thereof One or more selected from the group consisting of cargo, Cassia obtusifolia L. or Cassia tora L. sprout containing extract, or containing a fraction of the sprout containing it as an active ingredient, may be related to an antioxidant composition. One or more naphthopyrone derivatives selected from the group consisting of naphthopyrone derivatives of Formulas 1 to 5 may be selected from the group consisting of naphthopyrone derivatives of Formula 1 and naphthopyrone derivatives of Formulas 2 to 5 It may be a species of more than one naphthopyrone derivative. Descriptions of the naphthopyrone derivatives of Formulas 1 to 5, pharmaceutically acceptable salts, isomers, hydrates, solvates, modified buds, modified bud extracts or fractions, and contents are as described above.

The composition of the present invention may be a pharmaceutical composition, a cosmetic composition or a food composition.

Pharmaceutical compositions according to one aspect of the invention may be formulated in oral or parenteral dosage forms. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, soft or hard capsules, etc. These solid preparations include at least one excipient in one or more compounds, such as starch, calcium carbonate, sucrose. Or it is prepared by mixing lactose, gelatin, and the like. Also, in addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspending agents, intravenous solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used as diluents, various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, can be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. As the non-aqueous solvent and suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin may be used.

The pharmaceutical dosage form of the composition according to one aspect of the present invention may be used in the form of their pharmaceutically acceptable salts, and may also be used alone or in combination with other pharmaceutically active compounds as a suitable set. The salt is not particularly limited as long as it is pharmaceutically acceptable, for example, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid, hydrobromic acid, acetic formic acid, tartaric acid, lactic acid, citric acid, fumaric acid, maleic acid, succinic acid, methanesulfonic acid , Benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid, and the like. In addition, the parenteral dosage form can be a dermal patch, ointment, cream, eye drop, spray or injection.

In another aspect, the present invention may be a method for protecting a neuron of an individual, comprising administering the pharmaceutical composition to the individual.

The subject can be a mammal, for example, a human, a cow, a horse, a pig, a dog, a sheep, a goat, or a cat, and the mammal can be a human, and an effective dose of the compound of the present invention to the human body May vary depending on the patient's age, weight, gender, dosage form, health condition and degree of disease.

The administration may be administered in various dosage forms for oral administration or parenteral administration such as intravenous, intraperitoneal, intradermal, subcutaneous, epithelial or intramuscular administration, and when formulated, usually used fillers, extenders, binders, wetting agents, and disintegrants It is prepared using diluents or excipients such as release agents and surfactants.

The administration can be administered by methods known in the art. Administration can be administered directly to a subject by any means, for example, by intravenous, intramuscular, oral, or subcutaneous administration. The administration can be administered systemically or locally. The administration may be administered locally to a region where neurons are present or are expected to occur.

The food composition according to one aspect of the present invention may be a health functional food composition.

The formulation of the food composition is not particularly limited, but may be formulated in a form of ingesting orally or by directly or diluting a concentrate or powder, for example, tablets, granules, powders, liquids such as drinks, caramels , Gels, bars, and the like. The food composition of each formulation may be formulated by appropriately selecting the ingredients commonly used in the field in addition to the active ingredients according to the formulation or purpose of use without difficulty, and synergistic effects may occur when applied simultaneously with other raw materials. Specifically, the food composition may contain various flavoring agents or natural carbohydrates as additional components. The natural carbohydrate may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as taumatin and stevia extract, and synthetic sweeteners such as saccharin and aspartame can be used. The ratio of the natural carbohydrate can be selected from 0.01 to 0.04 parts by weight per 100 parts by weight of the composition, specifically, about 0.02 to 0.03 parts by weight. In addition, the food composition is a variety of nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonic acid And carbonated agents used in beverages. In addition, the functional food of the present invention may contain flesh for the production of natural fruit juice, fruit juice beverage and vegetable beverage. These ingredients can be used independently or in combination. The proportion of these additives is not so critical, but is generally included in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition herein.

In the food composition, the determination of the dosage of the active ingredient is within the level of those skilled in the art, the daily dosage of which is, for example, 0.1 mg/kg/day to 5000 mg/kg/day, more specifically 50 mg/kg. /Day to 500 mg/kg/day, but is not limited thereto, and may vary according to various factors such as age, health condition, and complications of the subject to be administered.

Food composition according to an aspect of the present invention, for example, chewing gum, caramel products, candy, ice cream, confectionery, various foods, soft drinks, mineral water, alcoholic beverages, beverage products, vitamins or minerals, etc. Functional foods.

The composition of the present invention may be a quasi-drug composition.

The'non-drug product' refers to drugs that are used for the purpose of drugs in accordance with the Pharmaceutical Affairs Act, and are drugs based on a classification standard separately set by the Ministry of Health and Welfare for products that have less action on the human body than drugs used to treat or prevent diseases. do. Therefore, fiber or rubber products used for the treatment or prevention of human or animal diseases, and the action on the human body may be slight or indirect, similar to that which is not an instrument or machine, or sterilization and pesticides to prevent infectious diseases. Can be. The quasi-drug composition of the present invention may be for improving, protecting or treating damage to nerve cells due to oxidative stress, and may be for preventing or improving neuro-damaging diseases caused by glutamate neurotoxicity.

The quasi-drug composition may further include a quasi-drugally acceptable excipient or carrier.

The quasi-drug composition may be formulated in the form of skin smears, creams, pars, eye drops, sprays.

Hereinafter, the present invention will be described in more detail through examples and test examples. However, these examples and test examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples and test examples.

[ Example One] Determination Preparation of sprout extract

After purchasing undisinfected domestic Cassia obtusifolia L. and Cassia tora L. (Dooson Aecho Herbs Co., Ltd., January 2016), bleached powder (CaOCl ₂ ) was sterilized at a concentration of 20 mg/mL for 15 minutes and washed thoroughly with water. After immersion in purified water for 4 hours, it was cultivated for 6 days in light-shielding conditions. The resulting buds obtained through the above cultivation were dried and warmed at 32° C. for 22 hours (38° C.) and pulverized to obtain 698.4 g of dried plants. Then, the dried plants were placed in an extraction container, and 5.5 L of ethanol was added, stirred at room temperature and stirred to extract for 7 days, and the mixture was Whatman paper filter paper having a film thickness of 0.34 mm and a diameter of 30 The process of gravity filtration using a cm glass funnel was repeated twice ('extraction-filtration' 2 times) to obtain a filtered extract. The filtered extract was put in a reduced pressure concentrator and concentrated by evaporating the solvent completely at 35° C. under reduced pressure to obtain 66.7 g (hereinafter referred to as “STS”) of the bud extract (yield 9.55%).

[ Comparative example 1] Preparation of Cassia tora extract

Purchase undisinfected domestic Cassia tora ( Cassia obtusifolia L. and Cassia tora L.) ( Dusson Aecho Herbs Co., Ltd., January 2016), pulverize, place in a 15 mL extraction container, add 11 mL of ethanol and shake at room temperature After stirring and extracting for 7 days, the mixture was subjected to gravity filtration using a Whatman paper filter paper having a film thickness of 0.34 mm and a 10 cm diameter glass funnel ('extraction-filtration' twice), followed by filtration. Obtained extract was obtained (hereinafter referred to as'ST'). The filtered extract was placed in a reduced pressure concentrator and concentrated by evaporating the solvent completely at 35° C. under reduced pressure (yield 3.34%).

[ Example 2] Various light Under conditions Cultivated Determination Preparation of sprout extract

In order to compare and analyze the components of the sprouted sprout extract (STS) grown under the light-shielding conditions of Example 1 and the ingredients of the sprouted sprout extract grown under specific light conditions, the sprouted shoots were grown with different light wavelength conditions. Cultivated buds were grown in the same manner as in Example 1, but the light conditions at the time of cultivation of the buds were not light-shielding conditions, but the fluorescent light, LED 385 nm, 465 nm, 645 nm, and 780 nm were selected with a large amount of wavelength, respectively. It was cultivated for 6 days each by irradiating light. Each of the bud extracts was prepared in the same manner as in Example 1 using 5 buds obtained through the cultivation, and the extracts obtained by cultivating under fluorescent light conditions were'STS-C', under the light condition of LED 385 nm. The extract obtained by cultivation of'STS-385', the extract obtained by cultivation under light conditions of LED 465 nm is'STS-465', and the extract of cultivation under light conditions of LED 645 nm is'STS-645', LED 780 nm The extract obtained by cultivation under the light condition of'STS-780' was called.

[ Example 3] Determination Sprout extract( STS )of Fraction Produce

After suspending 66.7 g of the bud extract of Example 1 (STS) in 600 mL of water, 1.8 L of ethyl acetate was added, stirred at room temperature and stirred to perform fractionation over 4 times for 2 hours, followed by ethyl acetate fraction (STS- EA) 23.5 g were obtained. The ethyl acetate fraction is methylene chloride (or ethyl acetate) alone or mixed solvent of n -hexane, methylene chloride (or ethyl acetate), methanol (or ethanol) ( n -hexane: methylene chloride mixing ratio (volume ratio) 1: 1, methylene chloride: the mixing ratio (volume ratio) of methanol is 200:1, 50:1, 10:1, 5:1, 3:1 and 1:1) by using 2.4L each as a fractional solvent of the STS-EA Fractions were prepared, ie fractions of bearer sprout extract (STS).

Specifically, the ethyl acetate fraction (STS-EA) was brought into contact with the 8 fraction solvents, stirred in celite, and the solvent was evaporated, and silica (silica) filled in a column having a diameter of 10 cm and a length of 20 cm was evaporated. ) Developed in resin. Chromatography eluting with each of the solvents was performed to obtain 20 fractions (STS-EA-fraction 1 to STS-EA-fraction 20).

[ Example 4] Determination Isolation of compound 1 from bud extract

STS-EA-fraction 12 prepared in Example 3 (the second fraction of 3 fractions in which the mixing ratio of the elution solvent in Example 3 is methylene chloride (or ethyl acetate): methanol (or ethanol) is 10:1. After concentration under reduced pressure), Compound 1 having the structure of Formula 1 was isolated at a retention time of about 221 minutes by performing preparative column chromatography under the conditions of Separation Method 1 below. (14.0 mg, 0.002% by weight of dry plant; 0.021% by weight of dry extract)

Separation method 1

Equipment used: YMC LC-Forte R

Column: Waters Delta-Pak C-18 RP HPLC column (30.0x300 mm, 15 μm)

Solvent: (a) acetonitrile with 0.02% TFA

(b) Water with 0.02% TFA

Mobile phase composition: starting elution at 0: (a) solvent: (b) solvent volume ratio 40:60

0 to 60 minutes (a) Increased the proportion of solvent from 40% to 100%

Mobile phase flow rate: 10.0 mL/min

Detector: ultraviolet 254 nm; ELSD

[ Example 5] Determination Separation of compounds 2 to 5 from bud extract

(1) Separation of compound 3

STS-EA-fraction 12 prepared in Example 3 (the elution solvent in Example 3 is methylene chloride (or ethyl acetate): the second fraction of the three fractions in which the mixing ratio of methanol (or ethanol) is 10:1 ) Was concentrated under reduced pressure, and then, under the conditions of separation method 1 of Example 4, preparative column chromatography was performed to separate 1.2 mg of Compound 3 having the structure of Chemical Formula 3 at a retention time of about 24 minutes.

(2) Separation of Compound 2

STS-EA-fraction 8 of Example 3 (the first fraction of the three fractions in which the mixing ratio of methylene chloride (or ethyl acetate): methanol (or ethanol) in Example 3 is 50:1) is: After concentration under reduced pressure, preparative column chromatography was performed under the condition of the following separation method 2 to separate 7.5 mg of compound 2 having the structure of Chemical Formula 2 at a retention time of about 36 minutes.

Separation method 2

Equipment used: YMC LC-Forte R

Column: Waters Delta-Pak C-18 RP HPLC column (30.0x300 mm, 15 μm)

Solvent: (a) acetonitrile with 0.02% TFA

(b) Water with 0.02% TFA

Mobile phase composition: start elution at 0: (a) solvent: (b) solvent volume ratio 60:40

0 to 60 minutes (a) Increased the proportion of solvent from 60% to 100%

Mobile phase flow rate: 10.0 mL/min

Detector: ultraviolet 254 nm; ELSD

(3) Separation of compounds 4 and 5

In Example 3, STS-EA-fraction 13 (the third fraction of the three fractions in which the mixing ratio of the elution solvent in Example 3 is methylene chloride (or ethyl acetate): methanol (or ethanol) is 10:1) After concentration under reduced pressure, preparative column chromatography was performed under the following separation method 3 to separate 0.6 mg of compound 4 having the structure of Formula 4 between retention time of about 71 minutes and retention time of about 120 minutes. 2.6 mg of Compound 5 having the structure of Formula 5 was isolated.

Separation method 3

Equipment used: JASCO LC-2000PLUS

Column: Phenomenex Luna C-18 RP HPLC column (21.2x250 mm, 10 μm)

Solvent: (a) acetonitrile with 0.02% TFA

(b) Water with 0.02% TFA

Mobile phase composition: Elution starts from 0 min to (a) solvent: (b) solvent volume ratio 10:90

0 to 60 minutes (a) Increased the proportion of solvent from 10% to 50%

60 to 80 minutes (a) Increased the proportion of solvents from 50% to 51%

80 to 140 minutes (a) Increased solvent ratio from 51% to 58%

140 to 180 minutes (a) Increased solvent ratio from 58% to 100%

Mobile phase flow rate: 8 mL/min

Detector: ultraviolet 254 nm

[ Comparative example 2] Determination Separation of compounds A to X from bud extract

(1) Separation of compounds F, G and H

After concentration of the STS-EA-fraction 12 of Example 3 under reduced pressure, preparative column chromatography was performed under the conditions of the separation method 1 of Example 4 to perform component separation for the fraction. As a result, 0.8 mg of compound F corresponding to peak F in FIG. 4, 0.9 mg of compound G corresponding to peak G in FIG. 4, and 0.5 mg of compound H corresponding to peak H in FIG. 4 were separated.

(2) Separation of compounds C, D and E

After concentration of the STS-EA-fraction 8 of Example 3 under reduced pressure, preparative column chromatography was performed under the conditions of the separation method 2 of Example 4 to separate the components contained in the fraction. As a result, 7.2 mg of compound C corresponding to peak C in FIG. 4, 0.8 mg of compound D corresponding to peak D in FIG. 4, and 2.5 mg of compound E corresponding to peak E in FIG. 4 were separated.

(3) Isolation of compound I

STS-EA-fraction 15 obtained in Example 3 (the elution solvent in Example 3 is methylene chloride (or ethyl acetate): the second fraction of the three fractions in which the mixing ratio of methanol (or ethanol) is 5: 1 ) Was concentrated under reduced pressure, and the components containing the fractions were separated by HPLC separation under the conditions of separation method 4 below. As a result, 0.7 mg of compound I corresponding to peak I in FIG. 4 was isolated.

Separation method 4

Equipment used: JASCO LC-2000PLUS

Column: Phenomenex Luna C-18 RP HPLC column (21.2x250 mm, 10 μm)

Solvent: (a) acetonitrile with 0.02% TFA

(b) Water with 0.02% TFA

Mobile phase composition: starting elution at 0: (a) solvent: (b) solvent volume ratio 30:70

0 to 240 minutes (a) Maintain the solvent ratio at 30%

Mobile phase flow rate: 8 mL/min

Detector: ultraviolet 254 nm

(4) Separation of compounds J, K, L, M, N, O, A and P

STS-EA-fraction 16 obtained in Example 3 (third fraction out of three fractions in which the mixing ratio of elution solvent in Example 3 is methylene chloride (or ethyl acetate): methanol (or ethanol) 5:1) ) Was concentrated under reduced pressure, and subjected to silica gel column chromatography under the conditions of the following separation method 5 to fractionate into 14 small fractions.

Separation method 5

Equipment used: Silica Flash Column

Column: Glass column (25.0x200 mm)

Solvent: (a) methylene chloride (methylene chloride)

(b) methanol

Mobile phase composition: (a) solvent: (b) solvent volume ratio 20:1 begins elution

(a) Solvent: (b) Solvent volume ratio changed to 10:1, 7:1, 4:1, 2:1, 1:1

Solvent volume at each stage: 300 mL

The peak of FIG. 4 from the small fraction 3 (the second fraction of the three small fractions in which the mixing ratio of the methylene chloride:methanol in the elution solvent in the separation method 5 is 10:1) is determined under the conditions of the separation method 6 below. 1.8 mg of compound J corresponding to J, 1.9 mg of compound K corresponding to peak K in FIG. 4, and 1.0 mg of compound L corresponding to peak L in FIG. 4 were isolated.

Separation method 6

Equipment used: Gilson 321 HPLC

Column: Phenomenex Luna C-18 RP HPLC column (21.2x250 mm, 10 μm)

Solvent: (a) acetonitrile with 0.02% TFA

(b) Water with 0.02% TFA

Mobile phase composition: starting elution at 0: (a) solvent: (b) solvent volume ratio 20:80

0 to 80 minutes (a) Increased the proportion of solvent from 20% to 40%

80 to 120 minutes (a) Increased the proportion of solvent from 40% to 100%

Mobile phase flow rate: 8 mL/min

Detector: ultraviolet 254 nm

Also, from the small fraction 4 (the third fraction of the three small fractions in which the mixing ratio of the methylene chloride:methanol in the elution solvent in the separation method 5 is 10:1) is shown in Figure 4 through HPLC separation under the conditions of the separation method 7 below. 2.5 mg of compound M corresponding to peak M, 2.1 mg of compound N corresponding to peak N in FIG. 4, and 0.5 mg of compound O corresponding to peak O in FIG. 4 were separated.

Separation method 7

Equipment used: Gilson 321 HPLC

Column: Phenomenex Luna C-18 RP HPLC column (21.2x250 mm, 10 μm)

Solvent: (a) acetonitrile with 0.02% TFA

(b) Water with 0.02% TFA

Mobile phase composition: starting elution at 0: (a) solvent: (b) solvent volume ratio 20:80

0 to 100 minutes (a) Increased the proportion of solvent from 20% to 80%

100 to 160 minutes (a) Increased the proportion of solvent from 80% to 100%

Mobile phase flow rate: 8 mL/min

Detector: ultraviolet 254 nm

The peak of FIG. 4 from the small fraction 6 (the second fraction of the three small fractions in which the elution solvent in the separation method 5 has a methylene chloride:methanol mixing ratio of 7:1) is separated under the conditions of the separation method 8 2.0 mg of Compound A corresponding to A was isolated.

Also, from the small fraction 7 (the third fraction of the three small fractions in which the elution solvent in the separation method 5 has a methylene chloride:methanol mixing ratio of 7:1) is separated through the HPLC separation method of Figure 4 4.1 mg of the compound P corresponding to the peak P was isolated.

Separation method 8

Equipment used: Gilson 321 HPLC

Column: Phenomenex Luna C-18 RP HPLC column (21.2x250 mm, 10 μm)

Solvent: (a) acetonitrile with 0.02% TFA

(b) Water with 0.02% TFA

Mobile phase composition: starting from 0 min (a) solvent: (b) solvent volume ratio 17:83

0 to 50 minutes (a) Maintain the solvent ratio at 17%

50 to 70 minutes (a) Increased the proportion of solvents from 17% to 25%

70 to 150 minutes (a) Increased the proportion of solvents from 25% to 30%

150 to 180 minutes (a) Increased the proportion of solvent from 30% to 100%

Mobile phase flow rate: 6.5 mL/min

Detector: ultraviolet 254 nm

(5) Separation of compound Q

STS-fraction 17 obtained in Example 3 (the first fraction of three fractions in which the mixing ratio of methylene chloride (or ethyl acetate):methanol (or ethanol) in Example 3 is 3:1) is reduced. After concentration, the components containing the fractions were separated by HPLC separation under the conditions of Separation Method 9 below. As a result, 1.7 mg of compound Q corresponding to peak Q in FIG. 4 was isolated.

Separation method 9

Equipment used: JASCO LC-2000PLUS

Column: Phenomenex Luna C-18 RP HPLC column (21.2x250 mm, 10 μm)

Solvent: (a) acetonitrile with 0.02% TFA

(b) Water with 0.02% TFA

Mobile phase composition: Elution starts from 0 min to (a) solvent: (b) solvent volume ratio 10:90

0 to 40 minutes (a) Maintain the solvent ratio at 10%

40 to 70 minutes (a) Increased the solvent ratio from 10% to 18%

70 to 180 minutes (a) Increased the percentage of solvents from 18% to 22%

180 to 260 minutes (a) Increased the percentage of solvents from 22% to 100%

Mobile phase flow rate: 6.5 mL/min

Detector: ultraviolet 254 nm

(6) Separation of compounds B, R, S, T, U, V, W and X

STS-EA-fraction 18 obtained in Example 3 (the second fraction out of three fractions in which the mixing ratio of elution solvent in Example 3 is methylene chloride (or ethyl acetate): methanol (or ethanol) 3:1) After concentration under reduced pressure, silica gel column chromatography was performed under the conditions of the following separation method 10 to fractionate into 24 small fractions.

Separation method 10

Equipment used: Silica Flash Column

Column: Glass column (25.0x200 mm)

Solvent: (a) methylene chloride (methylene chloride)

(b) methanol

Mobile phase composition: (a) solvent: (b) solvent volume ratio 20:1 begins elution

(a) Solvent: (b) Solvent volume ratio changed to 10:1, 7:1, 5:1, 3:1, 2:1, 1:1

Solvent volume at each stage: 400 mL

10:1 (small fractions 3 to 6), 7:1 (small fractions 7 to 10), 5:1 (small fractions 11 to 14), 3:1 (small fractions 15 to 18), 2:1 (small fractions) 19-22)

The small fractions 17 to 21 (the third and fourth fractions of the four small fractions in which the elution solvent in the separation method 10 has a methylene chloride:methanol mixing ratio of 3:1 and the elution solvent have a methylene chloride:methanol mixing ratio of 2 The first to third fractions of four small fractions per person) were separated under the conditions of the following separation method 11 by HPLC separation. As a result, 1.6 mg of compound B corresponding to peak B in FIG. 4, 24.8 mg of compound R corresponding to peak R in FIG. 4, 5.5 mg of compound S corresponding to peak S in FIG. 4, and peak T corresponding to peak T in FIG. Compound T 2.3 mg, Compound U corresponding to Peak U in FIG. 4, 9.3 mg Compound V corresponding to Peak V in FIG. 4, Compound W 4.1 mg corresponding to Peak W in FIG. 4, and Peak X in FIG. 4 The corresponding compound X 0.6 mg was isolated.

Separation method 11

Equipment used: JASCO LC-2000PLUS

Column: Phenomenex Luna C-18 RP HPLC column (21.2x250 mm, 10 μm)

Solvent: (a) acetonitrile with 0.02% TFA

(b) Water with 0.02% TFA

Mobile phase composition: Elution starts from 0 min to (a) solvent: (b) solvent volume ratio 10:90

0 to 40 minutes (a) Maintain the solvent ratio at 10%

40 to 70 minutes (a) Increased the solvent ratio from 10% to 18%

70 to 180 minutes (a) Increased the percentage of solvents from 18% to 22%

180 to 260 minutes (a) Increased the percentage of solvents from 22% to 100%

Mobile phase flow rate: 6.5 mL/min

The HPLC peaks corresponding to all the substances (Compounds 1 to 5, and Compounds A to X) separated in Examples 4 and 5 and Comparative Example 2 are as indicated for each peak of the named bud extract HPLC chromatogram in FIG. 4. A total of 29 substances including naphthopyrone derivatives having the structures of Formulas 1 to 5 (Compounds 1 to 5) are components that can be separated from the extract of the seedling sprout obtained in Example 1. In addition, according to the HPLC analytical chromatogram, the 29 kinds of substances correspond to the main constituents of the extract of the sprouted shoot.

[ Example 6] Determination Structural identification of compounds 1-5 isolated from bud extract

In order to identify the structures of the compounds 1 to 5 separated in Examples 4 and 5, analysis was performed using a mass spectrometer (MS) and a nuclear magnetic resonance spectroscopy (NMR) for each compound.

(1) Identification of compound 1

The molecular weight of Compound 1 isolated in Example 4 was determined to be 664 through MS measurement using an Agilent 1100 high-speed fluid chromatography-mass spectrometer (HPLC-ESI-MS), ultraviolet (PerkinElmer 343 Polarimeter), infrared (Thermo Scientific) Nicolet iS50), non-linear (PerkinElmer Lambda 35) spectral data and ¹ H and ¹³ C NMR spectrum analysis using a nuclear magnetic resonator (Bruker 400 MHz, 100 MHz NMR). Eggplant was determined to be a naphthopyrone derivative, 7-hydroxymushizinyl-rubrufusarin-8'-O-glucoside. The compound 1 is a novel compound that has not been reported so far, and has been isolated as one of the main components only in the seedling sprout, which is not found in the tracer or the fully grown plant, or in a very small amount.

[Formula 1]

Molecular formula C ₃₄ H ₃₂ O ₁₄ ; ESI-MS: m/z 665 [M+H] ⁺ ; Infrared absorption band ν _max 3384, 2936, 1631, 1373, 1204, 1059 cm ^-1 ; Ultraviolet absorption band (MeOH) λ _max (log ε) 240 (4.3), 282 (4.2); Non-linearity [α] ²² _D- 20 (c 0.05, MeOH);

¹ H NMR (methanol- d ₄ , 400 MHz): δ 7.18 (1H, s, H-10), 6.97 (1H, d, J = 2.0 Hz, H-5), 6.92 (1H, s, H-9 ), 6.34 (1H, br s, H-5'), 6.12 (1H, s, H-3), 5.12 (1H, dd, J = 7.5, 3.5 Hz, H-1``), 3.98 (1H, dd, J =12.0, 2.0 Hz, H-6``a), 3.78 (1H, dd, J = 12.0, 5.5 Hz, H-6''b), 3.75 (3H, s, OMe-8), 3.58 (1H, t, J = 8.0 Hz, H-2'), 3.55 (1H, dd, J =5.0, 2.0 Hz, H-5``), 3.51 (1H, overlapped, H-3''), 3.48 (1H, t, J = 9.0 Hz, H-4``), 2.63 (3H, s, Me-10'), 2.41 (3H, s, Me-11), 1.98 (3H, s, Me-11' ), ¹³ C NMR (CDCl ₃ , 500 MHz): δ 208.1 (C-9'), 184.1 (C-4), 170.1 (C-2), 161.3 (C-8), 160.9 (C-5), 156.2 (C-6'), 156.1 (C-8'), 154.7 (C-6), 152.4 (C-10a), 150.9 (C-1'), 139.8 (C-9a), 137.1 (C-4 'a), 132.4 (C-3'), 122.4 (C-2'), 120.6 (C-4'), 111.9 (C-7), 108.4 (C-8'a), 106.2 (C-5a) , 105.8 (C-3), 103.1 (C-1''), 102.9 (C-5'), 102.8 (C-7'), 102.5 (C-4a), 101.4 (C-10), 97.0 (C -9), 77.4 (C-5``), 76.7 (C-3''), 73.5 (C-2''), 69.7 (C-4''), 61.0 (C-6''), 54.8 (OMe-8), 31.4 (C-10'), 19.4 (C-11), 16.1 (C-11')

(2) Identification of compound 2

The molecular weight of Compound 2 isolated in Example 5 was determined to be 272 through MS measurement using an Agilent 1100 high-speed fluid chromatography-mass spectrometer (HPLC-ESI-MS), using a nuclear magnetic resonance (Bruker 400 MHz NMR). ^{Through 1} H NMR spectrum analysis, it was estimated to be an isotoralactone, a naphthopyrone derivative having the structure of Chemical Formula 2 below. In addition, the structure was identified by comparing NMR data with data from existing literature (Kitanaka, S. et al. Phytochemistry, 1981, 20, 1951-1953).

[Formula 2]

Molecular formula C ₁₅ H ₁₂ O ₅ ; ESI-MS: m/z 273 [M+H] ⁺ ;

¹ H NMR (CDCl ₃ , 400 MHz): δ 13.30 (1H, s, OH-10), 9.41 (1H, s, OH-9), 6.92 (1H, s, H-5), 6.59 (1H, d , J = 2.5 Hz, H-6), 6.56 (1H, d, J = 2.0 Hz, H-8), 4.93 (1H, d, J = 2.0 Hz, H-11), 4.62 (1H, d, J = 2.0 Hz, H-11), 3.90 (3H, s, OMe-7), 3.80 (1H, s, H-4)

(3) Identification of compound 3

The molecular weight of Compound 3 isolated in Example 5 was determined to be 272 through MS measurement using an Agilent 1100 high-speed fluid chromatography-mass spectrometer (HPLC-ESI-MS), and a nuclear magnetic resonance machine (Bruker 400 MHz NMR) was used. ^{Through 1} H NMR spectral analysis, the structure was estimated as toraactone, a naphthopyrone derivative having the structure shown in Chemical Formula 3. In addition, the structure was identified by comparing NMR data with data from existing literature (Newman, AG et al. J Am Chem Soc 2016, 138, 4219-4228).

[Formula 3]

Molecular formula C ₁₅ H ₁₂ O ₅ ; ESI-MS: m/z 273 [M+H] ⁺ ;

¹ H NMR (CDCl ₃ , 400 MHz): δ 13.56 (1H, s, OH-5), 9.44 (1H, s, OH-9), 7.00 (1H, s, H-5), 6.64 (1H, d , J = 2.5 Hz, H-6), 6.55 (1H, d, J = 2.5 Hz, H-8), 6.25 (1H, s, H-4), 3.92 (3H, s, OMe-7), 2.28 (3H, s, Me-11)

(4) Identification of compound 4

The molecular weight of Compound 4 isolated in Example 5 was determined to be 288 through MS measurement using an Agilent 1100 high-speed fluid chromatography-mass spectrometer (HPLC-ESI-MS), using a nuclear magnetic resonance machine (Bruker 400 MHz NMR). ^{Through 1} H NMR spectral analysis, the structure was estimated to be torosachrysone, a naphthopyrone derivative having the structure shown in Chemical Formula 4. In addition, the structure was identified by comparing the NMR data with those of the existing literature (Gill, M. et al. Aust J Chem 2000, 53, 213-220).

[Formula 4]

Molecular formula C ₁₆ H ₁₆ O ₅ ; ESI-MS: m/z 289 [M+H] ⁺ ;

¹ H NMR (methanol-d ₄ , 400 MHz): δ 9.80 (1H, s, OH-9), 6.90 (1H, s, H-5), 6.57 (1H, d, J = 2.0 Hz, H-6 ), 6.51 (1H, d, J = 2.0 Hz, H-8), 3.91 (3H, s, OMe-7), 3.08 (2H, br s, H-4), 2.86 (2H, br s, H- 2), 1.44 (3H, s, Me-11)

(5) Identification of compound 5

The molecular weight of Compound 5 isolated in Example 5 was determined to be 288 through MS measurement using an Agilent 1100 high-speed fluid chromatography-mass spectrometer (HPLC-ESI-MS), using a nuclear magnetic resonator (Bruker 400 MHz NMR). ^{Through 1} H NMR spectral analysis, the structure was estimated as a rubrofusarin, a naphthopyrone derivative having the structure of Chemical Formula 5 below. In addition, the structure was identified by comparing the NMR data with those of the existing literature (Alemayehu, G. et al. Phytochemistry, 1993, 32, 1273-1277).

[Formula 5]

Molecular formula C ₁₅ H ₁₂ O ₅ ; ESI-MS: m/z 273 [M+H] ⁺ ;

¹ H NMR (CDCl ₃ , 400 MHz): δ 9.68 (1H, s, OH-9), 7.00 (1H, s, H-10), 6.59 (1H, d, J = 2.5 Hz, H-9), 6.48 (1H, d, J = 2.0 Hz, H-7), 6.04 (1H, s, H-3), 3.91 (3H, s, OMe-7), 2.41 (3H, s, Me-11)

[ Test example One] Determination Evaluation of Antioxidative Effect of Sprout Extract

(1) of the Cassia tora extract and gyeolmyeong sprout extract DPPH Comparison of free radical scavenging efficacy

After dissolving the Cassia tora extract (ST) prepared in Comparative Example 1 and Cassia tora sprout extract (STS) prepared in Example 1 in methanol at concentrations of 1000 ppm, 500 ppm, 250 ppm, 125 ppm, respectively, and comparing Absorbance at 517 nm was measured by reacting 100 μL of the extract solution of Example 1 and 100 μL of the extract of the seedling sprout of Example 1 with 100 μL of DPPH for 40 minutes in dark condition, and ascorbic acid (L-ascorbic acid) as a positive control. ) Was used. The DPPH free radical scavenging efficacy derived by the following formula through the absorbance measurement is shown in FIG. 1.

As shown in FIG. 1, it was found that the DPPH free radical scavenging effect of the budding extract (STS) of Example 1 was 1.7 times higher in each concentration than the extractor extract (ST) of Comparative Example 1`. .

(2) The Cassia tora extract and gyeolmyeong sprout extract the sprocket Comparison of ABTS online antioxidant chromatography measurement results

Absolute extract (ST) prepared in Comparative Example 1 and Cascaded bud extract (STS) prepared in Example 1 were dissolved in an aqueous alcohol solution at the same concentration, and ABTS online antioxidant high performance liquid chromatography (HPLC) according to the following analysis conditions. ).

ABTS Online antioxidant HPLC Analysis conditions

Equipment used: Agilent 1200 system

Column: RP C-18 HPLC column (4.6x150 mm, 5 μm)

Solvent: (a) acetonitrile with 0.02% trifluoroacetic acid (TFA)

(b) Water with 0.02% TFA

Mobile phase composition: 0 min (a) Solvent: (b) Solvent volume ratio 10:90 Start elution

0 to 30 minutes (a) Increased solvent ratio from 10% to 100%

30 to 37 minutes (a) Elution with 100% solvent

Mobile phase flow rate: 0.7 mL/min

ABTS composition: water containing 0.08 mM ABTS and 0.12 mM potassium persulfate

ABTS flow rate: 0.35 mL/min

Detector detection wavelength: ultraviolet 254 nm; 734 nm

As a result, the ginseng extract (ST) and the ginseng sprout extract (STS) showed very different antioxidant components. Ingredients composed of flavonoids and naphthalene derivatives with sulfated activity were observed 15 minutes before the retention time of both the terminus extract (ST) and the terminus sprout extract (STS), but compared to the terminus extract (ST) in the terminus sprout extract (STS) Naphthalene and anthraquinone derivatives having antioxidant activity after 15 minutes while the contents of the above components were increased were additionally observed. (A) and (B) of Figure 2 is the result of performing the ABTS on-line antioxidant HPLC of the ginseng and the bud sprout extract, ultraviolet (UV) detector 254 nm (blue line chromatogram indicated upward), 734 nm (downward direction) Chromatogram of the red line indicated by) is a chromatogram obtained by detecting each wavelength.

[ Test example 2] Cassia tora extract (ST) and various lights Under conditions Cultivated Determination Of sprout extract ABTS Comparison of online antioxidant chromatography measurement results

The sprouted sprout extract (STS) prepared in Example 1 and the sprouted sprout extract (STS-C, STS-385, STS-465, STS-645) cultivated under 7 different light conditions prepared in Example 2, and STS-780) was dissolved in an aqueous alcohol solution to perform ABTS online antioxidant high performance liquid chromatography (HPLC) according to the same analysis conditions as in Test Example 1.

As a result, the constituent components having antioxidant activity were observed in common in the extract of shoot sprout cultivated under five different light conditions of Example 2, but the content of the components was changed according to the light conditions. A, B, C, D, E, and F of FIG. 3 are ABTS online antioxidants of STS, STS-C, STS-385, STS-465, STS-645, and STS-780 extracts, respectively, of the above six named sprout extracts. As a result of performing HPLC, the ultraviolet (UV) detector 254 nm (shows the peaks of the components absorbing UV 254 nm) and 734 nm wavelength (the peaks of the components showing the antioxidant action against ABTS and the area of the antioxidants of the peaks It is a chromatogram obtained by detecting each).

Based on the chromatograms of FIG. 3, the antioxidant component profiles of the bud extracts grown under light-shielding conditions, fluorescent lamps, and light conditions of each specific wavelength were examined, and the content and composition of antioxidant components differed by light wavelengths irradiated during the cultivation process However, no ingredients were found that disappeared or appeared under certain conditions. The novel naphthopyrone component (Compound 1 isolated in Example 4) having the structure of Formula 1 of the present invention in all chromatograms of FIGS. 3A to 3F is the most notable antioxidant peak or major antioxidant peak. 4 naphthopyrone components having the structures of Formulas 2 to 5 (Compounds 2 to 5 isolated in Example 5) were also antioxidant in the chromatograms of (A) to (F) of FIG. 3 It was detected as an ingredient. Except for the chromatogram (B) of the cultivated shoot extract (STS-C) grown under normal light conditions and the chromatogram (D) of the cultivated shoot extract (STS-465) grown under 465 nm LED illumination, (A), In the chromatograms of (C), (E) and (F), active ingredients of compounds 1 to 5 isolated in Examples 4 and 5 were detected in a relatively high content.

[ Test example 3] Example 7 of 1 and 2 Determination Of sprout extract Retinal progenitor cells (R28) Protective efficacy

The extract extract (ST) and the extract shoots (STS) obtained in Comparative Example 1 and Example 1, respectively, and the extract shoots grown under various light conditions obtained in Example 2 (STS-C, STS-385) , STS-465, STS-645, STS-780) was performed as follows to confirm the protective effect of retinal progenitor cells (R28).

Specifically, R28 cells are 10° C. in a Dulbecco's modified eagle's medium (DMEM)/low glucose medium containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. After incubation under 5% CO ₂ conditions in an incubator, after passage by using 0.05% trypsin every 2 days, 96 plates were inoculated at a density of 1*?*10 ⁴ and cultured for 24 hours. To the cultured R28 cells, the 7 extracts were treated with the concentrations of FIGS. 5(A) to (C), and after 2 hours, 10 mM glutamate and 0.5 mM BSO (buthionine sulphoximine) were added for 22 hours. Cultured. Then, to measure cell viability, 10 μL EZ-cytox was treated and maintained for 2 hours, and UV absorbance was measured at 450 nm.

In FIG. 5, (A) is the ginseng extract of the comparative example 1 (ST) at 3 concentrations from the oxidative stress caused by glutamate, the sprouting extract of the embodiment 1 (STS), and light conditions of the example 2 It is a result showing the cell protection efficiency of the R28 cell protection efficacy of five different sprouted shoots (STS-C, STS-385, STS-465, STS-645, STS-780) cultivated differently. In FIG. 5, (B) and (C) are R28 cells of the Cassia tora extract (ST) of Comparative Example 1 and Cassia tora sprout extract (STS) of Example 1 from oxidative stress induced by glutamate at 5 concentrations, respectively. It is a result showing the protective efficacy as a cell survival rate.

As shown in FIG. 5(A), in all extract treatment groups, it showed an effect of protecting HT-22 cells from oxidative stress caused by glutamate, but compared to the extract of shooter (STS, STS-C) , STS-385, STS-465, STS-645 and STS-780) showed a better effect of protecting R28 cells from oxidative stress caused by glutamate at most concentrations. As shown in B) and (C), it was confirmed that the seedling sprout extract (STS) of Example 1 was significantly superior in protecting R28 cells compared to the extractor extract (ST) of Comparative Example 1.

[ Test example 4] Example 5's and 6's Determination Compounds 1 to 5 isolated from bud extract Retinal progenitor cells (R28) Confirmation of protective efficacy

To confirm why the retinal progenitor cell (R28) protective efficacy of the modified shoot extract (STS) of Test Example 3 is superior to that of the deleted extract (ST), compounds 1 to 5 of Examples 5 and 6 for R28 cells The protective efficacy was compared.

In the same manner as in Test Example 3, the cell viability of R28 was measured, but the extract extract (ST), the extract extract of shoot (STS, STS-C, STS-385, STS-465) of Comparative Example 1, Examples 1 and 2, STS-645 and STS-780) instead of compounds 1 to 5 of Examples 5 and 6 were treated with 50 μM, 16.6 μM, and 5.55 μM, and the results are shown in FIG. 6.

As shown in FIG. 6, compounds 1 to 5 showed the effect of protecting R28 cells from neurotoxicity induced by glutamate, and the compounds have a very high relative content to the sprouted shoot extract (STS) or the seeded shoot extract Since it is a compound mainly present in (STS), it was confirmed that the R28 cell protection efficacy of the budding shoot extract (STS) is superior to that of the shooter extract (ST).

[ Test example 5] Example 7 of 1 and 2 Determination Efficacy of protecting hippocampal nerve cells (HT-22) from the bud extract

Seedling extract (ST) and seedling sprout extract (STS) obtained in Comparative Example 1 and Example 1, and seedling sprout extract (STS-C, STS-385) grown under various light conditions obtained in Example 2, STS-465, STS-645, STS-780) in order to confirm the protective efficacy of hippocampal neurons (HT-22) was performed as follows.

Specifically, HT-22 cells were 5% in a 37.5 °C incubator in a Dulbecco's modified eagle's medium (DMEM)/low glucose medium containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. Incubated under CO ₂ conditions, subcultured with 0.05% trypsin every 2 days, inoculated at a density of 3*?*10 ³ in 96 plates and cultured for 24 hours. The 7 extracts were treated with the cultured HT-22 cells at the concentrations shown in FIGS. 7(A) to (C), and after 2 hours, 5 mM glutamate was added and cultured for 22 hours. Then, to measure cell viability, 10 μL EZ-cytox was treated and maintained for 2 hours, and UV absorbance was measured at 450 nm.

In FIG. 7, (A) is the ginseng extract (ST) of the comparative example 1 at three concentrations from the oxidative stress caused by glutamate, the sprouted shoot extract of the first embodiment (STS), and light conditions of the second embodiment. It is a result showing the cell protection rate of HT-22 cell protection efficacy of five different sprouted shoots (STS-C, STS-385, STS-465, STS-645, STS-780) cultivated differently. In FIG. 7, (B) and (C) are HT-22 of the Cassia tora extract (ST) of Comparative Example 1 and Cassia tora sprout extract (STS) of Example 1 from oxidative stress induced by glutamate at 5 concentrations. It is a result showing the cell protection efficacy as a cell survival rate.

As shown in FIG. 7(A), in all extract treatment groups, it showed an effect of protecting HT-22 cells from oxidative stress caused by glutamate, but in particular, shown in FIGS. 7(B), (C) As described above, it was confirmed that the sprouted shoot extract (STS) of Example 1 exhibits superior HT-22 cell protection efficacy compared to the shooter extract (ST) of Comparative Example 1.

[ Test example 6] Example 5's and 6's Determination Confirmation of the protective efficacy of hippocampal nerve cells (HT-22) of compounds 1 to 5 isolated from the sprout extract

In order to confirm the reason that the protective efficacy of the hippocampal nerve cell (HT-22) of the budding bud extract (STS) of Test Example 5 is superior to that of the budding extract (ST), compounds 1 to 5 of Examples 5 and 6 for R28 cells And the protective efficacy of Compounds A to X of Comparative Example 2.

The HT-22 cell viability was measured in the same manner as in Test Example 5, except that the extract of extracts (ST) and extracts of shoots (STS, STS-C, STS-385, STS-465) of Comparative Example 1 and Examples 1 and 2 were used. , STS-645 and STS-780), compounds 1 to 5 of Examples 5 and 6 were treated with 50 μM, 16.6 μM, and 5.55 μM, and the results are shown in FIG. 8.

As shown in FIG. 8, compound K and compound T corresponding to peaks K and T of compounds 1 to 5 and Comparative Example 2 of Examples 5 and 6, respectively, from neurotoxicity induced by glutamate, HT-22 cells It showed a protective effect. That is, since the compounds 1 to 5 are compounds having a very high relative content to the named sprout extract (STS) or mainly present in the named sprout extract (STS), the efficacy of protecting the HT-22 cells of the named sprout extract (STS) is determined Reasons superior to the efficacy of the extract (ST) were confirmed.

The physiological activity of each component evaluated in Test Example 1, Test Example 4 and Test Example 6 is shown in Table 1 below.

[Table 1] Physiological activity of components isolated from the bud extract (STS) of Example 1

Claims (17)

A naphthopyrone derivative of Formula 1, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof.
[Formula 1]

A method for preparing a naphthopyrone derivative of Formula 1, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof, from a Cassia obtusifolia L. or Cassia tora L. sprout A method for producing a naphthopyrone derivative, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof, comprising the step of separating the naphthopyrone derivative.
[Formula 1]

According to claim 2, The manufacturing method further comprises the step of extracting the solvent selected from the group consisting of water, alcohol having 1 to 6 carbon atoms, and mixed solvents of them from the sprout. The method of claim 2, wherein the manufacturing method further comprises a process of fractionating one or more selected from the group consisting of water, ethyl acetate, hexane, methylene chloride, chloroform, methanol, ethanol, acetone, and mixed solvents thereof. , Manufacturing method. One or more selected from the group consisting of a naphthopyrone derivative of Formula 1, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof, and a name comprising the same ( Cassia obtusifolia L. or Cassia tora L. ) Sprout extract, or a new sprout fraction containing it as an active ingredient, for protecting neurons from oxidative stress or for suppressing neuronal cell death food composition.
[Formula 1]

The food composition of claim 5, wherein the oxidative stress is caused by glutamate. The food composition of claim 5, wherein the nerve cells are retinal nerve cells or hippocampal nerve cells. One or more selected from the group consisting of a naphthopyrone derivative of Formula 1, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof, and a name comprising the same ( Cassia obtusifolia L. or Cassia tora L. ) A composition for the prevention or treatment of neuro-damage diseases caused by damage or death of retinal nerve cells or hippocampal nerve cells, comprising a bud extract or a fraction fraction of a bud containing it as an active ingredient.
[Formula 1]

9. The method of claim 8, wherein the neurodegenerative diseases are decreased memory, decreased learning ability, depressive disorder, amyotrophic lateral sclerosis (ALS), Parkinson's disease, ischemic neuropathy, diabetic neuropathy, macular degeneration, retinal cell damage. By at least one selected from the group consisting of vision impairment, retinitis pigmentosa, retinal detachment, retinal vessel occlusion and glaucoma, the composition. The composition of claim 8, wherein the damage or death of the retinal nerve cells or hippocampal nerve cells is caused by glutamate neurotoxicity. 9. The composition of claim 8, wherein the naphthopyrone derivative is isolated or purified from the bud of Cassia obtusifolia L. or Cassia tora L. 12. The composition of claim 11, wherein the naphthopyrone derivative is isolated or purified from the named ethanol extract. 10. The method of claim 8, wherein the naphthopyrone derivative is fractionated with ethanol extract of budding buds with ethyl acetate, and then the ethyl acetate fraction is separated by chromatography or the ethyl acetate fraction is again mixed solvent of hexane, methylene chloride and methanol. Fractionated and separated by chromatography. The composition of claim 8, wherein the extract is extracted with a solvent selected from the group consisting of water, alcohols having 1 to 6 carbon atoms, and mixed solvents thereof. The composition of claim 8, wherein the fraction is fractionated by one or more selected from the group consisting of water, ethyl acetate, hexane, methylene chloride, chloroform, methanol, ethanol, acetone, and mixed solvents thereof. The composition of claim 8, wherein the composition is a pharmaceutical composition. One or more selected from the group consisting of a naphthopyrone derivative of Formula 1, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof, and a name comprising the same ( Cassia obtusifolia L. or Cassia tora L. ) Sprout extract, or containing a fraction of a sprout sprout containing it as an active ingredient, antioxidant composition.
[Formula 1]

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