CN112996799A - Composition for protecting nerve cells comprising naphthopyrone derivative derived from sprout of cassia seed - Google Patents

Composition for protecting nerve cells comprising naphthopyrone derivative derived from sprout of cassia seed Download PDF

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CN112996799A
CN112996799A CN201980072747.9A CN201980072747A CN112996799A CN 112996799 A CN112996799 A CN 112996799A CN 201980072747 A CN201980072747 A CN 201980072747A CN 112996799 A CN112996799 A CN 112996799A
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cassia
naphthopyrone
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weight
extract
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权学哲
朴真洙
权宰英
黄浩成
李政奂
李宰旭
金东会
金亨锡
张诚律
郑相勋
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Korea Advanced Institute of Science and Technology KAIST
Korea Institute of Science and Technology KIST
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
    • C07C49/587—Unsaturated compounds containing a keto groups being part of a ring
    • C07C49/613—Unsaturated compounds containing a keto groups being part of a ring polycyclic
    • C07C49/617—Unsaturated compounds containing a keto groups being part of a ring polycyclic a keto group being part of a condensed ring system
    • C07C49/643—Unsaturated compounds containing a keto groups being part of a ring polycyclic a keto group being part of a condensed ring system having three rings
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/78—Ring systems having three or more relevant rings
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    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
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    • C07H1/08—Separation; Purification from natural products
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0621—Eye cells, e.g. cornea, iris pigmented cells
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2300/00—Processes
    • A23V2300/14—Extraction
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere

Abstract

The present invention relates to a naphthopyrone derivative and a composition containing a cassia seed sprout extract containing the naphthopyrone derivative 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 nerve cell death, and particularly, an effect of inhibiting damage or death of retinal nerve cells or hippocampal nerve cells due to glutamate toxicity, and thus can be used as a pharmaceutical or food composition for the treatment or prevention of visual deterioration and decline caused by optic nerve damage and eye diseases, memory deterioration, learning ability deterioration, onset and deterioration of depression, and degenerative cranial nerve diseases due to cranial nerve damage.

Description

Composition for protecting nerve cells comprising naphthopyrone derivative derived from sprout of cassia seed
Technical Field
The present specification relates to naphthopyrone derivatives and compositions containing cassia tora sprout extracts containing the same as an active ingredient.
Background
The brain hippocampus (hippopopus) plays an important role in cognitive function in humans and animals as a structural body of the internal temporal lobe of the brain. Hippocampus is known to be a central tissue that is vulnerable to physiological/oxidative stress stimuli and can cause impairment of cognitive function due to stress. Stress can cause structural and brain cytopoiesis, synaptic plasticity, and behavioural changes associated with the hippocampus (Eun-Joo Kim, korean journal of psychology: cognition and biology, 2012,24, 65-88). It is known that when oxidative stress in nerve cells is induced in the hippocampus, pituitary gland, striatum, substantia nigra, forebrain cortex or hypothalamus, nerve cell death increases, reducing neurons and growth factors, leading to acute or chronic neurological diseases such as amyotrophic lateral sclerosis (lugeriger's disease), parkinson's disease or cerebral ischemic nerve injury (Rahman, t.et al.adv.biosci.biotechnol.2012,3,997-.
According to the health insurance data of the korean national health insurance business, retinal disease patients increased from about 83 to about 125 in 2010 to about 2015, with an average increase of about 8% per year. The retina is a thin nerve membrane inside the eye similar to the camera film. In the retina, there are one hundred million or more photoreceptor cells (cells that sense light) and one million or more optic nerve cells, so that light is converted into an electrical signal, and an image of what is seen with the eyes is transmitted to the brain through nerves. If such nerve damage or nerve function abnormality of the retina occurs, problems occur in the vision and visual field. Representative retinal diseases include visual deterioration, visual field disorders and blindness, and include diabetic retinopathy, age-related macular degeneration, retinitis pigmentosa, retinal detachment and retinal vascular occlusion. Glaucoma, a representative ocular disease caused by damage to the optic nerve of the retina, damages the optic nerve due to increased intraocular pressure, ischemia, oxidative stress, and the like (Kim, N.Y.et al.J.Korea Ophalmol. Soc.2015,56, 70-79; Kang, J.H.et al.J.Korea Ophthalmol. Soc.2003,44, 965-.
Glutamate is an excitatory neurotransmitter that plays an important role in the central nervous system of vertebrates. Glutamate acts on quisqualate (quisqualate) receptors, NMDA (N-methyl-D-aspartate) receptors and caine acid (Kainite) receptors, which are centrally distributed in the cerebellar amygdala and hippocampus, which are known to be involved in memory and learning in brain tissues, and is involved in various physiological manifestations. Conversely, when the release of glutamate from inside and outside nerve cells increases and the concentration of glutamate outside the cells increases sharply, it acts as an oxidative neurotoxic substance (Hyunjeong Kim et al, J.Life Sci.2009,19, 963-967). Excess Glutamate (Glutamate) is known to be associated with various acute neurological disorders including anemia, hypoxia, hypoglycemia, trauma, various chronic degenerative neurological diseases. Glutamate in the eye is associated with acute impairment and death of retinal ganglion cells (Otori, Y.et al. invest. Ophthalmol. Vis. Sci.1998,39, 972-981). It is known that overproduction of Reactive Oxygen Species (ROS) is stimulated at the same time as glutamate is produced in large amounts to activate presynaptic glutamate receptors (Tarasenko a.et al. neurochem. int.2012,61, 1044-1051). Furthermore, it is known that the oxidative action exerted in response to glutamate is mediated by activation of NMDA receptors, showing calcium ion dependence. Such NMDA receptor over-activation and mitochondrial uptake of calcium ions (uptake) are reported to contribute to ROS production (Reynolds I.J.et al.J.Neurosci.1995,15, 3318-3327). Reactive Oxygen Species (ROS) are continuously generated by oxidation/reduction of Oxygen based on respiration and immune reactions in mitochondria in living cells. Since ROS are chemically unstable and highly reactive, they can react with lipids, nucleic acids, proteins, etc. in vivo, causing DNA damage, increased intracellular concentrations of free calcium and iron, damage to the ion transport system of the biofilm, etc. (Kiselyov, K.et al.cell calcium.2016,60, 108-114). Furthermore, light is known to also induce ROS production, thereby inducing photooxidative damage of the optic nerve (Masuda, t.et al.oxid.med.cell.rangevision.2017; article ID9208489,14 pages).
Cassia seed is known to be conventionally used for eye health and has an excellent antioxidant effect. The Cassia seed is a mature seed of Cassia seed (Cassia obtusifolia L.) or Longgang nan tea (Cassia tora L.) belonging to the family Leguminosae, cultivated in Korea, and a row of moist seeds is contained in approximately 10cm of pod of Bow-shaped bean (Yen, G. — C.et al.J.Agric.food chem.1998,46, 820-. Studies report that the cassia seed extract has the efficacies of improving diabetes, improving dyslipidemia, protecting liver, resisting bacteria and reducing blood pressure (Dong, X.et al.mol.Med.Rep.2017,16, 2331-.
However, the neuroprotective effect of cassia tora sprout extract or 7-hydroxyRufuscin-rubrofusarin-8 '-O-glucopyranoside (7-hydroxymesityl-rubrin-8' -O-glucopyranoside) as a naphthopyrone derivative derived therefrom, isoctoralactone (isoctoralone), toralactone (toralactone), torachrysone (torachrysone) and rubrofusarin (rubrin) on glutamic acid-induced neurotoxicity has not been reported, and the techniques for the antioxidation of cassia tora sprout extract and the composition for neuroprotection have not been known.
In addition, no case of the development of the health functional and medicinal components using the extract of the sprout of cassia seed has been reported, and the present inventors confirmed that the extract of the sprout of cassia seed produces a novel antioxidant component and the antioxidant active component is increased as compared with the extract of cassia seed, thereby remarkably increasing the antioxidant effect and the neuronal protection effect, and completed the present invention by confirming that the naphthopyrone component containing the novel compound 7-hydroxydock-rubrofusarin-8' -O-glucopyranoside isolated from the extract of the sprout of cassia seed shows the effect of protecting retinal nerve cells and hippocampal nerve cells from the oxidative stress induced by glutamic acid.
[ Prior art documents ]
[ patent document ]
(patent document 1) KR10-1503429B1
(patent document 2) KR10-2010-0082054A
(patent document 3) KR10-0877371B1
(patent document 4) KR10-1807367B1
(patent document 5) KR10-2016-0058613A
[ non-patent document ]
(non-patent document 1) Jung, H.A. journal of Ethnopharmacology,2016, vol191, 152-160; the inhibitorships activities of major anthropogenic and other consistencies from the group of castia obtusifolia against beta-secretase and cholinesterase.
(non-patent document 2) Shrestha, S.et al. archives pharmaceutical Research,2018, online publication https:// doi.org/10.1007/s 12272-018-1044-0; two new naphthalene lactic acid glycosides from Cassia obtusifolia L.
Disclosure of Invention
An object of the present specification is to provide a naphthopyrone derivative of the following chemical formula 1, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof.
[ chemical formula 1]
Figure BDA0003050095460000041
Another object of the present specification is to provide a composition showing an effect of protecting nerve cells from damage by oxidative stress or inhibiting death of nerve cells.
Another object of the present specification is to provide a composition showing an effect of preventing or treating a nerve injury disease induced by damage or death of retinal nerve cells or hippocampal nerve cells.
It is another object of the present specification to provide a composition which exhibits an antioxidant effect.
In order to achieve the above objects, one aspect of the present invention provides a naphthopyrone derivative of the following chemical formula 1, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof.
[ chemical formula 1]
Figure BDA0003050095460000042
In another aspect, the present invention provides a method for producing 1 or more naphthopyrone derivatives selected from the naphthopyrone derivatives of the following chemical formulas 1 to 5, stereoisomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, or solvates thereof.
[ chemical formula 1]
Figure BDA0003050095460000051
[ chemical formula 2]
Figure BDA0003050095460000052
[ chemical formula 3]
Figure BDA0003050095460000053
[ chemical formula 4]
Figure BDA0003050095460000054
[ chemical formula 5]
Figure BDA0003050095460000055
In another aspect, the present invention provides a composition for protecting nerve cells from oxidative stress or for inhibiting nerve cell death, comprising, as an active ingredient, one or more selected from the group consisting of a naphthopyrone derivative, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof, a cassia seed (cassia obtusifolia or cassia tora) bud extract comprising the same, or a cassia seed bud fraction comprising the same, wherein the naphthopyrone derivative is 1 or more selected from the group consisting of the naphthopyrone derivatives of chemical formulae 1 to 5.
In another aspect, the present invention provides a composition for preventing or treating a nerve damage disease induced by damage or death of retinal nerve cells or hippocampal nerve cells, comprising as an active ingredient at least one selected from the group consisting of a naphthopyrone derivative, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof, and a cassia seed (cassia obtusifolia or cassia tora) bud extract comprising the same, or a cassia seed bud fraction comprising the same, wherein the naphthopyrone derivative is at least 1 selected from the group consisting of naphthopyrone derivatives of chemical formulae 1 to 5.
In another aspect, the present invention provides an antioxidant composition comprising, as an active ingredient, at least one selected from the group consisting of a naphthopyrone derivative selected from the group consisting of 1 or more naphthopyrone derivatives of the following chemical formulae 1 to 5, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, and a solvate thereof, and an extract of a sprout of cassia seed (cassia obtusifolia or cassia tora) containing the same, or a fraction of the sprout of cassia seed containing the same.
Effects of the invention
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 nerve cell death, and particularly, an effect of inhibiting damage or death of retinal nerve cells or hippocampal nerve cells due to glutamate toxicity. Accordingly, the composition according to one aspect of the present invention protects retinal nerve cells, and thus can be used as a treatment or prevention of visual deterioration and decline accompanying optic nerve damage and eye diseases, protects hippocampal nerve cells, and thus can be used as a treatment or prevention of hypomnesis, learning ability decline, onset and exacerbation of depression and degenerative cranial nerve diseases caused by cranial nerve damage, and further, can be used for mental health for improving memory and learning ability, relieving stress, etc., and for protecting optic nerve and improving eye health for visual deterioration, etc., and thus can be used as a pharmaceutical or food composition.
Drawings
FIG. 1 is a graph showing DPPH (α, α -diphenyl- β -piperidinozyl, α, α -diphenyl- β -pyridohydrazino) radical scavenging activity of cassia seed extract (ST) and cassia seed sprout extract (STS).
FIG. 2 is a graph showing ABTS on-line antioxidant HPLC chromatograms of Cassia tora extract (ST) and Cassia tora sprout extract (STS). The chromatogram of the blue line marked upward is a graph showing components detected at Ultraviolet (UV)254nm, and the chromatogram of the red line marked downward is a graph showing antioxidant components that react with the ABTS reagent and are detected at UV/VIS (ultraviolet/visible) 734 nm.
FIG. 3 is a graph showing ABTS on-line antioxidant HPLC chromatograms of cassia seed bud extracts (STS-C, STS-385, STS-465, STS-645, and STS-780) cultivated under various light conditions. In each of the chromatograms (a) to (F), the upward-labeled chromatogram is a graph showing components detected at Ultraviolet (UV)254nm, and the downward-labeled chromatogram is a graph showing antioxidant components detected at UV/VIS (ultraviolet/visible) 734nm by reaction with ABTS reagent. (A) The sample is the cassia seed sprout extract cultivated under shading conditions for 2 weeks (STS of example 1), (B) the sample is the cassia seed sprout extract cultivated under general light conditions for 2 weeks (STS-C of example 2), (C) the sample is the cassia seed sprout extract cultivated under LED illumination of 385nm for 2 weeks (STS-385 of example 2), (D) the sample is the cassia seed sprout extract cultivated under LED illumination of 465nm for 2 weeks (STS-465 of example 2), (E) the sample is the cassia seed sprout extract cultivated under LED illumination of 645nm for 2 weeks (STS-645 of example 2), (F) the sample is the cassia seed sprout extract cultivated under LED illumination of 780nm for 2 weeks (STS-780 of example 2).
Fig. 4 is an HPLC chromatogram marking the peak of the main component separated from the cassia seed bud extract (STS) of example 1 as an embodiment of the present invention.
FIG. 5 is a graph showing the effect of the Cassia tora extract (ST) according to comparative example 1, the Cassia tora bud extract cultivated under light-shielding conditions (STS according to example 1) and the Cassia tora bud extracts cultivated under various light conditions (STS, STS-C, STS-385, STS-465, STS-645 and STS-780 according to example 2) on the protection of retinal progenitor cell (R28) damage induced by glutamic acid (glutamate) toxicity.
Fig. 6 is a graph showing the effect of protecting retinal progenitor cell (R28) damage induced by glutamate toxicity according to the concentration of each compound (compound 1 to compound 5 of examples 4 and 5, and compound a to compound X of comparative example 2) isolated from cassia seed bud extract (STS).
FIG. 7 is a graph showing the effects of the cassia seed extract (ST) of comparative example 1, the cassia seed bud extract cultivated under light-shielding conditions (STS of example 1), and the cassia seed bud extract cultivated under various light conditions (STS, STS-C, STS-385, STS-465, STS-645, and STS-780 of example 2) on the protection of hippocampal neuronal cell (HT-22) damage induced by glutamic acid (glutamate) toxicity.
Fig. 8 is a graph showing the effect of protecting hippocampal neural cell (HT-22) damage induced by glutamate toxicity according to the concentration of each compound (compound 1 to compound 5 of examples 4 and 5, and compound a to compound X of comparative example 2) isolated from cassia tora sprout extract (STS).
Detailed Description
One aspect of the present invention may relate to a naphthopyrone derivative of the following chemical formula 1, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof.
[ chemical formula 1]
Figure BDA0003050095460000081
The naphthopyrone derivative of chemical formula 1 may be 7-hydroxyiminorubicin-8 '-O-glucopyranoside (7-hydroxymesityl-rubrin-8' -O-glucopyranoside).
In the present specification, "pharmaceutically acceptable" means that the most toxic effect is avoided when used in a general medical dose (medicinal dose), and thus the compound can be used in animals, more specifically, in humans, and can be used in the approval or acquisition of a government or a regulatory agency approved by the government or can be listed in pharmacopeia or can be known and described in other general pharmacopeias.
In the present specification, "pharmaceutically acceptable salt" refers to a salt according to an aspect of the present invention which is pharmaceutically acceptable and has a preferred pharmacological activity of the parent compound (parent compound). The above salts may include: (1) formed from inorganic salts such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., or from acetic acid, propionic acid, caproic 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-ethanedisulfonic acid, 2-isethionic 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, glucoheptoic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, etc, Acid addition salts (acid addition salts) formed with organic acids such as salicylic acid, stearic acid, and muconic acid; or (2) a salt formed when an acidic proton present in the parent compound is substituted.
In particular, in the present specification, "isomers" include not only optical isomers (e.g., substantially pure enantiomers (essentiauomers), substantially pure diastereomers (essentiauomers), or mixtures thereof), but also conformational isomers (i.e., isomers that differ only in the angle of one or more chemical bonds), positional isomers (particularly tautomers), or geometric isomers (e.g., cis-trans isomers).
In the present specification, "substantially pure" means that, for example, when used in association with an enantiomer or a diastereomer, the enantiomer or the diastereomer may be present in an amount of about 90% or more, preferably about 95% or more, more preferably about 97% or more or about 98% or more, further preferably about 99% or more, and still further preferably about 99.5% or more (w/w) of the specific compound exemplified.
In the present specification, "hydrate" refers to a compound to which water is bonded, and is a broad concept including a clathrate compound having no chemical bond between water and the compound.
In the present specification, "solvate" refers to a high-order compound generated between a molecule or ion of a solute and a molecule or ion of a solvent
Figure BDA0003050095460000091
One aspect of the present invention may relate to a method for producing a naphthopyrone derivative, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof, which is a method for producing 1 or more naphthopyrone derivatives, stereoisomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, or solvates thereof selected from the naphthopyrone derivatives of the following chemical formulae 1 to 5, including a process of separating one or more selected from the naphthopyrone derivatives, stereoisomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, or solvates thereof from Cassia tora (Cassia obtusifolia L.) or Cassia tora L) buds (sprout).
[ chemical formula 1]
Figure BDA0003050095460000101
[ chemical formula 2]
Figure BDA0003050095460000102
[ chemical formula 3]
Figure BDA0003050095460000103
[ chemical formula 4]
Figure BDA0003050095460000104
[ chemical formula 5]
Figure BDA0003050095460000105
The compound of chemical formula 1 (naphthopyrone derivative) may be 7-hydroxyruminomycin-8' -O-glucopyranoside, the compound of chemical formula 2 (naphthopyrone derivative) may be isoctoralactone (isotoralone), the compound of chemical formula 3 (naphthopyrone derivative) may be toralactone (toralone), the compound of chemical formula 4 (naphthopyrone derivative) may be torachrysone, and the compound of chemical formula 5 (naphthopyrone derivative) may be rubrofusarin.
The sprout is a sprout to be cultivated in a short time by germinating the seed, and may be a sprout cultivated by germinating the seed indoors or a sprout generally sold.
The Cassia seed sprout (sprout) or the Cassia seed sprout plant of the above production method may be a sprout or young plant of Cassia seed (Cassia obtusifolia L.)) or Longgang nan tea (Cassia tora L.) or Cassia occidentalis (Senna tora) that sprouts and naturally grows or is cultivated for 2 to 31 days, specifically, a sprout or young plant that naturally grows or is cultivated for 2 to 31 days, specifically, for 2 to 3 days or more, for 4 to 5 days or more, for 6 to 6 days or more, for 7 days or more, for 8 days or more, for 9 days or more, for 10 days or more, for 11 days or more, for 12 days or more, for 13 days or more, for 14 days or more, for 15 days or more, for 16 days or more, for 17 days or more, for 18 days or more, for 19 days or more, for 20 days or more, for 21 days or more, for 22 days or more, for 23 days or more, for 24 days or more, for 25 days or more, for 26 days or more, for 27 days or more, for 28 days or 29 days or 30 days or more, the bud or plantlet may be a bud or plantlet which is naturally grown or cultivated for 31 days or less, 30 days or less, 29 days or less, 28 days 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 or less, 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, and more specifically, may be a bud or plantlet which is naturally grown or cultivated for a period selected from a period of 2 days to 21 days.
The Cassia seed (Cassia obtusifolia L.) or Cassia tora (Cassia tora L.) is a plant of leguminosae of rosales of dicotyledonae of angiosperma, and is a mature seed of the Cassia seed (Cassia obtusifolia L.) or dragon holnan tea (Cassia tora L) or Cassia occidentalis (Senna tora), although the Cassia seed extract is known to improve diabetes, improve dyslipidemia, protect liver, resist bacteria, and reduce blood pressure (Dong, x.et al.mol.med.rep.2017,16, 2331-membered 2346), it has not been known to improve the effect of oxidative stress or drug toxicity, particularly, the effect of preventing damage or death of retinal nerve cells or hippocampal nerve cells caused by glutamic acid.
The said sprout or plantlet of Cassia tora can be whole or part of sprout or plantlet of Cassia tora. The above-mentioned parts may be front tips or aerial parts or underground parts. The above-mentioned aerial parts may be stems, leaves, flowers or a combination thereof. The underground portion may be a root. The plants can be grown naturally or cultivated artificially. The cassia buds of the present invention can be easily cultivated indoors, and can be cultivated and used in a short period of several weeks without supplying extra nutrients, and therefore, have an advantage of great industrial applicability.
The above cassia sprout extract comprises crude extract (fraction extract) or further fraction obtained by fractionating the extract (fractionation). Specifically, the above cassia sprout extract can be crude extract, fraction, or combination thereof of cassia sprout. The crude extract is obtained by contacting the buds of Cassia obtusifolia with an extraction solvent. The fraction is obtained by separating a substance containing a specific component from the crude extract. The above extract, or fraction thereof, may be included in the composition of the present invention in the form of an extract of a cassia seed bud plant, a fraction thereof, or a small fraction, each or a mixture thereof. The above-mentioned small fraction can be obtained by an ultrafiltration membrane having a cut-off value, and can be obtained by column chromatography or solvent fractionation. The cassia seed sprout extract or fraction may contain 1 or more naphthopyrone derivatives selected from the naphthopyrone derivatives of the chemical formulas 1 to 5.
The separation may be by filtration, precipitation, centrifugation, solvent fractionation, chromatography, or a combination thereof. The chromatography is performed for separation according to various conditions, i.e., size, charge, hydrophobicity or affinity, and may be ion exchange chromatography, affinity chromatography, size exclusion chromatography, HPLC, high performance liquid chromatography, column chromatography, reverse phase column chromatography or a combination thereof.
The extraction may comprise incubating the plant in a solvent for a period of time. The extraction may be carried out with or without stirring, or may include heating. The incubation may be carried out with or without stirring at room temperature or reflux temperature. The incubation temperature may be appropriately selected depending on the selected solvent. For example, it may be room temperature to reflux temperature, 30 ℃ to reflux temperature, 40 ℃ to reflux temperature. The heating may include heating to 50 deg.C, 60 deg.C, 70 deg.C, 80 deg.C, or reflux temperature. The heating may be to 50 ℃ to reflux temperature, 60 ℃ to reflux temperature, 70 ℃ to reflux temperature, 80 ℃ to reflux temperature, or reflux temperature. The above extraction time may vary depending on the selected temperature, and may be 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, 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 hours 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 time, 2 times, 5 times, 10 times or 15 times or more relative to the weight of the plant. 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 relative to the weight of the plant. The plant may be dried in a shade or shade device or in a warm air or drying apparatus.
The extraction method may be a method generally used in the art, such as filtration, hot water extraction, precipitation extraction, cold precipitation, microwave extraction, reflux cooling extraction, pressure extraction, subcritical extraction, supercritical extraction, and ultrasonic extraction. For example, a precipitation extraction method may be used. The precipitation can be warm precipitation or precipitation at room temperature, and can be extracted for 1-5 times. The above cassia buds may be contacted with 0.1 to 10 times or 1 to 6 times of the extraction solvent. The extraction temperature of the cold precipitation can be 20 ℃ to 40 ℃. The warm precipitation or heat extraction temperature may be 40 ℃ to 100 ℃. The extraction time of the cold precipitation can be 24 hours to 120 hours, and the extraction time of the warm precipitation or the heating can be 0.5 hour to 48 hours.
The above extraction may further comprise removing the solvent from the obtained extract by a known method such as evaporation or concentration under reduced pressure. The above extraction may further comprise drying the obtained extract by lyophilization or the like to produce a dried extract. The vacuum concentration may be performed by a vacuum concentrator or a vacuum rotary evaporator. Further, the drying may be reduced pressure drying, vacuum drying, boiling drying, spray drying or freeze drying.
The method may further comprise extracting the sprout of the cassia seed with a solvent selected from water, C1-C6 alcohol and a mixed solvent thereof. The above alcohol may be an alcohol of C1 to C3, an alcohol of C1 to C4, an alcohol of C1 to C5, or an alcohol of C1 to C6. The alcohol may be a primary alcohol. The above alcohol of C1 to C6 may be methanol, ethanol, propanol, isopropanol, butanol or a mixture thereof.
The above production method may further comprise a step of fractionating the extract with at least one solvent selected from the group consisting of water, ethyl acetate, hexane, dichloromethane, chloroform, methanol, ethanol, acetone, and a mixture thereof. The above-mentioned production method may further comprise a step of producing a fraction of the sprout of cassia seed containing 1 to 20 wt% of at least one naphthopyrone derivative selected from the naphthopyrone derivatives of chemical formulae 1 to 5, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof, based on the total weight of the extract obtained by the step of extracting the sprout of cassia seed, and specifically may comprise a step of producing a fraction of the sprout of cassia seed containing at least 1 wt%, at least 2 wt%, at least 3 wt%, at least 4 wt%, at least 5 wt%, at least 6 wt%, at least 7 wt%, at least 8 wt%, at least 9 wt%, at least 10 wt%, at least 11 wt%, at least 12 wt%, at least 13 wt%, at least 14 wt%, at least 15 wt%, at least 16 wt%, at least 17 wt%, at least, The process of producing the cassia sprout fraction of 18 wt% or more or 19 wt% or more may include a process of producing a cassia sprout fraction containing 20 wt% or less, 19 wt% or less, 18 wt% or less, 17 wt% or less, 16 wt% or less, 15 wt% or less, 14 wt% or less, 13 wt% or less, 12 wt% or less, 11 wt% or less, 10 wt% or less, 9 wt% or less, 8 wt% or less, 7 wt% or less, 6 wt% or less, 5 wt% or less, 4 wt% or less, 3 wt% or less, or 2 wt% or less.
The naphthopyrone derivative of 1 or more species selected from the naphthopyrone derivatives of the chemical formulae 1 to 5, stereoisomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, or solvates thereof may be contained in an amount of 1 to 50% by weight, 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% by weight or more, 7% by weight or more, 8% by weight or more, 9% by weight or more, 10% by weight or more, 11% by weight or more, 12% by weight or more, 13% by weight or more, 14% by weight or more, 15% by weight or more, 16% by weight or more, 17% by weight or more, 18% by weight or more, 19% by weight or more, 20% by weight or more, 21% by weight or more, 22% or more, based on the total weight of the cassia seed extract or fraction, 23% by weight or more, 24% by weight or more, 25% by weight or more, 26% by weight or more, 27% by weight or more, 28% by weight or more, 29% by weight or more, 30% by weight or more, 31% by weight or more, 32% by weight or more, 33% by weight or more, 34% by weight or more, 35% by weight or more, 36% by weight or more, 37% by weight or more, 38% by weight or more, 39% by weight or more, 40% by weight or more, 41% by weight or more, 42% by weight or more, 43% by weight or more, 44% by weight or more, 45% by weight or more, 46% by weight or more, 47% by weight or more, 48% by weight or more, or 49% by weight or more, and may include 50% by weight or less, 49% by weight or less, 48% by weight or less, 47% by weight or less, 46% or less, 45% by weight or less, 44% or less, 43% by weight or less, 42% or less, or less, 41% by weight or less, 40% by weight or less, 39% by weight or less, 38% by weight or less, 37% by weight or less, 36% by weight or less, 35% by weight or less, 34% by weight or less, 33% by weight or less, 32% by weight or less, 31% by weight or less, 30% by weight or less, 29% by weight or less, 28% by weight or less, 27% by weight or less, 26% by weight or less, 25% by weight or less, 24% by weight or less, 23% by weight or less, 22% by weight or less, 21% by weight or less, 20% by weight or less, 19% by weight or less, 18% by weight or less, 17% by weight or less, 16% by weight or less, 15% by weight or less, 14% by weight or less, 13% by weight or less, 12% by weight or less, 11% by weight or less, 10% by weight or less, 9% by weight or less, 8% or less, 7% by weight or less, 6% by weight or less, 4% by weight or less, 3% by weight or less, or 2% by weight or less.
One aspect of the present invention may relate to a composition for protecting nerve cells from oxidative stress or for inhibiting nerve cell death, comprising, as an active ingredient, one or more selected from the group consisting of a naphthopyrone derivative, which is 1 or more selected from the naphthopyrone derivatives of chemical formulae 1 to 5, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof, and a Cassia seed (Cassia obtusifolia L.) or Cassia tora L bud extract comprising the same, or a Cassia seed bud fraction comprising the same. The 1 or more naphthopyrone derivatives selected from the naphthopyrone derivatives of chemical formula 1 to chemical formula 5 may be 1 or more naphthopyrone derivatives selected from the naphthopyrone derivatives of chemical formula 1 and the naphthopyrone derivatives of chemical formula 2 to chemical formula 5. The explanation about the naphthopyrone derivative of the above chemical formula 1 to chemical formula 5, pharmaceutically acceptable salt, stereoisomer, hydrate, solvate, cassia buds, cassia bud extract or fraction is as described above.
The composition may be a composition for protecting nerve cells or for inhibiting nerve cell death, and specifically, may be a composition for protecting nerve cells from oxidative stress induced by metabolic toxicity, neurotoxicity, chemical causes, or the like or for inhibiting nerve cell death. The above oxidative stress may be induced by glutamic acid (glutamate), glutamate toxicity or glutamate neurotoxicity, or may be oxidative stress caused by calcium homeostasis disorders, mitochondrial dysfunction, excitotoxicity, trophic factor depletion, or the like, similar to the oxidative stress induced by glutamate neurotoxicity. Specifically, the composition can reduce, inhibit, ameliorate, or prevent damage to retinal or hippocampal nerve cells induced by glutamate, glutamate toxicity, or glutamate neurotoxicity, or resuscitate or regenerate dead retinal or hippocampal nerve cells, and can protect nerve cells or inhibit nerve cell death by antioxidant activation, glutamate metabolism regulation, or the like, which is a defense mechanism against oxidative stress induced by glutamate neurotoxicity, calcium homeostasis disorder, mitochondrial dysfunction, excitotoxicity, trophic factor depletion, or the like.
According to an embodiment of the present invention, the effect of DPPH radical scavenging is more than 1.7 times superior to that of the extract of cassia buds (test example 1), the compound of the above chemical formula 1 to chemical formula 5 (naphthopyrone derivative) corresponding to the content of the antioxidant component is higher, and the more superior antioxidant effect is exhibited (test example 2), and the antioxidant effect as a defense mechanism against oxidative stress of the composition of the present invention is activated, and the effect of protecting nerve cells or inhibiting nerve cell death is confirmed to be superior.
The composition can be administered before, simultaneously with, or after the occurrence of nerve cell injury or death. The composition may contain 0.001 to 80% by weight, relative to the total weight of the composition, of at least one selected from the group consisting of a naphthopyrone derivative, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof, a cassia seed (cassia obtusifolia or cassia tora) bud extract containing the same, or a cassia seed bud fraction containing the same, wherein the naphthopyrone derivative is at least 1 selected from the group consisting of naphthopyrone derivatives of chemical formulas 1 to 5, and specifically, may contain at least 0.001% by weight, at least 0.01% by weight, at least 0.05% by weight, at least 0.1% by weight, at least 1% by weight, at least 2% by weight, at least 3% by weight, at least 4% by weight, at least 5% by weight, at least 10% by weight, at least 20% by weight, at least 30% by weight, at least 40% by weight, or at least 60% by weight, may comprise 80 wt% or less, 60 wt% or less, 40 wt% or less, 30 wt% or less, 20 wt% or less, 10 wt% or less, 5 wt% or less, 4 wt% or less, 3 wt% or less, 2 wt% or less, or 1 wt% or less.
The cassia seed sprout extract or fraction may contain 1 or more species of naphthopyrone derivatives selected from the naphthopyrone derivatives of chemical formulae 1 to 5, stereoisomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, or solvates thereof in an amount of 1 to 50 wt% based on the total weight of the cassia seed sprout extract or fraction, and specifically may contain 1 wt% or more, 2 wt% or more, 3 wt% or more, 4 wt% or more, 5 wt% or more, 6 wt% or more, 7 wt% or more, 8 wt% or more, 9 wt% or more, 10 wt% or more, 11 wt% or more, 12 wt% 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, 19 wt% or more, 20 wt% or more, or less, 21% by weight or more, 22% by weight or more, 23% by weight or more, 24% by weight or more, 25% by weight or more, 26% by weight or more, 27% by weight or more, 28% by weight or more, 29% by weight or more, 30% by weight or more, 31% by weight or more, 32% by weight or more, 33% by weight or more, 34% by weight or more, 35% by weight or more, 36% by weight or more, 37% by weight or more, 38% by weight or more, 39% by weight or more, 40% by weight or more, 41% by weight or more, 42% by weight or more, 43% by weight or more, 44% by weight or more, 45% by weight or more, 46% by weight or more, 47% by weight or more, 48% by weight or more, or 49% by weight or less, 48% by weight or less, 47% by weight or less, 46% by weight or less, 45% by weight or less, 44% or less, 43% or less, or less by weight, or less, 42% by weight or less, 41% by weight or less, 40% by weight or less, 39% by weight or less, 38% by weight or less, 37% by weight or less, 36% by weight or less, 35% by weight or less, 34% by weight or less, 33% by weight or less, 32% by weight or less, 31% by weight or less, 30% by weight or less, 29% by weight or less, 28% by weight or less, 27% by weight or less, 26% by weight or less, 25% by weight or less, 24% by weight or less, 23% by weight or less, 22% by weight or less, 21% by weight or less, 20% by weight or less, 19% by weight or less, 18% by weight or less, 17% by weight or less, 16% by weight or less, 15% by weight or less, 14% by weight or less, 13% by weight or less, 12% by weight or less, 11% by weight or less, 10% by weight or less, 9% by weight or less, 8% by weight or less, 7% by weight or, 5% by weight or less, 4% by weight or less, 3% by weight or less, or 2% by weight or less.
The nerve cell may be a central nerve, peripheral nerve or optic nerve associated with the brain hippocampus or retina of the eye, and specifically may be a retinal nerve cell or hippocampal nerve cell.
The retinal nerve cell may be one or more cells selected from rod cells (rod cells), cone cells (cone cells), bipolar cells (bipolar cells), retinal amacrine cells (retinal amacrine cells), horizontal cells (horizontal cells), and retinal ganglion cells (retinal ganglion cells). The rod cells and the cone cells are nerve cells for sensing light and recognizing things, the bipolar cells, the retinal amacrine cells and the horizontal cells are nerve cells for transmitting visual information to retinal ganglion cells for transmitting visual information of the retina to the brain. The R28 retinal progenitor cells or R28 retinal neural progenitor cells of one embodiment of the present invention are characteristic of various constituent cells expressing the retina, and are viable even when transplanted to the retina, and can be effectively used for cell death and cytotoxicity studies associated with expression of glutamate or GABA receptors associated with glaucoma, for cell death caused by hypoxia or serum deficiency. In addition, the R28 cells can differentiate into retinal ganglion cells from retinal progenitor cells according to culture conditions, and can be used not only for basic cytological studies of the retina but also for basic studies of retinal ganglion cells (Jungil Lee, Jaewoo Kim, journal of the korean society of ophthalmology, 2009,50, 919-.
The hippocampal nerve cells are nerve cells of the hippocampus (hippopus) which is a structure of the medial temporal lobe of the brain, and the hippocampus is weak to physiological/oxidative stress stimuli, causing impairment of cognitive functions due to stress, known as central tissues, which can cause changes in the kinematics associated with hippocampus structure and brain cell generation, synaptic plasticity, and hippocampus (Kim Eun-joo, korean journal of psychology: cognition and biology 2012,24, 65-88). HT-22 hippocampal neurons of one embodiment of the present invention are sensitive to glutamate (glutamate) and can be used as an in vitro model system for studying neurotoxicity induced by oxidative stress (Liu, J.et al. Life Science,2009,84, 267-19; Kim Jihyun Kim, Soonsil Jeon, Korea journal of food nutrition Science, 2017,46, 886-890).
According to an embodiment of the present invention, when R28 cells induced oxidative stress by glutamic acid treatment were treated with cassia seed sprout extract and the naphthopyrone derivatives of the above chemical formulas 1 to 5, respectively, the survival rate of cells decreased by glutamic acid was increased to a level corresponding to the control group, and the effect of protecting retinal neurons was more excellent than that of the cassia seed extract as a comparative example, confirming that the composition of the present invention has an excellent effect of protecting retinal neurons from damage caused by oxidative stress or inhibiting retinal neuron death (test examples 3 and 4).
In addition, according to an embodiment of the present invention, when HT-22 cells induced oxidative stress by glutamate treatment were treated with cassia seed sprout extract and naphthopyrone derivatives of the above chemical formulae 1 to 5, respectively, the survival rate of cells decreased by glutamate increased to a level corresponding to the control group, and such a protective effect on hippocampal neurons was more excellent than that of the cassia seed extract as a comparative example, confirming that the composition of the present invention has an excellent effect of protecting hippocampal neurons from damage due to oxidative stress or inhibiting hippocampal neuronal death (test examples 5 and 6).
An aspect of the present invention may relate to a composition for preventing or treating a nerve injury disease induced by damage or death of retinal nerve cells or hippocampal nerve cells, which includes one or more naphthopyrone derivatives selected from the group consisting of 1 or more naphthopyrone derivatives of the above-described chemical formula 1 to chemical formula 5, stereoisomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, or solvates thereof, a cassia seed (cassia obtusifolia or cassia tora) bud extract including the same, or a cassia seed bud fraction including the same as an active ingredient, or may relate to a composition for preventing or treating a nerve injury disease induced by glutamate neurotoxicity. The 1 or more naphthopyrone derivatives selected from the naphthopyrone derivatives of chemical formula 1 to chemical formula 5 may be 1 or more naphthopyrone derivatives selected from the naphthopyrone derivatives of chemical formula 1 and the naphthopyrone derivatives of chemical formula 2 to chemical formula 5. The explanation about the naphthopyrone derivatives, pharmaceutically acceptable salts, stereoisomers, hydrates, solvates, cassia buds, cassia bud extracts or fractions, contents of the above-mentioned chemical formula 1 to chemical formula 5 is as described above.
The above-mentioned nerve injury diseases induced by the damage or death of retinal nerve cells or hippocampal nerve cells can be caused by glutamic neurotoxicity, and the explanation on the above-mentioned retinal nerve cells and hippocampal nerve cells is as described above. The nerve injury disease may be one or more selected from diabetic retinopathy caused by damage or death of retinal nerve cells, macular degeneration, visual impairment caused by damage of retinal cells, retinitis pigmentosa, retinal detachment, retinal vascular occlusion, and glaucoma, and may be one or more selected from hypomnesis, learning ability deterioration, depression, amyotrophic lateral sclerosis (lugeriger's disease), parkinson's disease, and cerebral ischemic nerve injury caused by damage or death of hippocampal nerve cells, but is not limited as long as it is a disease caused by damage or death of retinal nerve cells or hippocampal nerve cells caused by glutamate neurotoxicity.
The glutamic acid neurotoxicity refers to toxicity exhibited by the action of glutamic acid as an oxidative neurotoxic substance due to a sharp increase in the concentration of glutamic acid outside nerve cells. The glutamic acid neurotoxicity may be induced by glutamic acid influx from the outside of an individual, and the glutamic acid influx may be induced by ingestion or administration of a food containing glutamic acid, a therapeutic agent containing glutamic acid, a prophylactic agent containing glutamic acid, an antibiotic containing glutamic acid, or the like, but is not limited thereto.
The naphthopyrone derivative of 1 or more selected from the naphthopyrone derivatives of the chemical formulas 1 to 5 may be separated or purified from the bud of cassia seed (cassia obtusifolia or cassia tora), specifically, may be separated or purified from the ethanol extract of the bud of cassia seed, and may be a derivative obtained by fractionating the ethanol extract of the bud of cassia seed with ethyl acetate, then chromatographically separating the ethyl acetate fraction, or fractionating the ethyl acetate fraction again with a mixed solvent of hexane, dichloromethane and methanol, and chromatographically passing the same.
The above cassia sprout extract may be an extract obtained by extracting cassia sprouts with a solvent selected from the group consisting of water, C1 to C6 alcohols and a mixed solvent thereof. The above alcohol may be an alcohol of C1 to C3, an alcohol of C1 to C4, an alcohol of C1 to C5, or an alcohol of C1 to C6. The alcohol may be a primary alcohol. The above alcohol of C1 to C6 may be methanol, ethanol, propanol, isopropanol, butanol or a mixture thereof.
The fractionation may further comprise a process of fractionating with one or more selected from the group consisting of water, ethyl acetate, hexane, dichloromethane, chloroform, methanol, ethanol, acetone, and a mixed solvent thereof.
According to an embodiment of the present invention, 1 or more naphthopyrone derivatives selected from the naphthopyrone derivatives of chemical formulas 1 to 5 described above as an antioxidant ingredient are contained in a higher amount in a cassia seed sprout extract than a cassia seed extract (test example 2), and the naphthopyrone derivatives have an excellent effect of protecting retinal nerve cells (R28) and hippocampal nerve cells (HT-22) damaged or even dead by glutamate treatment (test examples 4 and 6), confirming that the naphthopyrone derivatives of chemical formulas 1 to 5 described above are effective ingredients of cassia seed sprouts having an effect of preventing or treating a nerve injury disease induced by glutamate neurotoxicity.
One aspect of the present invention may relate to an antioxidant composition comprising, as an active ingredient, at least one naphthopyrone derivative selected from the group consisting of at least 1 naphthopyrone derivative selected from the naphthopyrone derivatives of the above chemical formulae 1 to 5, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof, and a cassia seed (cassia obtusifolia or cassia tora) bud extract comprising the same, or a cassia seed bud fraction comprising the same. The 1 or more naphthopyrone derivatives selected from the naphthopyrone derivatives of chemical formula 1 to chemical formula 5 may be 1 or more naphthopyrone derivatives selected from the naphthopyrone derivatives of chemical formula 1 and the naphthopyrone derivatives of chemical formula 2 to chemical formula 5. The explanation about the naphthopyrone derivatives, pharmaceutically acceptable salts, isomers, hydrates, solvates, cassia buds, cassia bud extracts or fractions, contents of the above chemical formula 1 to chemical formula 5 are as described above.
In another aspect of the present invention, there may be provided a method for protecting nerve cells from oxidative stress or inhibiting nerve cell death, comprising administering to an individual in need of protection of nerve cells from oxidative stress or inhibition of nerve cell death, one or more naphthopyrone derivatives selected from the group consisting of 1 or more naphthopyrone derivatives of the above chemical formula 1 to chemical formula 5, stereoisomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, or solvates thereof, a cassia seed (cassia obtusifolia or cassia tora) bud extract comprising the same, or a cassia seed bud fraction comprising the same. In one aspect of the present invention, the application of the above method may be carried out according to the application method and application amount described in the present specification.
In another aspect of the present invention, may relate to a method for preventing or treating a nerve injury disease induced by damage or death of retinal or hippocampal neurons, including administering to a subject in need of prevention or treatment of a nerve injury disease induced by damage or death of retinal or hippocampal neurons one or more naphthopyrone derivatives selected from the group consisting of 1 or more naphthopyrone derivatives of the above chemical formula 1 to 5, stereoisomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, or solvates thereof, a cassia seed (cassia obtusifolia or cassia tora) bud extract containing the same, or a cassia seed bud fraction containing the same. In one aspect of the present invention, the application of the above method may be carried out according to the application method and application amount described in the present specification.
In another aspect of the present invention, may relate to an antioxidant method comprising administering to a subject in need of antioxidant treatment one or more naphthopyrone derivatives selected from the group consisting of 1 or more naphthopyrone derivatives of the above chemical formula 1 to chemical formula 5, stereoisomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, or solvates thereof, a cassia seed (cassia obtusifolia or cassia tora) bud extract comprising the same, or a cassia seed bud fraction comprising the same. In one aspect of the present invention, the application of the above method may be carried out according to the application method and application amount described in the present specification.
In another aspect of the present invention, may relate to a use of one or more naphthopyrone derivatives selected from the group consisting of 1 or more naphthopyrone derivatives selected from the group consisting of the naphthopyrone derivatives of the above chemical formula 1 to chemical formula 5, stereoisomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, or solvates thereof, a cassia seed (cassia obtusifolia or cassia tora) bud extract comprising the same, or a cassia seed bud fraction comprising the same, for manufacturing a pharmaceutical composition for protecting nerve cells from oxidative stress or for inhibiting nerve cell death.
In another aspect of the present invention, there may be involved a use of one or more naphthopyrone derivatives selected from the group consisting of 1 or more naphthopyrone derivatives of the above chemical formula 1 to chemical formula 5, stereoisomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, or solvates thereof, a cassia seed (cassia obtusifolia or cassia tora) bud extract containing the same, or a cassia seed bud fraction containing the same, for manufacturing a pharmaceutical composition for preventing or treating a nerve injury disease induced by damage or death of retinal nerve cells or hippocampal nerve cells.
In another aspect of the present invention, there may be involved the use of one or more naphthopyrone derivatives selected from the group consisting of 1 or more naphthopyrone derivatives selected from the group consisting of the naphthopyrone derivatives of the above chemical formulae 1 to 5, stereoisomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, or solvates thereof, a cassia seed (cassia obtusifolia or cassia tora) bud extract comprising the same, or a cassia seed bud fraction comprising the same, for manufacturing a pharmaceutical composition for antioxidation.
In another aspect of the present invention, may relate to a use of a naphthopyrone derivative selected from the group consisting of 1 or more naphthopyrone derivatives selected from the group consisting of the naphthopyrone derivatives of the above chemical formula 1 to chemical formula 5, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof, a cassia seed (cassia obtusifolia or cassia tora) bud extract comprising the same, or a cassia seed bud fraction comprising the same, for manufacturing a cosmetic composition for protecting nerve cells from oxidative stress or for inhibiting nerve cell death.
In another aspect of the present invention, there may be involved a use of one or more naphthopyrone derivatives selected from the group consisting of 1 or more naphthopyrone derivatives of the above chemical formula 1 to chemical formula 5, stereoisomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, or solvates thereof, a cassia seed (obtusifolia cassia tora or cassia tora) bud extract containing the same, or a cassia seed bud fraction containing the same, for the manufacture of a cosmetic composition for improving, preventing, or treating a nerve injury disease induced by damage or death of retinal nerve cells or hippocampal nerve cells.
In another aspect of the present invention, there may be involved the use of one or more naphthopyrone derivatives selected from the group consisting of 1 or more naphthopyrone derivatives selected from the above-mentioned naphthopyrone derivatives of chemical formula 1 to chemical formula 5, stereoisomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, or solvates thereof, a cassia seed (cassia obtusifolia or cassia tora) bud extract comprising the same, or a cassia seed bud fraction comprising the same for manufacturing an antioxidant cosmetic composition.
In another aspect of the present invention, may relate to a use of a naphthopyrone derivative selected from 1 or more species of naphthopyrone derivatives selected from the group consisting of the naphthopyrone derivatives of the above chemical formula 1 to chemical formula 5, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof, a cassia seed (cassia obtusifolia or cassia tora) bud extract containing the same, or a cassia seed bud fraction containing the same, for manufacturing a food composition for protecting nerve cells from oxidative stress or for inhibiting nerve cell death.
In another aspect of the present invention, may relate to a use of one or more naphthopyrone derivatives selected from the group consisting of 1 or more naphthopyrone derivatives of the above chemical formula 1 to chemical formula 5, stereoisomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, or solvates thereof, a cassia seed (cassia obtusifolia or cassia tora) bud extract containing the same, or a cassia seed bud fraction containing the same, for manufacturing a food composition for improving, preventing, or treating a nerve injury disease induced by damage or death of retinal nerve cells or hippocampal nerve cells.
In another aspect of the present invention, the present invention may relate to the use of one or more naphthopyrone derivatives selected from the group consisting of 1 or more naphthopyrone derivatives selected from the group consisting of the naphthopyrone derivatives of the above chemical formulae 1 to 5, stereoisomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, or solvates thereof, a cassia seed (cassia obtusifolia or cassia tora) bud extract comprising the same, or a cassia seed bud fraction comprising the same, for manufacturing a food composition for antioxidation.
In another aspect of the present invention, may relate to a use of a naphthopyrone derivative selected from the group consisting of 1 or more naphthopyrone derivatives selected from the group consisting of the naphthopyrone derivatives of the above chemical formula 1 to chemical formula 5, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof, a cassia seed (cassia obtusifolia or cassia tora) bud extract comprising the same, or a cassia seed bud fraction comprising the same, for manufacturing a quasi-pharmaceutical composition for protecting nerve cells from oxidative stress or for inhibiting nerve cell death.
In another aspect of the present invention, may relate to a use of one or more naphthopyrone derivatives selected from the group consisting of 1 or more naphthopyrone derivatives of the above chemical formula 1 to chemical formula 5, stereoisomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, or solvates thereof, a cassia seed (cassia obtusifolia or cassia tora) bud extract containing the same, or a cassia seed bud fraction containing the same, for manufacturing a quasi-pharmaceutical composition for improving, preventing, or treating a nerve injury disease induced by damage or death of retinal nerve cells or hippocampal nerve cells.
In another aspect of the present invention, there may be involved the use of one or more naphthopyrone derivatives selected from the group consisting of 1 or more naphthopyrone derivatives selected from the group consisting of the naphthopyrone derivatives of the above chemical formulae 1 to 5, stereoisomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, or solvates thereof, a cassia seed (cassia obtusifolia or cassia tora) bud extract comprising the same, or a cassia seed bud fraction comprising the same, for the manufacture of a quasi-pharmaceutical composition for antioxidation.
In another aspect of the present invention, there may be involved the use of 1 or more naphthopyrone derivatives selected from the naphthopyrone derivatives of the above chemical formulae 1 to 5, one or more stereoisomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, or solvates thereof, a cassia seed (cassia obtusifolia or cassia tora) bud extract containing the same, or a cassia seed bud fraction containing the same for protecting nerve cells from oxidative stress or inhibiting nerve cell death.
In another aspect of the present invention, there may be involved the use of one or more naphthopyrone derivatives selected from the group consisting of 1 or more naphthopyrone derivatives of the above-mentioned chemical formula 1 to chemical formula 5, stereoisomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, or solvates thereof, a cassia seed (cassia obtusifolia or cassia tora) bud extract comprising the same, or a cassia seed bud fraction comprising the same for the prevention or treatment of a nerve injury disease induced by damage or death of retinal nerve cells or hippocampal nerve cells.
In another aspect of the present invention, there may be provided an antioxidant use of 1 or more naphthopyrone derivatives selected from the naphthopyrone derivatives of the above chemical formulae 1 to 5, one or more stereoisomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, or solvates thereof, a cassia seed (cassia obtusifolia or cassia tora) bud extract containing the same, or a cassia seed bud fraction containing the same.
In another aspect of the present invention, there may be involved the use of one or more naphthopyrone derivatives selected from the group consisting of 1 or more naphthopyrone derivatives selected from the group consisting of the naphthopyrone derivatives of the above chemical formula 1 to chemical formula 5, stereoisomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, or solvates thereof, a cassia seed (cassia obtusifolia or cassia tora) bud extract containing the same, or a cassia seed bud fraction containing the same, for protecting nerve cells from oxidative stress or inhibiting death of nerve cells.
In another aspect of the present invention, may relate to the use of one or more naphthopyrone derivatives selected from the group consisting of 1 or more naphthopyrone derivatives of the above-mentioned chemical formula 1 to chemical formula 5, stereoisomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, or solvates thereof, a cassia seed (cassia obtusifolia or cassia tora) bud extract comprising the same, or a cassia seed bud fraction comprising the same, for preventing or treating a nerve injury disease induced by damage or death of retinal nerve cells or hippocampal nerve cells.
In another aspect of the present invention, there is provided a use of a naphthopyrone derivative selected from the group consisting of 1 or more naphthopyrone derivatives of the above chemical formulae 1 to 5, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof, a cassia seed (cassia obtusifolia or cassia tora) bud extract containing the same, or a cassia seed bud fraction containing the same for antioxidation.
The composition of the present invention may be a pharmaceutical composition, a cosmetic composition or a food composition.
The pharmaceutical composition according to one aspect of the present invention may be formulated for oral or non-oral administration. For formulation, the pharmaceutical composition can be prepared by using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant, which is generally used. Solid preparations for oral administration include tablets, pills, powders, granules, soft or hard capsules and the like, and such solid preparations are prepared by mixing at least one or more excipients, for example, starch, calcium carbonate, sucrose (sucrose) or lactose (lactose), gelatin and the like, in one or more compounds. In addition, lubricants such as magnesium stearate and talc have been used in addition to simple excipients. The liquid preparations for oral administration correspond to suspensions, liquid solutions, emulsions, syrups and the like, and may include various excipients such as wetting agents, sweeteners, flavoring agents, preservatives and the like, in addition to water and liquid paraffin, which are commonly used as simple diluents. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, suppositories. As the nonaqueous solvent and the suspending solvent, vegetable oils such as propylene glycol (propylene glycol), polyethylene glycol, and olive oil; injectable esters such as ethyl oleate, and the like. As the base of the suppository, synthetic fatty acid ester (witepsol), polyethylene glycol, tween (tween)61, cacao butter, laurate, glycerogelatin, and the like can be used.
The pharmaceutical administration forms of the compositions according to one aspect of the invention can also be used in the form of their pharmaceutically acceptable salts, alone or in combination with other pharmaceutically active compounds, as well as in appropriate combinations. The salt is not particularly limited as long as it is a pharmaceutically acceptable salt, and examples thereof include hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid, hydrobromic acid, formic acid, acetic 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 non-oral administration form may be a skin patch, ointment, cream, eye drop, spray or injection.
In another aspect, the present invention may be a method for protecting nerve cells of an individual comprising the step of administering to the individual a pharmaceutical composition as described above.
The subject may be a mammal, for example, a human, a cow, a horse, a pig, a dog, a sheep, a goat or a cat, the mammal may be a human, and the effective administration amount of the compound of the present invention to the human may vary depending on the age, body weight, sex, administration mode, health state and disease degree of the patient.
The administration can be carried out in various dosage forms for oral administration or non-oral administration such as intravenous, film-coating, intradermal, subcutaneous, epithelial or intramuscular administration, and the preparation can be carried out using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc., which are generally used.
The above administration can be carried out by methods known in the art. Administration may be suitably carried out to the individual by any method, for example, by intravenous, intramuscular, oral, or subcutaneous administration. The administration can be systemic or local. Such administration can be minimally administered to the site where nerve cells are expected to be present or generated.
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, and the food composition may be formulated into a concentrated solution or powder for ingestion as it is or by dilution, or may be formulated into a form for oral ingestion, for example, into a liquid drug such as a tablet, granule, powder, or oral liquid, caramel, gel, stick, or the like. In the food composition of each dosage form, ingredients generally used in the art may be appropriately selected and mixed by those skilled in the art according to the dosage form or the purpose of use, in addition to the active ingredient, and when used together with other raw materials, the effect of gain may be brought about. Specifically, the food composition may contain various flavors or natural carbohydrates as additional ingredients. The natural carbohydrate may be monosaccharide such as glucose and fructose; disaccharides such as maltose and sucrose; and polysaccharides such as dextrin, cyclodextrin; xylitol, sorbitol, erythritol and other sugar alcohols. As the sweetener, natural sweeteners such as thaumatin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like can be used. The proportion of the natural carbohydrate may be selected from the range of 0.01 to 0.04 parts by weight, specifically, about 0.02 to 0.03 parts by weight per 100 parts by weight of the composition. The food composition may further contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acids and salts thereof, alginic acids and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonating agents used for carbonated beverages, and the like. In addition, the present invention may contain pulp for use in the manufacture of natural fruit juices, fruit juice beverages, and vegetable beverages. Such ingredients may be used alone or in combination. Although the proportion of such additives is not critical, it is generally included in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present specification.
In the above food composition, the determination of the amount of the above active ingredient to be administered is within the level of those skilled in the art, and for example, the amount to be administered for 1 day may be 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 be changed depending on various factors such as age, health condition, complications, and the like of the subject to be administered.
The food composition according to one aspect of the present invention may be, for example, various foods such as chewing gum, caramel product, candy, ice fruit, puffed food, and the like; beverage products such as refreshing beverages, mineral water, alcoholic beverages, etc.; health functional food containing vitamins or minerals.
The compositions of the invention may be quasi-drugs
Figure BDA0003050095460000251
A composition is provided.
The "quasi-drug" is a drug excluding an article used as a drug according to a pharmaceutical act, and means a drug according to a classification standard individually defined by a health care and welfare department for an article having a weaker action on the human body than a drug used for the treatment or prevention of a disease. Thus, fibers, rubber articles for use in the treatment or prevention of diseases in humans or animals; slight or no direct effect on the human body, instruments or non-instruments and the like; or antibacterial and pesticide for blocking infectious diseases. The quasi-drug composition can be used for improving, protecting or treating nerve cells from being damaged by oxidative stress, and can be used for preventing or improving nerve injury diseases induced by glutamic acid neurotoxicity.
The quasi-pharmaceutical composition may further comprise an excipient or carrier that is quasi-pharmaceutically acceptable.
The quasi-drug composition can be prepared into the forms of skin smearing preparation, cream, ointment, eye drops and spray.
The present invention will be described in more detail below with reference to examples and test examples. However, these examples and test examples are intended to illustrate the present invention, and the scope of the present invention is not limited to these examples and test examples.
EXAMPLE 1 preparation of extract of sprout of Cassia seed
Non-sterilized domestic semen Cassiae (Cassia obtusifolia L.) ((L.) HANDSherb, 2016 (1 month) and 2016) was purchased and washed with 20mg/mL bleaching powder (CaOCl)2) After 15 minutes of sterilization, the seeds were thoroughly washed with water, soaked in purified water for 4 hours, and then cultivated under shade conditions for 6 days. The cassia buds obtained by the above cultivation were dried with hot air (38 ℃) at 32 ℃ for 22 days, and ground to obtain 698.4g of dried plant. Then, after the above dried plants were put in an extraction vessel, 5.5L of ethanol was added, and mixed and stirred at normal temperature for extraction for 7 days, the above mixture was gravity-filtered using a Wohtman (whatman) filter paper having a film thickness of 0.34mm and a glass funnel having a diameter of 30cm, and this process was repeated 2 times ("extraction-filtration" 2 times) to obtain a filtered extract. The extract was put into a vacuum concentrator, and the solvent was completely evaporated and concentrated at 35 ℃ under reduced pressure, thereby obtaining 66.7g (hereinafter referred to as "STS") of a cassia bud extract (yield 9.55%).
Comparative example 1 production of Cassia seed extract
Non-sterilized domestic Cassia seed (Cassia obtusifolia L.) ((L.) handsherb, 2016 (1 month)), pulverized, put into a 15mL extraction vessel, added with 11mL of ethanol, mixed and stirred at normal temperature, extracted for 7 days, and the mixture was subjected to a gravity filtration process ("extract-filtration" 2 times) using a Woltmann filter paper having a film thickness of 0.34mm and a glass funnel having a diameter of 10cm to obtain a filtered extract (hereinafter referred to as "ST"). The extract was concentrated by completely evaporating the solvent at 35 ℃ under reduced pressure in a vacuum concentrator. (yield 3.34%).
EXAMPLE 2 production of extract of sprout of Cassia obtusifolia cultivated under various light conditions
In order to compare and analyze the components of the cassia seed sprout extract (STS) cultivated under the light-shielding condition of the above example 1 and the components of the cassia seed sprout extract cultivated under the specific light condition, the cassia seed sprout was cultivated under different light wavelength conditions. Cassia buds were cultivated by the same method as in example 1, and were cultivated for 6 days under light conditions in which fluorescent lamps were irradiated and light of LEDs 385nm, 465nm, 645nm and 780nm were irradiated as selective light qualities with a large number of wavelengths, respectively, without light-shielding conditions. Using the 5 types of sprouts obtained by the above cultivation, extracts of each cassia sprout were produced in the same manner as in example 1, and the extract obtained by cultivation under fluorescent lamp conditions was referred to as "STS-C", the extract obtained by cultivation under LED 385nm light conditions was referred to as "STS-385", the extract obtained by cultivation under LED 465nm light conditions was referred to as "STS-465", the extract obtained by cultivation under LED 645nm light conditions was referred to as "STS-645", and the extract obtained by cultivation under LED 780nm light conditions was referred to as "STS-780".
EXAMPLE 3 production of fraction of Cassia seed sprout extract (STS)
66.7g of the cassia seed sprout extract (STS) of example 1 was suspended in 600mL of water, and 1.8L of ethyl acetate was added thereto, followed by stirring with shaking at room temperature and fractionation for 4 times per 2 hours to obtain 23.5g of an ethyl acetate fraction (STS-EA). The fraction of STS-EA, i.e., the fraction of the cassia bud extract (STS), was prepared by using dichloromethane (or ethyl acetate) alone or a mixed solvent of 2.4L portions of n-hexane, dichloromethane (or ethyl acetate), and methanol (or ethanol) (the mixed ratio (volume ratio) of n-hexane to dichloromethane, and the mixed ratio (volume ratio) of dichloromethane to methanol were 200:1, 50:1, 10:1, 5:1, 3:1, and 1:1) as the fractionation solvent.
Specifically, the ethyl acetate fraction (STS-EA) was brought into contact with the 8-fraction solvent, stirred in celite (celite), the solvent was evaporated, and the mixture was developed with a silica (silica) resin packed in a column having a diameter of 10cm and a length of 20 cm. Chromatography using the above-mentioned solvents was performed to obtain 20 fractions (STS-EA-fraction 1 to STS-EA-fraction 20).
EXAMPLE 4 isolation of Compound 1 from extract of Cassia Torae semen sprout
After concentrating the STS-EA fraction 12 produced in example 3 (the 2 nd fraction of 3 fractions in example 3 in which the elution solvent was dichloromethane (or ethyl acetate): methanol (or ethanol) at a mixing ratio of 10: 1) under reduced pressure, chromatography for separation was performed under the following separation method 1, and compound 1 having the structure of the above chemical formula 1(14.0mg, 0.002% of the weight of the dried plant; 0.021% of the weight of the dried extract) was separated with a retention time of about 221 minutes.
Separation method 1
The equipment used was: YMC LC-Forte R
A chromatographic column: waters Delta-Pak C-18RP HPLC column (30.0X 300mm, 15 μm)
Solvent: (a) acetonitrile containing 0.02% TFA
(b) Water containing 0.02% TFA
Mobile phase composition: starting elution with (a) solvent (b) solvent at a volume ratio of 40:60 in 0 min
Increasing the proportion of the solvent (a) from 40% to 100% from 0 to 60 minutes
Flow rate of mobile phase: 10.0mL/min
A detector: ultraviolet ray 254 nm; ELSD
EXAMPLE 5 isolation of Compounds 2 to 5 from Cassia Torae semen sprout extract
(1)Isolation of Compound 3
After concentrating the STS-EA fraction 12 produced in example 3 (the 2 nd fraction of 3 fractions having a mixing ratio of dichloromethane (or ethyl acetate): methanol (or ethanol) of 10:1 as an eluting solvent in example 3) under reduced pressure, chromatography for separation was performed under the conditions of the separation method 1 in example 4, and 1.2mg of compound 3 having the structure of chemical formula 3 was separated with a retention time of about 24 minutes.
(2)Isolation of Compound 2
After concentrating the STS-EA fraction 8 of example 3 (the 1 st fraction of 3 fractions having a mixing ratio of dichloromethane (or ethyl acetate): methanol (or ethanol) of 50:1 as the elution solvent in example 3) under reduced pressure, chromatography for separation was performed under the following separation method 2, and 7.5mg of compound 2 having the structure of chemical formula 2 was separated under a retention time of about 36 minutes.
Separation method 2
The equipment used was: YMC LC-Forte R
A chromatographic column: waters Delta-Pak C-18RP HPLC column (30.0X 300mm, 15 μm)
Solvent: (a) acetonitrile containing 0.02% TFA
(b) Water containing 0.02% TFA
Mobile phase composition: starting elution with (a) solvent (b) solvent at a volume ratio of 60:40 in 0 min
Increasing the proportion of the solvent (a) from 60% to 100% from 0 to 60 minutes
Flow rate of mobile phase: 10.0mL/min
A detector: ultraviolet ray 254 nm; ELSD
(3)Isolation of Compounds 4 and 5
After concentrating STS-EA fraction 13 of example 3(3 rd fraction of 3 fractions having a mixing ratio of dichloromethane (or ethyl acetate): methanol (or ethanol) of 10:1 as an eluting solvent in example 3) under reduced pressure, chromatography for separation was performed under the conditions of separation method 3 described below, and about 71 minutes was kept for a retention time, 0.6mg of compound 4 having the structure of chemical formula 4 was separated, and about 120 minutes was kept for a retention time, 2.6mg of compound 5 having the structure of chemical formula 5 was separated.
Separation method 3
The equipment used was: JASCO LC-2000PLUS
A chromatographic column: phenomenex Luna C-18RP HPLC column (21.2X 250mm, 10 μm)
Solvent: (a) acetonitrile containing 0.02% TFA
(b) Water containing 0.02% TFA
Mobile phase composition: starting elution with (a) solvent (b) solvent at a volume ratio of 10:90 in 0 min
Increasing the proportion of the solvent (a) from 10% to 50% from 0 to 60 minutes
Increasing the proportion of the solvent (a) from 50% to 51% from 60 minutes to 80 minutes
Increasing the proportion of the solvent (a) from 51% to 58% from 80 to 140 minutes
From 140 minutes to 180 minutes, the proportion of the solvent (a) is increased from 58% to 100%
Flow rate of mobile phase: 8mL/min
A detector: ultraviolet ray 254nm
Comparative example 2 isolation of Compounds A to X from Cassia seed sprout extract
(1)Isolation of Compound F, G and H
The STS-EA fraction 12 of example 3 was concentrated under reduced pressure, and then subjected to chromatography for separation under the conditions of the separation method 1 of example 4, and the fraction was subjected to component separation. As a result, 0.8mg of compound F corresponding to peak F in fig. 4, 0.9mg of compound G corresponding to peak G in fig. 4, and 0.5mg of compound H corresponding to peak H in fig. 4 were isolated.
(2)Isolation of Compounds C, D and E
The STS-EA fraction 8 of example 3 was concentrated under reduced pressure, and then subjected to chromatography for separation under the conditions of the separation method 2 of example 4, thereby separating components contained in the fraction. As a result, 7.2mg of compound C corresponding to peak C of fig. 4, 0.8mg of compound D corresponding to peak D of fig. 4, and 2.5mg of compound E corresponding to peak E of fig. 4 were isolated.
(3)Isolation of Compound I
The STS-EA fraction 15 obtained in example 3 (the 2 nd fraction of 3 fractions in example 3 in which the elution solvent was dichloromethane (or ethyl acetate): methanol (or ethanol) at a mixing ratio of 5: 1) was concentrated under reduced pressure, and then the components contained in the fraction were separated by HPLC separation under the conditions of the following separation method 4. As a result, 0.7mg of Compound I corresponding to peak I in FIG. 4 was isolated.
Separation method 4
The equipment used was: JASCO LC-2000PLUS
A chromatographic column: phenomenex Luna C-18RP HPLC column (21.2X 250mm, 10 μm)
Solvent: (a) acetonitrile containing 0.02% TFA
(b) Water containing 0.02% TFA
Mobile phase composition: starting elution with (a) solvent (b) solvent at a volume ratio of 30:70 in 0 min
The solvent (a) is maintained at 30% from 0 to 240 minutes
Flow rate of mobile phase: 8mL/min
A detector: ultraviolet ray 254nm
(4)Isolation of Compounds J, K, L, M, N, O, A and P
The STS-EA fraction 16 obtained in example 3 (the 3 rd fraction of 3 fractions in example 3 in which the elution solvent was dichloromethane (or ethyl acetate): methanol (or ethanol) at a mixing ratio of 5: 1) was concentrated under reduced pressure, and then subjected to silica gel column chromatography under the following separation method 5 to be fractionated into 14 small fractions.
Separation method 5
The equipment used was: quick silica gel column
A chromatographic column: glass column (25.0X 200mm)
Solvent: (a) methylene chloride (methylene chloride)
(b) Methanol mobile phase composition: starting elution with (a) solvent (b) solvent at a volume ratio of 20:1
(a) Solvent (b) the volume ratio of the solvent is 10:1, 7:1, 4:1, 2:1, 1:1, and the solvent amount is divided into steps: 300mL
From the above-mentioned small fraction 3 (the 2 nd fraction of the 3 small fractions having a mixture ratio of dichloromethane to methanol of 10:1 as an eluting solvent in the above-mentioned separation method 5), 1.8mg of the compound J corresponding to the peak J of fig. 4, 1.9mg of the compound K corresponding to the peak K of fig. 4, and 1.0mg of the compound L corresponding to the peak L of fig. 4 were separated by HPLC separation method under the conditions of the following separation method 6.
Separation method 6
The equipment used was: gilson 321HPLC
A chromatographic column: phenomenex Luna C-18RP HPLC column (21.2X 250mm, 10 μm)
Solvent: (a) acetonitrile containing 0.02% TFA
(b) Water containing 0.02% TFA
Mobile phase composition: starting elution with (a) solvent (b) solvent at a volume ratio of 20:80 in 0 min
Increasing the proportion of the solvent (a) from 20% to 40% from 0 to 80 minutes
From 80 minutes to 120 minutes, the proportion of the solvent (a) is increased from 40% to 100%
Flow rate of mobile phase: 8mL/min
A detector: ultraviolet ray 254nm
From the above-mentioned small fraction 4 (the 3 rd fraction of 3 small fractions having a mixture ratio of dichloromethane to methanol of 10:1 as an eluting solvent in the above-mentioned separation method 5), 2.5mg of the compound M corresponding to the peak M in fig. 4, 2.1mg of the compound N corresponding to the peak N in fig. 4, and 0.5mg of the compound O corresponding to the peak O in fig. 4 were separated by HPLC separation method under the conditions of the following separation method 7.
Separation method 7
The equipment used was: gilson 321HPLC
A chromatographic column: phenomenex Luna C-18RP HPLC column (21.2X 250mm, 10 μm)
Solvent: (a) acetonitrile containing 0.02% TFA
(b) Water containing 0.02% TFA
Mobile phase composition: starting elution with (a) solvent (b) solvent at a volume ratio of 20:80 in 0 min
Increasing the proportion of the solvent (a) from 20% to 80% from 0 min to 100 min
From 100 minutes to 160 minutes, the proportion of the solvent (a) is increased from 80% to 100%
Flow rate of mobile phase: 8mL/min
A detector: ultraviolet ray 254nm
From the above-mentioned small fraction 6 (the 2 nd fraction of 3 small fractions having a mixture ratio of dichloromethane to methanol of 7:1 as an eluting solvent in the above-mentioned separation method 5), 2.0mg of compound a corresponding to peak a in fig. 4 was separated by HPLC separation method under the conditions of the following separation method 8.
From the above-mentioned small fraction 7 (the 3 rd fraction of 3 small fractions having a mixture ratio of dichloromethane to methanol of 7:1 as an eluting solvent in the above-mentioned separation method 5), 4.1mg of the compound P corresponding to the peak P in fig. 4 was separated by the HPLC separation method under the conditions of the following separation method 8.
Separation method 8
The equipment used was: gilson 321HPLC
A chromatographic column: phenomenex Luna C-18RP HPLC column (21.2X 250mm, 10 μm)
Solvent: (a) acetonitrile containing 0.02% TFA
(b) Water containing 0.02% TFA
Mobile phase composition: starting elution with (a) solvent (b) solvent at a volume ratio of 17:83 in 0 min
The solvent (a) is maintained at a ratio of 17% from 0 to 50 minutes
Increasing the proportion of the solvent (a) from 17% to 25% from 50 minutes to 70 minutes
Increasing the proportion of the solvent (a) from 25% to 30% from 70 minutes to 150 minutes
Increasing the proportion of the solvent (a) from 30% to 100% from 150 to 180 minutes
Flow rate of mobile phase: 6.5mL/min
A detector: ultraviolet ray 254nm
(5)Isolation of Compound Q
STS-fraction 17 obtained in example 3 (the 1 st fraction of 3 fractions having a mixing ratio of dichloromethane (or ethyl acetate): methanol (or ethanol) of 3:1 as the eluting solvent in example 3) was concentrated under reduced pressure, and then the components contained in the fraction were separated by HPLC separation under the conditions of separation method 9 described below. As a result, 1.7mg of compound Q corresponding to peak Q of FIG. 4 was isolated.
Separation method 9
The equipment used was: JASCO LC-2000PLUS
A chromatographic column: phenomenex Luna C-18RP HPLC column (21.2X 250mm, 10 μm)
Solvent: (a) acetonitrile containing 0.02% TFA
(b) Water containing 0.02% TFA
Mobile phase composition: starting elution with (a) solvent (b) solvent at a volume ratio of 10:90 in 0 min
The solvent (a) is maintained at a ratio of 10% from 0 to 40 minutes
Increasing the proportion of the solvent (a) from 10% to 18% from 40 minutes to 70 minutes
Increasing the proportion of the solvent (a) from 18% to 22% from 70 minutes to 180 minutes
Increasing the proportion of the solvent (a) from 22% to 100% from 180 to 260 minutes
Flow rate of mobile phase: 6.5mL/min
A detector: ultraviolet ray 254nm
(6)Isolation of Compounds B, R, S, T, U, V, W and X
The STS-EA fraction 18 obtained in example 3 (the 2 nd fraction of 3 fractions in example 3 in which the elution solvent was dichloromethane (or ethyl acetate): methanol (or ethanol) at a mixing ratio of 3: 1) was concentrated under reduced pressure and subjected to silica gel column chromatography under the conditions of the following separation method 10 to be fractionated into 24 small fractions.
Separation method 10
The equipment used was: quick silica gel column
A chromatographic column: glass column (25.0X 200mm)
Solvent: (a) methylene chloride (methylene chloride)
(b) Methanol
Mobile phase composition: (a) solvent (b) starting elution with a solvent volume ratio of 20:1
(a) Solvent (b) the volume ratio of the solvent is changed according to 10:1, 7:1, 5:1, 3:1, 2:1 and 1:1
Step-by-step solvent amount: 400mL
10:1 ( fraction 3,4, 5, 6), 7:1 (fraction 7, 8, 9, 10), 5:1 (fraction 11, 12, 13, 14), 3:1 ( fraction 15, 16, 17, 18), 2:1 (fraction 19, 20, 21, 22)
The fractions 17 to 21 (the 3 rd and 4 th fractions of the 4 small fractions having a mixture ratio of dichloromethane to methanol of 3:1 as the elution solvent in the separation method 10, and the 2 nd to 4 th fractions of the 4 small fractions having a mixture ratio of dichloromethane to methanol of 2:1 as the elution solvent) were separated by HPLC under the conditions of the separation method 11 described below to separate the components contained in the fractions. As a result, 1.6mg of compound B corresponding to peak B of fig. 4, 24.8mg of compound R corresponding to peak R of fig. 4, 5.5mg of compound S corresponding to peak S of fig. 4, 2.3mg of compound T corresponding to peak T of fig. 4, 9.3mg of compound U corresponding to peak U of fig. 4, 6.4mg of compound V corresponding to peak V of fig. 4, 4.1mg of compound W corresponding to peak W of fig. 4, and 0.6mg of compound X corresponding to peak X of fig. 4 were separated.
Separation method 11
The equipment used was: JASCO LC-2000PLUS
A chromatographic column: phenomenex Luna C-18RP HPLC column (21.2X 250mm, 10 μm)
Solvent: (a) acetonitrile containing 0.02% TFA
(b) Water containing 0.02% TFA
Mobile phase composition: starting elution with (a) solvent (b) solvent at a volume ratio of 10:90 in 0 min
The solvent (a) is maintained at a ratio of 10% from 0 to 40 minutes
Increasing the proportion of the solvent (a) from 10% to 18% from 40 minutes to 70 minutes
Increasing the proportion of the solvent (a) from 18% to 22% from 70 minutes to 180 minutes
Increasing the proportion of the solvent (a) from 22% to 100% from 180 to 260 minutes
Flow rate of mobile phase: 6.5mL/min
HPLC peaks corresponding to all the substances (compounds 1 to 5, and compounds C to X) separated from the above examples 4 and 5 and comparative example 2 are shown as marks of each peak of the cassia seed sprout extract HPLC chromatogram of fig. 4. A total of 29 substances including naphthopyrone derivatives (compounds 1 to 5) having the structures of the above chemical formulas 1 to 5 are components isolated from the cassia bud extract in the amount obtainable from the above example 1. Furthermore, the 29 substances correspond to the main constituents of the cassia sprout extract according to HPLC analysis chromatography.
Example 6 structural identification of Compounds 1 to 5 isolated from Cassia tora sprout extract
In order to identify the structures of the compounds 1 to 5 isolated in the above examples 4 and 5, each compound was analyzed using a Mass Spectrometer (MS) and a Nuclear Magnetic Resonance (NMR).
(1)Identification of Compound 1
The molecular weight of Compound 1 isolated in example 4 was determined to be 664 by MS measurement using an Agilent 1100 high performance liquid chromatography-Mass spectrometer (HPLC-ESI-MS), and specific optical rotation (PerkinElmer Lambda 35) spectroscopic data by ultraviolet light (PerkinElmer 343 polarometer), infrared light (Thermo Scientific Nicolet iS50), and nuclear magnetic resonance apparatus (Bruker 400MHz,100MHz NMR)1H and13c NMR spectrum (spectrum) analysis, and the structure was identified as 7-hydroxyruminomycin-rubrofusarin-8' -O-glucopyranoside, which is a naphthopyrone derivative having the structure of the following chemical formula 1. The compound 1 is a novel compound which has not been reported so far, and is isolated as one of the main components only in the sprout of cassia seed, which is not found because it is not contained or contained in a very small amount in cassia seed or a fully grown plant of cassia seed.
[ chemical formula 1]
Figure BDA0003050095460000371
Molecular formula C34H32O14；ESI-MS：m/z 665[M+H]+(ii) a Infrared absorption band vmax3384、2936、1631、1373、1204、1059cm-1(ii) a Ultraviolet absorption band (MeOH) lambdamax(log ε)240(4.3), 282 (4.2); specific optical rotation [ alpha ]]22 D-20(c 0.05，MeOH)；
1H NMR (methanol-d)4400 MHz): δ 7.18(1H, s, H-10), 6.97(1H, d, J ═ 2.0Hz, 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, 3.5Hz, H-1 "), 3.98(1H, dd, J ═ 12.0, 2.0Hz, H-6" a), 3.78(1H, dd, J ═ 12.0, 5.5Hz, H-6 "b), 3.75(3H, s, OMe-8), 3.58(1H, t, J ═ 8.0Hz, H-2'), 3.55(1H, dd, J ═ 5.0, J ═ 2H-5H, OMe-8), 3.51H-5H ═ 5H, t ═ 3.51, 3.51H ═ 5, 3.51 ″, 3.5H, H-5 ═ 3.5Hz, 3.51, 3.5H ″, 3.51, 3.5H, J ═ 5 ═ 3.5 ″, 3.5, 2.41(3H, s, Me-11), 1.98(3H, s, Me-11'),13C NMR(CDCl3，500MHz)：δ208.1(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 by MS measurement using an Agilent 1100 high Performance liquid chromatography-Mass spectrometer (HPLC-ESI-MS) and 272 by nuclear magnetic resonance apparatus (Bruker 400MHz NMR)1H NMR spectrum (spectrum) analysis concluded to be an isoctoralactone (isotoralaclone) which is a naphthopyrone derivative having the structure shown in the following chemical formula 2. Furthermore, the structure was identified by comparing NMR data with data from the prior art (Kitanaka, S.et al. phytochemistry,1981,20, 1951-1953).
[ chemical formula 2]
Figure BDA0003050095460000381
Molecular formula C15H12O5；ESI-MS：m/z 273[M+H]+；
1H NMR(CDCl3，400MHz)：δ13.30(1H，s，OH-10)，9.41(1H，s，OH-9)，6.92(1H，s，H-5)，6.59(1H，d，J＝2.5Hz，H-6)，6.56(1H，d，J＝2.0Hz，H-8)，4.93(1H，d，J＝2.0Hz，H-11)，4.62(1H，d，J＝2.0Hz，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 by MS measurement using an Agilent 1100 high Performance liquid chromatography-Mass spectrometer (HPLC-ESI-MS) and 272 by nuclear magnetic resonance apparatus (Bruker 400MHz NMR)1H NMR spectroscopy analysis indicated that the structure was a toralactone (toralactone) which is a naphthopyrone derivative having a structure shown in the following chemical formula 3. In addition, the structure was identified by comparing the NMR data with that of the existing literature (Newman, A.G.et al.J.Am Chem Soc 2016,138, 4219-4228).
[ chemical formula 3]
Figure BDA0003050095460000391
Molecular formula C15H12O5；ESI-MS：m/z 273[M+H]+；
1H NMR(CDCl3，400MHz)：δ13.56(1H，s，OH-5)，9.44(1H，s，OH-9)，7.00(1H，s，H-5)，6.64(1H，d，J＝2.5Hz，H-6)，6.55(1H，d，J＝2.5Hz，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 above was determined to be 288 by MS measurement using an Agilent 1100 high Performance liquid chromatography-Mass Spectroscopy (HPLC-ESI-MS), and the molecular weight was determined to be 288 by MS measurement using an HPLC-ESI-MSBy nuclear magnetic resonance apparatus (Bruker 400MHz NMR)1H NMR spectroscopy analysis, and the structure is inferred to be cassia anthrone (torachrysone) which is a naphthopyrone derivative having the structure shown in the following chemical formula 4. In addition, the structure was identified by comparing NMR data with data from the prior art literature (Gill, M.et al. Aust Jchem 2000,53, 213-220).
[ chemical formula 4]
Figure BDA0003050095460000401
Molecular formula C16H16O5；ESI-MS：m/z 289[M+H]+；
1H NMR (methanol-d)4，400MHz)：δ9.80(1H，s，OH-9)，6.90(1H，s，H-5)，6.57(1H，d，J＝2.0Hz，H-6)，6.51(1H，d，J＝2.0Hz，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 288 as determined by MS measurement using an Agilent 1100 high Performance liquid chromatography-Mass spectrometer (HPLC-ESI-MS), and 288 as determined by NMR measurement using a Bruker 400MHz NMR spectrometer (Bruker NMR)1H NMR spectrum analysis, and its structure is inferred to be rubrofusarin (rubrofusarin) as a naphthopyrone derivative as shown in the following chemical formula 5. Furthermore, the structure was identified by comparing the NMR data with the data of the prior art (Alemayehu, g.et al. phytochemistry,1993,32, 1273-.
[ chemical formula 5]
Figure BDA0003050095460000411
Molecular formula C15H12O5；ESI-MS：m/z 273[M+H]+；
1H NMR(CDCl3，400MHz)：δ9.68(1H，s，OH-9)，7.00(1H，s，H-10)，6.59(1H，d，J＝2.5Hz，H-9)，6.48(1H，d，J＝2.0Hz，H-7)，6.04(1H，s，H-3)，3.91(3H，s，OMe-7)，2.41(3H，s，Me-11)
[ test example 1] evaluation of antioxidant Effect of Cassia seed sprout extract
(1)DPPH radical scavenging Effect of Cassia Torae semen extract and Cassia Torae semen sprout extract
After the cassia seed extract (ST) produced in the above comparative example 1 and the cassia seed sprout extract (STs) produced in the above example 1 were dissolved in methanol at concentrations of 1000ppm, 500ppm, 250ppm and 125ppm, 100 μ L of the cassia seed extract solution of the above comparative example 1 and 100 μ L of the cassia seed sprout extract of the above example 1 were reacted with 100 μ L of DPPH in the dark for 40 minutes, and the absorbance was measured at 517nm, and ascorbic acid (L-ascorbyl acid) was used as a positive control. The DPPH radical scavenging effect measured by the above absorbance and obtained by the following formula is shown in FIG. 1.
Figure BDA0003050095460000412
As shown in fig. 1, the DPPH radical scavenging effect of the cassia seed sprout extract (STS) of example 1 was found to be 1.7 times or more higher than that of the cassia seed extract (ST) of comparative example 1 at each concentration.
(2)Comparison of ABTS on-line antioxidant chromatography measurement results of semen Cassiae extract and semen Cassiae sprout extract
The cassia seed extract (ST) produced in the comparative example 1 and the cassia seed sprout extract (STs) produced in the example 1 were dissolved in an alcohol aqueous solution at the same concentration, and ABTS online antioxidant High Performance Liquid Chromatography (HPLC) was performed according to the following analysis conditions.
ABTS on-line antioxidation HPLC analysis condition
The equipment used was: agilent 1200 system
A chromatographic column: RP C-18HPLC column (4.6X 150mm, 5 μm)
Solvent: (a) acetonitrile containing 0.02% TFA (Trifluoroacetic acid)
(b) Water containing 0.02% TFA
Mobile phase composition: starting elution with (a) solvent (b) solvent at a volume ratio of 10:90 in 0 min
Increasing the proportion of the solvent (a) from 10% to 100% from 0 to 30 minutes
From 30 minutes to 37 minutes, 100% elution with (a) solvent
Flow rate of mobile phase: 0.7mL/min
ABTS composition: water containing 0.08mM ABTS and 0.12mM potassium persulfate (potassium persulfate)
ABTS flow rate: 0.35mL/min
The detector detects the wavelength: ultraviolet ray 254 nm; 734nm
As a result, the cassia seed extract (ST) and the cassia seed sprout extract (STs) show very different antioxidant ingredient patterns. While the cassia seed extract (ST) and the cassia seed sprout extract (STs) each had a content of flavonoids and naphthalene derivatives having antioxidant activity observed before the retention time of 15 minutes, naphthalene and anthraquinone derivatives having antioxidant activity were further observed after 15 minutes while the content of the above components was increased in the cassia seed sprout extract (STs) as compared with the cassia seed extract (ST). Fig. 2 (a) and (B) show ABTS on-line antioxidant HPLC results of cassia seed and cassia sprout extracts, and are chromatograms obtained by detecting wavelengths of 254nm (chromatogram of blue line labeled upward) and 734nm (chromatogram of red line labeled downward) with an Ultraviolet (UV) detector, respectively.
[ test example 2] comparison of ABTS on-line antioxidant chromatography measurement results of Cassia Torae semen extract (ST) and Cassia Torae semen sprout extract cultivated under various light conditions
The cassia seed sprout extract (STS) produced in the above example 1 and 7 types of the cassia seed sprout extracts (STS-C, STS-385, STS-465, STS-645, and STS-780) produced in the above example 2 and cultivated under various light conditions were dissolved in an alcohol aqueous solution, and ABTS on-line antioxidant High Performance Liquid Chromatography (HPLC) was performed according to the same analysis conditions as in the above test example 1.
As a result, the 5 types of cassia buds extracts of example 2, which were cultivated under various light conditions, exhibited a change in the content of the components, although the components having antioxidant activity were observed together. A, B, C, D, E, F in FIG. 3 is a chromatogram obtained by subjecting extracts of the 6 types of cassia buds, which are STS, STS-C, STS-385, STS-465, STS-645, and STS-780, respectively, to ABTS on-line antioxidant HPLC, and detecting a wavelength of 254nm (which indicates a peak of a UV component absorbing 254 nm) and a wavelength of 734nm (which indicates an antioxidant degree by a peak of a component exhibiting an antioxidant effect on ABTS and an area of the peak) with an Ultraviolet (UV) detector.
Based on the chromatogram of fig. 3, the antioxidant components of the cassia bud extract cultivated under the light-shielding condition and the fluorescent lamp and the light of each specific wavelength were observed in a general manner, and the content and composition of the antioxidant components were different depending on the wavelength of light irradiated during the cultivation, but no components disappeared or newly appeared under the specific conditions were found. In all chromatograms of fig. 3 (a) to (F), the novel naphthopyrone component having the structure of the above chemical formula 1 of the present invention (compound 1 isolated in example 4) was detected as the most significant antioxidant peak or main antioxidant peak, and 4 naphthopyrone components having the structures of the above chemical formulas 2 to 5 (compounds 2 to 5 isolated in example 5) were also detected as antioxidant components in chromatograms of fig. 3 (a) to (F). In addition to the chromatogram (B) of the cassia seed sprout extract (STS-C) cultivated under ordinary light conditions and the chromatogram (D) of the cassia seed sprout extract (STS-465) cultivated under LED illumination of 465nm, the effective components of the compounds 1 to 5 isolated in the above examples 4 and 5 were detected at relatively high contents in the chromatograms of (A), (C), (E) and (F).
Experimental example 3 protective effect of retinal progenitor cells (R28) of 7 kinds of cassia seed bud extracts of examples 1 and 2
In order to confirm the protective effects of the semen cassiae extract (ST) and the semen cassiae sprout extract (STS) obtained in the above comparative example 1 and example 1, respectively, and the retinal progenitor cells (R28) of the semen cassiae sprout extract (STS-C, STS-385, STS-465, STS-645, STS-780) obtained in the above example 2 cultivated under various light conditions, the following experiments were performed.
Specifically, R28 cells were cultured in DMEM (Dulbecco's modified eagle's medium) containing 10% FBS (total bone serum), 100U/mL penicillin (penicillin), and 100. mu.g/mL streptomycin (streptomycin) in a 37 ℃ incubator at 5% CO in a 5% CO low sugar (lowglucose) medium2The culture was performed under the condition of subculture with 0.05% Trypsin (Trypsin) every 2 days, and then in 96-well plates at 1X 104The culture was inoculated at the density of (1) and cultured for 24 hours. The above 7 extracts were treated at the concentrations of (a) to (C) of fig. 5 into the above cultured R28 cells, and after 2 hours, 10mM of glutamic acid (glutamate) and 0.5mM of BSO (buthionine sulphoxide) were added and cultured for 22 hours. Then, in order to determine the cell survival rate, 10. mu. LEZ-cytox was treated and maintained for 2 hours, and the UV absorbance was measured at 450 nm.
In FIG. 5, (A) is a result of showing the effect of protecting R28 cells from oxidative stress induced by glutamic acid by cell survival rate, based on the cell survival rate, of 5 types of cassia seed bud extracts (STS-C, STS-385, STS-465, STS-645, STS-780) cultivated under 3 types of concentration conditions, with the cassia seed extract (ST) of comparative example 1, the cassia seed bud extract (STS) of example 1, and the light conditions of example 2 being changed. In 5, (B) and (C) are results of showing the effect of the cassia seed extract (ST) of the above comparative example 1 and the cassia seed sprout extract (STs) of the above example 1 in protecting R28 cells from oxidative stress induced by glutamic acid at 5 concentrations of each by cell survival rate.
As shown in fig. 5 (a), all the extract-treated groups showed the effect of protecting HT-22 cells from oxidative stress induced by glutamic acid, but showed more excellent effect of protecting R28 cells from oxidative stress induced by glutamic acid at most concentration than the cassia seed extract (STS, STS-C, STS-385, STS-465, STS-645, and STS-780), and in particular, as shown in fig. 5 (B) and (C), it was confirmed that the cassia seed sprout extract (STS) of example 1 was significantly excellent in R28 cell-protecting effect as compared with the cassia seed extract (ST) of comparative example 1.
Experimental example 4 confirmation of protective effect of retinal progenitor cells (R28) of compounds 1 to 5 isolated from the cassia seed sprout extracts of examples 5 and 6
In order to confirm the reason why the effect of protecting retinal progenitor cells (R28) is more excellent than that of the cassia seed extract (ST) in the cassia seed sprout extract (STS) of test example 3, the protective effects of the compounds 1 to 5 of examples 5 and 6 on R28 cells were compared.
The survival rate of R28 cells was measured by the same method as in test example 3, and compounds 1 to 5 of examples 5 and 6 were treated with 50. mu.M, 16.6. mu.M and 5.55. mu.M in place of the cassia seed extract (ST) and the cassia seed sprout extract (STS, STS-C, STS-385, STS-465, STS-645 and STS-780) of comparative example 1, examples 1 and 2, and the results are shown in FIG. 6.
As shown in fig. 6, the effect of compounds 1 to 5, which are relatively high in the content of cassia sprout extract (STS) or are mainly present in cassia sprout extract (STS), on protecting R28 cells from glutamate-induced neurotoxicity is shown, and it can be confirmed that the effect of cassia sprout extract (STS) on protecting R28 cells is more excellent than that of cassia extract (ST).
[ test example 5] Hippocampus nerve cell (HT-22) protective effects of 7 kinds of cassia seed sprout extracts of examples 1 and 2
In order to confirm the protective effects of the cassia seed extract (ST) and the cassia seed sprout extract (STS) obtained in the above comparative example 1 and example 1, and the cassia seed sprout extract (STS-C, STS-385, STS-465, STS-645, STS-780) obtained in the above example 2 cultivated under various light conditions on the hippocampal nerve cells (HT-22), the following experiments were carried out.
Specifically, HT-22 cells were cultured in DMEM (Dulbecco's modified eagle's Medium)/Low-sugar Medium containing 10% FBS (fetal bovine serum), 100U/mL penicillin, and 100. mu.g/mL streptomycin at 37.5 ℃ in a 5% CO incubator2Culturing under the condition of 0.05% trypsin every 2 daysAfter subculture, 3X 10 cells were plated in 96-well plates3The cells were inoculated at the density of (1) and cultured for 24 hours. The above 7 extracts were treated at the concentrations of (A) to (C) in FIG. 7 to the above cultured HT-22 cells, and after 2 hours, 5mM glutamic acid was added thereto and the cells were cultured for 22 hours. Then, in order to determine the cell survival rate, 10. mu.L of EZ-cytox was treated for 2 hours, and the UV absorbance was measured at 450 nm.
In FIG. 7, (A) is a result of showing the effect of protecting HT-22 cells from oxidative stress induced by glutamic acid, which is obtained by changing the cassia seed extract (ST) of comparative example 1, the cassia seed sprout extract (STS) of example 1, and the 5 cassia seed sprout extracts (STS-C, STS-385, STS-465, STS-645, and STS-780) cultivated under the light conditions of example 2 at 3 concentrations, as a result of cell survival rate. In fig. 7, (B) and (C) are results of showing the effect of the cassia seed extract (ST) of the above comparative example 1 and the cassia seed sprout extract (STs) of the above example 1 at 5 concentrations to protect HT-22 cells from oxidative stress induced by glutamic acid by cell survival rate.
As shown in fig. 7 (a), the effect of protecting HT-22 cells from oxidative stress induced by glutamic acid was exhibited in all the extract-treated groups, and particularly, as shown in fig. 7 (B) and (C), it was confirmed that the cassia seed sprout extract (STS) of example 1 exhibited an excellent HT-22 cell-protecting effect as compared with the cassia seed extract (ST) of comparative example 1.
Test example 6 Hippocampus nerve cell (HT-22) protective effects of Compounds 1 to 5 isolated from the extract of sprout of Cassia tora L.in examples 5 and 6 were confirmed
In order to confirm the reason why the hippocampal nerve cell (HT-22) protective effect of the cassia seed sprout extract (STS) of the above test example 5 is more excellent than that of the cassia seed extract (ST), the protective effects of the compounds 1 to 5 of examples 5 and 6 and the compounds a to X of comparative example 2 on R28 cells were compared.
The survival rate of HT-22 cells was determined in the same manner as in test example 5, and compounds 1 to 5 of examples 5 and 6 were treated with 50. mu.M, 16.6. mu.M and 5.55. mu.M in place of the cassia seed extract (ST) and the cassia seed sprout extract (STS, STS-C, STS-385, STS-465, STS-645 and STS-780) of comparative example 1, examples 1 and 2, and the results are shown in FIG. 8.
As shown in fig. 8, the effects of compound K and compound T, which correspond to peak K and peak T of compounds 1 to 5 of examples 5 and 6 and comparative example 2, respectively, on protecting HT-22 cells from glutamate-induced neurotoxicity are shown. That is, the above-mentioned compounds 1 to 5 are compounds having a relatively very high content in the cassia seed sprout extract (STS) or mainly existing in the cassia seed sprout extract (STS), and thus it can be confirmed that the HT-22 cytoprotective effect of the cassia seed sprout extract (STS) is more excellent than the effect of the cassia seed extract (ST).
The physiological activities of the components evaluated in test example 1, test example 4, and test example 6 are shown in table 1 below. The following table 1 relates to physiological activities of components isolated from the cassia tora bud extract (STS) of example 1.
< Table 1> physiological Activity of ingredients isolated from Cassia tora sprout extract
Figure BDA0003050095460000471
O significant activity; x is inactive; w is weakly active.

Claims (17)

1. A naphthopyrone derivative of the following chemical formula 1, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof,
chemical formula 1
Figure FDA0003050095450000011
2. A process for producing a naphthopyrone derivative, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof, which comprises separating at least one member selected from the group consisting of a naphthopyrone derivative of the following chemical formulae 1 to 5, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, and a solvate thereof from the bud of Cassia obtusifolia (Cassia obtusifolia L.) or Cassia tora (Cassia tora L.),
chemical formula 1
Figure FDA0003050095450000012
Chemical formula 2
Figure FDA0003050095450000013
Chemical formula 3
Figure FDA0003050095450000021
Chemical formula 4
Figure FDA0003050095450000022
Chemical formula 5
Figure FDA0003050095450000023
3. The method according to claim 2, further comprising a step of extracting the sprout of the cassia seed with a solvent selected from the group consisting of water, an alcohol having 1 to 6 carbon atoms, and a mixed solvent thereof.
4. The production method according to claim 2, further comprising a step of fractionating the solvent with at least one selected from the group consisting of water, ethyl acetate, hexane, dichloromethane, chloroform, methanol, ethanol, acetone, and a mixed solvent thereof.
5. A composition for protecting nerve cells from oxidative stress or for inhibiting nerve cell death, comprising as an active ingredient one or more selected from naphthopyrone derivatives selected from the group consisting of 1 or more of naphthopyrone derivatives of the following chemical formulae 1 to 5, stereoisomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, or solvates thereof, a Cassia tora (Cassia obtusifolia L.) or Cassia tora L bud extract containing the same, or a Cassia tora bud fraction containing the same,
chemical formula 1
Figure FDA0003050095450000031
Chemical formula 2
Figure FDA0003050095450000032
Chemical formula 3
Figure FDA0003050095450000033
Chemical formula 4
Figure FDA0003050095450000034
Chemical formula 5
Figure FDA0003050095450000035
6. The composition of claim 5, wherein the oxidative stress is induced by glutamate.
7. The composition of claim 5, wherein the neural cell is a retinal neural cell or a hippocampal neural cell.
8. A composition for preventing or treating a nerve injury disease induced by damage or death of retinal nerve cells or hippocampal nerve cells, comprising, as an active ingredient, at least one member selected from the group consisting of a naphthopyrone derivative which is at least 1 member selected from the group consisting of naphthopyrone derivatives of the following chemical formulae 1 to 5, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof, and a Cassia tora (Cassia obtusifolia L.) or Cassia tora L) bud extract containing the same, or a Cassia tora bud fraction containing the same,
chemical formula 1
Figure FDA0003050095450000041
Chemical formula 2
Figure FDA0003050095450000042
Chemical formula 3
Figure FDA0003050095450000043
Chemical formula 4
Figure FDA0003050095450000044
Chemical formula 5
Figure FDA0003050095450000045
9. The composition according to claim 8, wherein the nerve injury disease is one or more selected from the group consisting of vision disorders due to memory impairment, learning deficit, depression, amyotrophic lateral sclerosis (lounge's disease), parkinson's disease, cerebral ischemic nerve injury, diabetic neuropathy, macular degeneration, retinal cell injury, retinitis pigmentosa, retinal detachment, retinal vascular occlusion, and glaucoma.
10. The composition of claim 8, wherein the damage or death of retinal or hippocampal neural cells is caused by glutamate neurotoxicity.
11. The composition of claim 8, wherein the naphthopyrone derivative is isolated or purified from the bud of Cassia tora (Cassia obtusifolia L.) or Cassia tora (Cassia tora L.)).
12. The composition of claim 11, wherein the naphthopyrone derivative is isolated or purified from an ethanol extract of cassia buds.
13. The composition as claimed in claim 8, wherein the naphthopyrone derivative is obtained by fractionating the ethanol extract of cassia buds with ethyl acetate, and then chromatographically separating the ethyl acetate fraction or fractionating the ethyl acetate fraction again with a mixed solvent of hexane, dichloromethane and methanol, and chromatographically separating it.
14. The composition as claimed in claim 8, wherein the extract is obtained by extracting with a solvent selected from the group consisting of water, alcohols having 1 to 6 carbon atoms, and mixed solvents thereof.
15. The composition according to claim 8, wherein the fraction is obtained by fractionating the fraction with at least one solvent selected from the group consisting of water, ethyl acetate, hexane, dichloromethane, chloroform, methanol, ethanol, acetone, and a mixture thereof.
16. The composition of claim 8, wherein the composition is a pharmaceutical or food composition.
17. An antioxidant composition comprising, as an active ingredient, at least one selected from the group consisting of a naphthopyrone derivative, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, and a solvate thereof, and at least one extract of the bud of Cassia obtusifolia (Cassia obtusifolia L.) or Cassia tora (Cassia tora L.) containing the same, wherein the naphthopyrone derivative is at least one selected from the group consisting of naphthopyrone derivatives represented by the following chemical formulae 1 to 5,
chemical formula 1
Figure FDA0003050095450000061
Chemical formula 2
Figure FDA0003050095450000062
Chemical formula 3
Figure FDA0003050095450000063
Chemical formula 4
Figure FDA0003050095450000064
Chemical formula 5
Figure FDA0003050095450000065
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