JP2008013448A - Cytoprotective agent of optic nerve - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a preventing/treating agent of eye diseases by using a specific lignin derivative. <P>SOLUTION: This cytoprotective agent contains 1 kind or ≥2 kinds selected from a group consisting of (a) a water-soluble lignin secondary derivative obtained by alkali-treating a primary derivative of the lignin obtained by adding and mixing an acid to a lignin-containing material solvated with a phenolic compound, (b) the secondary lignin derivative carboxyalkylated by a carboxyalkyl group equipped with a 1-5C lower alkyl group on the (a) primary lignin derivative and (c) a tertiary lignin derivative obtained by carboxyalkylating with the carboxyalkyl group equipped with the 1-5C lower alkyl group on the water-soluble and/or water-insoluble secondary lignin derivative obtained by alkali-treating the primary lignin derivative obtained by adding and mixing the acid to the lignin-containing material solvated with the phenol compound. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to the use of a lignin derivative, and more particularly to the use of an ophthalmic disease preventive / therapeutic agent and an optic nerve cell protecting agent.

  The retina plays an important role in visual function for receiving external light. The retina is composed of 10 layers including a retinal pigment epithelium layer, an optic nerve fiber layer (OFL), a nerve cell layer, an inner plexiform layer (IPL), an inner granule layer (INL), an outer reticular layer (OPL), and the like. The tissue is ˜0.5 mm. In the inner plexiform layer, there is a neuron that forms a synapse by pairing with a ganglion cell process called amacrine cell, but it responds well at the beginning and end of light irradiation. It is thought to work as a detector. A ganglion cell (hereinafter referred to as “RGC”) located in the innermost side of the retina is present in the nerve cell layer, and is deeply involved in motor vision, peripheral vision, color vision, morphological sense, and the like. In the nerve fiber layer, retinal blood vessels, which are branches of the central retinal arteriovenous vein, run and supply oxygen and nutrients to retinal neurons.

  By the way, when the retinal blood vessels are blocked or narrowed due to factors such as spasm, thrombus, arteriosclerosis, the retinal circulation is impaired, and the supply of oxygen and nutrients to retinal neurons is closed. If the supply of oxygen and nutrients is insufficient due to this retinal circulatory disorder, retinal nerve cells will die and optic neuropathy will be caused. Symptoms due to optic neuropathy include retinal vascular occlusion in which retinal veins and retinal arteries are blocked or narrowed, diabetic retinopathy that can lead to retinal detachment, and ischemic optic neuropathy in which visual dysfunction appears. In addition, retinal neuronal cell death is considered to be deeply involved in the onset of retinal diseases such as macular degeneration, retinitis pigmentosa, and Label disease.

  In glaucoma, which is one of eye diseases that lead to blindness, RGCs of retinal neurons are selectively damaged, causing optic nerve damage and progressing to visual field damage. For this reason, it is said that preventing or minimizing RGC disorders leads to glaucoma treatment (Non-patent Document 1). The detailed mechanism of glaucomatous optic neuropathy is not yet clear, but the optic nerve is compressed by increased intraocular pressure, resulting in a mechanical disorder due to optic nerve atrophy, and circulation due to optic nerve atrophy caused by circulatory disturbance of the optic nerve head There is an obstacle theory. Actually, these mechanical disorders and circulatory disorders are considered to be involved in a complicated manner, and both mechanical disorders and circulatory disorders cause optic nerve axonal transport disorders. And it is thought that the supply disruption of the neurotrophic factor due to this axonal transport disorder contributes to RGC disorder (Non-patent Document 2). In addition, glutamate is one of the neurotransmitters in the retina, and it is considered that excessive activation of the glutamate signal cascade for some reason is also a cause of RGC disorder (Non-patent Document 3). .

  Thus, it has been elucidated that apoptosis, which is a form of programmed cell death, is involved in the pathology of various eye diseases. For example, retinal damage caused by ischemia-reperfusion (Non-Patent Document 4), retinal detachment (Non-Patent Document 5), retinitis pigmentosa (Non-Patent Documents 6 and 7), retinal photopathy (Non-Patent Document 8), glaucoma (Non-patent Documents 9 and 10) all report RGC apoptosis. There are various causes of visual dysfunction, and as a result, visual dysfunction is highly likely due to apoptosis of neurons constituting the visual information network.

  Therefore, substances having a protective effect on retinal nerve cells are retinal vascular occlusion, diabetic retinopathy, ischemic optic neuropathy, macular degeneration, retinitis pigmentosa and label disease, and optic neuropathy. It is expected to be useful for the prevention and treatment of eye diseases such as glaucoma.

On the other hand, lignin is known as a main component constituting the cell wall of higher plants and as a natural phenolic polymer. Since natural lignin cannot be separated from a plant body simply by extraction operation, in order to obtain lignin, the plant components must be decomposed by some method and separated as a derivative derived from natural lignin. As such lignin derivatives derived from natural lignin, there are many types of lignin derivatives such as kraft lignin, steamed crushed lignin, and acetic acid lignin. In addition to this, lignophenol derivatives which are polyphenol compounds obtained by adding a phenol compound to a plant body and solvating it and then adding an acid are known (Patent Documents 1, 2 and 3). . In particular, regarding this lignophenol, the present inventors have found various bioactive functions in water-soluble derivatives subjected to chemical modification such as carboxymethylation. For example, water-soluble derivatives of lignophenol have been found to have non-physiological cell death suppression and cytoprotective effects caused by oxidative stress such as active oxygen, and prevention and treatment of neurological diseases such as Alzheimer's disease and Parkinson's disease (Patent Document 4).
JP-A-2-233701 JP-A-7-206601 Japanese Unexamined Patent Publication No. Hei 8-143587 JP 2004-24367 A Ophthalmology, 40, 251-273,1998 Ophthalmology, 44, 1413-1416,2002 Surv. Ophthalmol. 48, S38-S46, 2003 J. Ocul. Pharmacol. Ther., 11, 253-259, 1995 Arc. Ophthalmol., 113, 880-886, 1995 Proc. Natl. Acad. Sci. USA, 91, 974-978, 1994 Invest. Ophthalmol. Vis. Sci., 35, 2693-2699, 1994 Invest. Ophthalmol. Vis. Sci., 37, 775-782, 1996 Invest. Ophthalmol. Vis. Sci., 36, 774-786, 1995 Exp. EyeRes., 61, 33-44, 1995

  However, the effects of lignophenol derivatives that have cytoprotective effects on oxidative stress on optic nerve cells and on eye diseases such as retinal diseases and glaucoma that are thought to be caused by cell death are being investigated. Not.

  An object of the present invention is to provide a use of a lignophenol derivative as a preventive / therapeutic agent for mammalian eye diseases and an optic nerve cell protecting agent.

  The present inventors have deeply studied the relationship between the lignophenol derivatives and the cytoprotective action, and have conducted intensive studies on the effects on optic nerve cell death suppression and eye diseases such as retinal diseases and glaucoma. As a result, lignophenol derivatives have a protective function for retinal nerve cells such as RGC and are typified by retinal vascular occlusion, diabetic retinopathy, ischemic optic neuropathy, macular degeneration, retinitis pigmentosa and labeling disease. The present invention has been found to be useful as an agent for preventing or treating eye diseases associated with optic nerve disorders such as retinal diseases and glaucoma. According to the present invention, the following means are provided.

(1) (a) a water-soluble lignin secondary derivative obtained by subjecting a primary lignin derivative obtained by adding an acid to a lignin-containing material solvated with a phenol compound to an alkali treatment,
(B) A lignin secondary derivative carboxyalkylated with a carboxyalkyl group having a lower alkyl group having 1 to 5 carbon atoms with respect to the lignin primary derivative described in (a), and (c) a lignin solvated with a phenol compound Carboxy having a lower alkyl group having 1 to 5 carbon atoms with respect to a water-soluble and / or water-insoluble lignin secondary derivative obtained by alkali treatment of a primary derivative of lignin obtained by adding an acid to the contained material and mixing. A lignin tertiary derivative obtained by carboxyalkylation with an alkyl group,
A prophylactic / therapeutic agent for eye diseases comprising one or more lignin derivatives selected from the group consisting of as active ingredients.
(2) The preventive / therapeutic agent according to (1), wherein the phenol compound is a monovalent phenol compound.
(3) The preventive / therapeutic agent according to (1) or (2), wherein the phenol compound is cresol or vanillin.
(4) The prophylactic / therapeutic agent according to any one of (1) to (3), wherein the lignin derivative is water-soluble.
(5) The preventive / therapeutic agent according to any one of (1) to (4), comprising the lignin tertiary derivative according to (c) as an active ingredient.
(6) The preventive / therapeutic agent according to (5), wherein the phenol compound is cresol or vanillin.
(7) The prevention / regulation according to any one of (1) to (6), wherein the lignin-containing material is one or more lignocellulosic materials selected from woody plants and herbaceous plants. Therapeutic agent.
(8) The prophylactic / therapeutic agent according to any one of claims 1 to 7, wherein the lignin-containing material is bamboo.
(9) The prophylactic / therapeutic agent according to any one of (1) to (8), wherein the eye disease is an optic nerve cell death-related eye disease caused by hypoxia and / or low glucose.
(10) The prophylactic / therapeutic agent according to any one of (1) to (9), wherein the eye disease is an optic nerve cell death-related eye disease caused by endoplasmic reticulum stress.
(11) The prophylactic / therapeutic agent according to any one of (1) to (9), wherein the eye disease is an optic nerve cell death-related eye disease caused by an increase in intracellular Ca ²⁺ concentration.
(12) The prophylactic / therapeutic agent according to any one of (1) to (9), wherein the eye disease is a retinal disease.
(13) The prophylactic / therapeutic agent according to any one of (1) to (9), wherein the eye disease is glaucomatous optic neuropathy.
(14) (a) a water-soluble lignin secondary derivative obtained by subjecting a primary lignin derivative obtained by adding and mixing an acid to a lignin-containing material solvated with a phenol compound to alkali treatment;
(B) A lignin secondary derivative carboxyalkylated with a carboxyalkyl group having a lower alkyl group having 1 to 5 carbon atoms with respect to the lignin primary derivative described in (a), and (c) a lignin solvated with a phenol compound Carboxy having a lower alkyl group having 1 to 5 carbon atoms with respect to a water-soluble and / or water-insoluble lignin secondary derivative obtained by alkali treatment of a primary derivative of lignin obtained by adding an acid to the contained material and mixing. A lignin tertiary derivative obtained by carboxyalkylation with an alkyl group,
An optic neuroprotective agent comprising one or more lignin derivatives selected from the group consisting of as active ingredients.
(15) The protective agent according to (14), wherein the optic nerve includes retinal ganglion cells.
(16) A nutritional supplement containing the optic neuroprotective agent according to (14) or (15).
(17) A prophylactic / therapeutic agent for eye diseases, comprising the optic neuroprotective agent according to (14) or (15).

Hereinafter, embodiments of the present invention will be described in detail.
The preventive / therapeutic agent and optic neuroprotective agent of the present invention contain a lignophenol derivative obtained by separation from a lignin-containing material by a predetermined method. Examples of these lignophenol derivatives include the following lignin derivatives (a) to (c).

  The optic nerve cell protective agent and preventive / therapeutic agent of the present invention can be applied to mammals or optic nerve cells thereof, even if a decrease in the number of optic nerve cells, particularly the number of optic ganglion cells (RGC), the thickness of the inner reticulated layer, etc. is induced. Against this inducing action, the decrease in the number of optic ganglion cells and the thickness of the inner reticulated layer can be suppressed. Therefore, the optic nerve cell protecting agent and the preventive / therapeutic agent of the present invention are effective for the prevention / treatment of eye diseases based on such optic nerve cell injury. Further, according to the optic nerve cell protecting agent of the present invention, optic nerve cells can be protected by resisting various types of apoptosis induction. Further, in the optic nerve cell protective agent and the preventive / therapeutic agent of the present invention, the three-dimensional structure as a polymer derived from natural lignin is considered important. Therefore, it is presumed that the lignophenol derivative that maintains a structure derived from natural lignin at a high level and this derivative all have a protective effect on optic nerve cells. Lignin Hereinafter, these lignin derivatives will be described, and the use in the present invention will be further described.

(A) a water-soluble lignin secondary derivative obtained by subjecting a primary lignin derivative obtained by adding and mixing an acid to a lignin-containing material solvated with a phenol compound to an alkali;
(B) A lignin secondary derivative carboxyalkylated with a carboxyalkyl group having a lower alkyl group having 1 to 5 carbon atoms with respect to the lignin primary derivative described in (a), and (c) a lignin solvated with a phenol compound Carboxy having a lower alkyl group having 1 to 5 carbon atoms with respect to a water-soluble and / or water-insoluble lignin secondary derivative obtained by alkali treatment of a primary derivative of lignin obtained by adding an acid to the contained material and mixing. Tertiary derivatives of lignin obtained by carboxyalkylation with alkyl groups

(Primary derivative of lignin)
The lignin primary derivative used in the present invention is a lignin derivative (hereinafter also referred to as a lignophenol derivative) which is a phenol compound of lignin obtained by solvating a lignin-containing material with a phenol compound and then adding an acid and mixing. . Through this reaction process, a lignin derivative in which a phenol compound is grafted (introduced) to the benzyl position (side chain C1 position, hereinafter simply referred to as C1 position) of the arylpropane unit of lignin can be obtained. The phenol compound is bonded to the carbon atom at the C1 position in the ortho position or para position with respect to the phenolic hydroxyl group. As a result, 1,1-bis (aryl) propane units are formed in lignin. In this reaction, since the phenol compound is selectively introduced with respect to the C1 position, various bonds at the C1 position in the lignin-containing material as a starting material are released, reducing the diversity of the lignin matrix, and The molecular weight can be reduced. Furthermore, as a result, it is already known that various properties such as solubility in various solvents, thermal fluidity, and thermoplasticity, which were not found in conventional lignin, are exhibited. Here, solvating with a phenol compound means solvating by immersing a lignin-containing material in a liquid phenol compound, or dissolving a liquid or solid phenol compound in a solvent in which the phenol compound is dissolved. This can also be achieved by sorbing a phenolic compound onto the lignin-containing material by distilling off the solvent after applying it to the lignin-containing material.

  Separation of lignocellulosic materials into carbohydrates and lignophenol derivatives, combined with disruption of tissue structure due to carbohydrate swelling by concentrated acids and lignin solvation by phenolic compounds, while suppressing inactivation of lignin A method (JP-A-2-233701) is known. In addition to these, for more general description of lignophenol derivatives and the production process thereof, International Publication No. WO99 / 14223, JP 2001-64494 A, JP 2001-261839 A, JP 2001 2001 -131201, JP-A-2001-342353, and JP-A-2002-105240 (the contents described in these patent documents are all incorporated herein by reference). .

  An example of a structure conversion process in a system for obtaining a lignophenol derivative from a lignocellulosic material by this process will be described below. In this structure conversion process, the lignocellulosic material is solvated with a phenol compound in advance, and the lignocellulosic material is brought into contact with an acid to relax the lignin complex state of the lignin arylpropane unit. At the same time, the phenol derivative is selectively grafted to the C1 position (benzyl position) of the aryl propane unit of natural lignin to produce a lignophenol derivative, and at the same time, it can be separated into cellulose and lignophenol derivative.

  A lignophenol derivative itself is a mixture of lignin-derived polymers obtained by reaction and separation from a lignin-containing material such as a lignocellulosic material. For this reason, the amount and molecular weight of the introduced phenol derivative in the obtained polymer vary depending on the lignin structure and reaction conditions of the lignin-containing material as a raw material.

(Lignin-containing material)
The lignin-containing material in the present invention includes a lignocellulosic material containing natural lignin. Lignocellulosic materials are woody materials, various materials that are mainly wood, such as wood flour, chips, agricultural waste and industrial waste associated with woody plant resources such as waste wood, scraps, and waste paper. Can be mentioned. Moreover, as a kind of woody tree to be used, any kind can be used, such as conifers such as cedar and cypress, and broad-leaved trees such as beech. Furthermore, various herbaceous plants such as kenaf, jute, rice, bamboo, and related agricultural and industrial wastes such as rice straw and rice straw can be used. In the derivative used in the present invention, any lignocellulosic material of a woody plant or a herbaceous plant can be used, but it is particularly preferable to use a lignocellulosic material derived from a herbaceous plant. More preferred is bamboo. The lignophenol derivative derived from bamboo has a high cytoprotective action as described in JP-A-2004-244367. In addition, bamboo has the advantage that it is easy to secure resources and the use of bamboo is effective for the improvement of bamboo forests.

(Phenol compound)
The phenol compound may be a compound having at least one phenolic OH group. Specifically, a monovalent phenol compound, a divalent phenol compound, a trivalent phenol compound, or the like can be used. In the present invention, a monovalent phenol compound can be preferably used. Specific examples of the monovalent phenol compound include phenol that may have one or more substituents, naphthol that may have one or more substituents, and one or more substituents. A good anthrol, an anthroquinone all optionally having one or more substituents, and the like can be mentioned. Specific examples of the divalent phenol compound include catechol which may have one or more substituents, resorcinol which may have one or more substituents, and one or more substituents. Examples include good hydroquinone. Specific examples of the trivalent phenol compound include pyrogallol, which may have one or more substituents.

  As the phenol compound, aldehyde phenol can be used. As the aldehyde phenol, any phenolic OH group and aldehyde group may be used. Examples thereof include vanillin (4-hydroxy-3-methoxy-benzaldehyde), ethyl vanillin (3-ethoxy-4-hydroxy-benzaldehyde). 3,4-dihydroxybenzaldehyde and the like. In addition, aldehyde phenol shall be comprehensively expressed as a monovalent, divalent or trivalent phenol compound depending on the number of phenolic OH groups.

  In the primary derivative of the present invention, these phenol compounds can be used alone or in combination of two or more, but it is preferable to use one or more selected from monovalent phenol compounds. Moreover, 1 type, or 2 or more types can also be used among a bivalent phenol compound and a trivalent phenol compound.

  The kind of the substituent that the monovalent to trivalent phenol compound may have is not particularly limited, and may have any substituent, but preferably an electron-withdrawing group (halogen) A lower alkyl group-containing substituent having 1 to 4 carbon atoms, preferably 1 to 3 carbon atoms. Examples of the lower alkyl group-containing substituent include a lower alkyl group (such as a methyl group, an ethyl group, and a propyl group) and a lower alkoxy group (such as a methoxy group, an ethoxy group, and a propoxy group). Further, it may have an aromatic substituent such as an aryl group (such as a phenyl group). Further, it may be a hydroxyl group-containing substituent.

  In the present invention, preferably p-cresol, m-cresol, o-cresol, 2,6-dimethylphenol, 2,4-dimethylphenol, 2-methoxyphenol (Guaiacol), 2,6-dimethoxyphenol and the like are used. More preferred are p-cresol, phenol, and p-ethylphenol. Moreover, as a bivalent phenol compound, it is preferable to use resorcinol, catechol, homocatechol, hydroquinone, 1,3-naphthalenediol, and more preferably resorcinol and catechol. Moreover, as a trivalent phenol compound, Preferably, pyrogallol, phloroglucinol, 2-hydroxy-naphthoquinone, etc. can be used, More preferably, they are pyrogallol and phloroglucinol.

  In these phenol compounds, a 1,1-bis (aryl) propane unit is formed by bonding a carbon atom in the ortho-position or para-position to the C1-position carbon of the lignin arylpropane unit relative to the phenolic hydroxyl group. Will be. Therefore, in order to secure at least one introduction site, it is preferable that a substituent is not present in at least one of the ortho and para positions. A unit formed by bonding the ortho-position carbon atom of the phenolic hydroxyl group of the phenol compound to the C1-position is called an ortho-position binding unit, and the para-position carbon atom of the phenolic hydroxyl group of the phenol compound is bonded to the C1-position. The formed unit is called a para unit. As an example of the ortho-position binding unit and the para-position binding unit, p-cresol and 2,6-dimethylcresol can be used as the phenol compound.

  From the above, in the present invention, as the primary derivative, in addition to the unsubstituted phenol derivative, at least one phenol derivative in various substituted forms having at least one unsubstituted ortho-position or para-position is appropriately selected. Can be used.

  The ortho bond unit and the para bond unit express different functions in, for example, an alkali treatment step described later. The ortho-position binding unit eliminates the phenolic hydroxyl group in the phenolic compound introduced by mild alkali treatment and generates an arylcoumaran structure in the unit, and changes the molecular form as the aryl group moves by strong alkali treatment. . In any case, the ortho-position binding unit contributes to efficient molecular reduction of the lignophenol derivative by alkali treatment. On the other hand, the para-bonded unit does not cause an aryl coumaran structure or subsequent molecular morphology change by alkali treatment, and does not contribute to the reduction in molecular weight at the unit site.

  In order to obtain a lignophenol derivative having an ortho-position binding unit, a phenol compound having no substituent at at least one ortho-position (preferably all ortho-positions) is used. In addition, a phenol compound (typically a 2,4-position substituted monohydric phenol derivative) in which at least one ortho position (position 2 or 6) has no substituent and has a substituent in the para position (position 4) ) Is preferred. Most preferably, it is a phenol compound (typically a 4-position substituted monohydric phenol compound) in which all the ortho positions do not have a substituent and has a substituent in the para position. Accordingly, the 4-position substituted phenol compound and the 2,4-position substituted phenol compound can be used alone or in combination of two or more.

  In order to obtain a lignophenol derivative having a para-position binding unit, a phenol compound (typically a 2-position (or 6-position) substituted monohydric phenol compound) having no substituent at the para position is preferred, and more Preferably, at the same time, a phenol compound having a substituent at the ortho position (preferably, all ortho positions) (typically 2,6-substituted monohydric phenol compound) is used. That is, it is preferable to use one or two or more of 2-position (or 6-position) substituted phenol compound and 2,6-position substituted phenol.

(acid)
The acid to be brought into contact with the lignin-containing material is not particularly limited, and various inorganic acids and organic acids can be used as long as a lignophenol derivative can be generated. Therefore, in addition to inorganic acids such as sulfuric acid, phosphoric acid and hydrochloric acid, p-toluenesulfonic acid, trifluoroacetic acid, trichloroacetic acid, formic acid and the like can be used. When a lignocellulosic material is used as the lignin-containing material, it preferably has an action of swelling cellulose. For example, 65 wt% or more sulfuric acid (preferably 72 wt% sulfuric acid), 85 wt% or more phosphoric acid, 38 wt% or more hydrochloric acid, p-toluenesulfonic acid, trifluoroacetic acid, trichloroacetic acid, formic acid, etc. Can be mentioned. Preferred acids are 65% or more sulfuric acid (preferably 72% sulfuric acid), 85% or more (preferably 95% or more) phosphoric acid, trifluoroacetic acid or formic acid.

  Various methods can be adopted as a method of converting lignin in the lignin-containing material into a lignophenol derivative and separating it. As the reaction step for obtaining the primary derivative, for example, a liquid phenol compound (as described above, for example, p-cresol) is infiltrated into the lignin-containing material, lignin is solvated with the phenol derivative, and then, An acid (as described above, for example, 72% sulfuric acid) is added to the lignocellulosic material and mixed to dissolve the cellulose component (hereinafter also referred to as a one-step method). According to this method, lignin is reduced in molecular weight, and at the same time, a lignophenol derivative in which a phenol compound is introduced at the C1 position of the basic structural unit is generated in the phenol compound phase. From this phenolic compound phase, lignophenol derivatives are extracted. A lignophenol derivative is obtained as an aggregate of low molecular weight forms of lignin in which the benzyl aryl ether bond in lignin is cleaved to reduce the molecular weight. As another reaction step, a solvent (for example, ethanol or acetone) in which a solid or liquid phenol compound is dissolved is infiltrated into the lignin-containing material, and then the solvent is distilled off (sorption of the phenol derivative). In this case, as in the previous method, a lignophenol derivative is produced (hereinafter also referred to as a two-step method).

  Water solubility is imparted to the lignin primary derivative by subsequent carboxymethylation and / or alkali treatment. Therefore, as the primary derivative, it is effective to separate and extract the lignin primary derivative of the organic solvent classification. In order to separate and recover the lignin primary derivative of the organic solvent section, in the one-stage method, a precipitate obtained by adding a liquid phenol compound phase to a large excess of ethyl ether is collected and dissolved in acetone. The acetone insoluble part is removed by centrifugation, and the acetone soluble part is concentrated. The acetone soluble part is dropped into a large excess of ethyl ether, and the precipitate section is collected. The solvent is distilled off from this precipitation section to obtain a lignin primary derivative. The crude primary derivative can be obtained by simply removing the phenol compound phase or acetone-soluble fraction by distillation under reduced pressure.

  Further, in the two-stage method, the produced lignophenol derivative can be extracted and separated with a liquid phenol compound. Alternatively, the entire reaction solution is poured into excess water, the insoluble sections are collected by centrifugation, deacidified and then dried. Acenolic alcohol is added to the dried product to extract the lignophenol derivative. Further, as in the case of the one-stage method, this soluble segment can be dropped into excess ethyl ether or the like to obtain a lignophenol derivative as an insoluble segment. Specific examples of the method for preparing the primary derivative have been described above, but the present invention is not limited to these, and it can be prepared by a method in which these are appropriately improved.

  Since the lignophenol derivative (primary derivative) thus obtained has a higher-order structure derived from natural lignin, it is considered to exhibit an optic nerve protecting action. In addition, lignophenol derivatives can be applied as alkali derivatives or phenolic OH modified as described later, so that they can be applied as optic nerve cell protecting agents and prophylactic / therapeutic agents for ocular diseases, and the choice of dosage form is free. The degree can be improved. Next, the secondary and tertiary derivatives obtained by further processing the primary derivative will be described.

(Solubilization by alkali treatment)
The alkali treatment is performed by bringing the lignin primary derivative into contact with an alkali. Preferably it heats. In the alkali treatment, the C2-position carbon is attacked by the phenoxide ion of the introduced phenol compound in the ortho-position binding unit. That is, once this reaction occurs, the C2 aryl ether bond is cleaved. For example, in a mild alkali treatment, when the primary derivative has a first unit, the phenolic hydroxyl group of the introduced phenol derivative is cleaved, and the resulting phenoxide ion has an intramolecular C2 position constituting a C2 aryl ether bond. The nucleophilic reaction can be attacked to cleave the ether bond to reduce the molecular weight. Cleavage of the C2 aryl ether bond results in the formation of a phenolic hydroxyl group in the lignin nucleus, and the introduced phenol nucleus forms a coumaran skeleton with the phenylpropane unit into which it is introduced by the intramolecular nucleophilic reaction. The resulting structure (arylcoumaran unit) is expressed. As a result, the phenolic hydroxyl group on the phenol derivative side is in the same state as if it moved to the lignin mother nucleus side. For this reason, in lignophenol derivatives having an ortho-position binding unit, (1) low molecular weight by cleavage of C2 aryl ether bond, (2) expression of arylcoumaran structure, (3) C2 A phenolic hydroxyl group on the side of the lignin mother nucleus bonded with an aryl ether bond is expressed.

  Specifically, the alkali treatment is performed by dissolving a lignin primary derivative in an alkali solution, reacting for a predetermined time, and heating if necessary. The alkali solution that can be used for this treatment is not particularly limited as long as it can dissociate the phenolic hydroxyl group of the introduced phenol compound in the lignin primary derivative, and the type and concentration of alkali and the type of solvent are not particularly limited. . This is because if the phenolic hydroxyl group is dissociated under an alkali, a coumaran structure is formed due to the effect of neighboring group participation. The alkaline solution is not particularly limited. Any lignin derivative to be used for the treatment may be dissolved, and an appropriate organic solvent such as alcohol or a mixture of the organic solvent and water may be used. For example, a sodium hydroxide solution can be used for a lignin primary derivative into which p-cresol is introduced. For example, the alkali concentration range of the alkaline solution can be 0.5 to 2 N, and the treatment time can be about 1 to 5 hours. The lignin primary derivative in the alkaline solution easily develops a coumaran structure when heated. Conditions such as temperature and pressure during heating can be appropriately set without any particular limitation. For example, the molecular weight of the lignin primary derivative can be reduced by heating the alkaline solution to 100 ° C. or higher (for example, about 140 ° C.). Furthermore, the molecular weight of the primary derivative may be reduced by heating the alkaline solution to above its boiling point under pressure.

  It has been found that at the same concentration in the same alkaline solution, when the heating temperature is in the range of 120 ° C. to 140 ° C., the higher the heating temperature, the lower the molecular weight due to the cleavage of the C2-aryl ether bond. In addition, it is known that the phenolic hydroxyl group derived from the aromatic nucleus derived from the lignin matrix increases and the phenolic hydroxyl group derived from the introduced phenol compound decreases as the heating temperature is higher in this temperature range. Therefore, the degree of molecular weight reduction and the degree of conversion of the phenolic hydroxyl moiety from the C1-introduced phenol compound side to the phenol nucleus of the lignin matrix can be adjusted by the reaction temperature. That is, a reaction temperature of about 80 to 140 ° C. is preferred in order to obtain an aryl coumaran body in which low molecular weight is promoted or more phenolic hydroxyl sites are converted from the C1-introduced phenol derivative side to the lignin matrix. .

  Although the cleavage of C2-aryl ether by participation of adjacent group of C1 phenol nucleus is accompanied by the formation of aryl coumaran structure as described above, the reduction of the molecular weight of the cross-linked lignophenol derivative is not necessarily efficiently generated by aryl claman. It is not necessary to carry out under the conditions (around 140 ° C.), and it can be carried out at a higher temperature (eg, around 170 ° C.) In this case, once the generated claman ring is cleaved and the phenolic hydroxyl group is regenerated on the introduced phenol derivative side, a material having higher phenol activity than that of the 140 ° C. treated product can be induced. As the lignin derivative of the present invention, alkali treatment at 150 to 180 ° C., preferably 160 to 180 ° C., more preferably about 170 ° C. can be employed.

  From the above, the heating temperature in the alkali treatment is not particularly limited, but is preferably 80 ° C. or higher and 200 ° C. or lower. This is because the reaction does not proceed sufficiently when the temperature is greatly below 80 ° C., and an undesirable side reaction tends to be easily induced when the temperature is greatly above 200 ° C.

  A preferable example of the treatment for formation of the Kuraman structure and the accompanying reduction in molecular weight is a condition in which a 0.5N sodium hydroxide aqueous solution is used as an alkaline solution and the heating time is 140 ° C. for 60 minutes. In addition, a 0.5N sodium hydroxide aqueous solution is used as an alkaline solution, and the heating time is 60 minutes at 170 ° C.

  As a method for obtaining a water-soluble lignin secondary derivative by alkali treatment, after neutralizing the alkali-treated reaction solution, the water-soluble fraction is separated and collected by centrifugation, etc., desalted by dialysis or the like, and lyophilized. It is possible to adopt a method of equalizing. The liquid after desalting can be used as it is as a composition containing a water-soluble lignin secondary derivative. The water-insoluble fraction after neutralization with the alkali treatment reaction solution becomes a precursor of a carboxyalkylated tertiary derivative described later as a water-insoluble lignin secondary derivative. The water-soluble lignin secondary derivative preferably has a weight average molecular weight of 700 to 10,000, more preferably 700 to 6,000. The weight average molecular weight of the water-insoluble lignin secondary derivative is 700 to 10,000, preferably 700 to 6,000.

(Secondary derivatization and tertiary derivatization by carboxyalkylation)
A carboxyalkylated secondary derivative and a tertiary derivative can be obtained by introducing a carboxyalkyl group into the primary derivative and the water-soluble and / or water-insoluble lignin secondary derivative. The alkyl group in the carboxylalkyl group is preferably a linear or branched alkyl group having 1 to 5 carbon atoms, and more preferably a linear alkyl group having 1 to 3 carbon atoms. For example, a methyl group, an ethyl group, or a propyl group can be used. The carboxyl alkyl group is introduced into an alcoholic or phenolic hydroxyl group in the lignin derivative. Carboxyalkylation can generally be achieved by reacting with a monohalogenoalkyl carboxylic acid in the presence of an alkali. Various conventionally known methods can be adopted as the carboxyalkylation method of the lignin derivative. For example, a lignin primary derivative is dispersed in an organic solvent such as isopropanol in which the derivative can be dispersed, an aqueous alkaline solution is added, and an organic solvent such as isopropanol is further added as necessary. Monohalogenoacetic acid (carboxyalkylating agent) such as acetic acid can be added and reacted while stirring. Alternatively, after the lignin derivative is preliminarily immersed in an alkaline solution, the immersion product can be dispersed in an organic solvent and then reacted. The alkali may be a commonly used alkali reagent, but a strong alkali such as an alkali metal compound such as sodium hydroxide or potassium hydroxide is preferably used. Examples of the organic solvent for dispersing the lignin derivative and the like include alcohols such as methanol, ethanol, isopropanol and t-butanol, ketones such as acetone and methyl ethyl ketone, ethers such as diethyl ether, tetrahydrofuran and 1,4-dioxane, Halides such as dichloromethane and chloroform, and hydrocarbons such as hexane, cyclohexane, benzene, and toluene can be used, and these can be used alone or in admixture of two or more. Among these, 1 type, or 2 or more types of combinations of polar organic solvents, such as alcohol, ketones, ethers, are good, More preferably, isopropanol, acetone, 1, 4- dioxane is used.

As the monohalogenoalkylcarboxylic acid, those represented by the following general formula [I] are preferably used.
XRCOOH [I]
(In the above formula, X and R represent the following, respectively.
X: a halogen atom such as F, Cl, Br, or I; R: an alkyl group having 1 to 5 carbon atoms and a straight chain and a branched chain. )
Among the above compounds, those having a linear alkyl group having 1 to 3 carbon atoms and preferably having a Cl or Br atom are preferred.

  The monohalogenoalkylcarboxylic acid may be used in an amount that is equal to or greater than the amount of hydroxyl groups provided in the lignin derivative, and is preferably about 1 to 3 mol per hydroxyl group. As a method for adding the monohalogenoalkylcarboxylic acid, it may be added as a solid and / or as a solution of this organic solvent by batch or continuous dropping. The method of continuous dripping which took 0.5 to 3 hours as a solution of an organic solvent is preferable, and it is particularly preferable to continuously drop the solution in 1-2 hours. In addition, after completion of the addition of the monohalogenoalkylcarboxylic acid, it is preferable to continue the reaction at that temperature for 0.5 to 5 hours (more preferably 1 to 4 hours). In the reaction with the monohalogenoalkylcarboxylic acid, the reaction solution is preferably heated in the range of about 40 ° C to 60 ° C.

  Carboxyalkylated secondary and tertiary derivatives are obtained in a partially dissolved state in addition to the solid product. After completion of the reaction, the solid product is separated and collected by filtration or the like, dissolved in water, and neutralized with an acid such as dilute mineral acid such as dilute hydrochloric acid or dilute sulfuric acid. It can be obtained by desalting by electrodialysis and freeze-drying. The dissolved product can be recovered by distilling off the organic solvent in the filtrate, drying the dissolved product, and adding the dried product to the solid product before neutralization. In the present invention, a secondary derivative obtained by carboxyalkylating a primary derivative of a monovalent phenol compound is a preferred form, more preferably, the monovalent phenol compound is cresol or vanillin, and more preferably carboxymethylation. It is a form made. Furthermore, it is preferable to use bamboo as the lignocellulose material. Further, a water-soluble and / or water-insoluble secondary derivative carboxymethylated tertiary derivative obtained by alkali treatment of a primary derivative of a monovalent phenol compound is also a preferred form. A derivative obtained by carboxymethylating a derivative is preferred, and more preferred is a form in which the monovalent phenol compound is cresol, which is a carboxymethylated form.

(Ophthalmic disease prevention / treatment agent and optic nerve cell protecting agent)
The lignin derivative in the present invention described above, that is, the secondary derivative and tertiary derivative described above, imparts resistance to cell death of optic nerve cells that can cause eye diseases to optic nerve cells of mammals including humans. Can do. In other words, the optic nerve cells can be protected by exerting the cell death inhibitory action of optic nerve cells such as retinal ganglion cells. Thereby, it can be used as a prophylactic / therapeutic agent for eye diseases and also as an optic nerve cell protecting agent.

The optic nerve cell protective action of such a lignin derivative can be confirmed by inducing optic nerve cell death under various conditions and measuring the cell death inhibitory activity of the lignin derivative. Such optic nerve cell death induction conditions include, for example, optic nerve cell death conditions due to oxygen and / or glucose deficiency, optic nerve cell death conditions induced by endoplasmic reticulum stress by tunicamycin, and N-methyl-D-aspartic acid (NMDA). Conditions for inducing optic nerve cell death by inducing endoplasmic reticulum stress and / or increasing intracellular Ca ²⁺ concentration can be used. Therefore, the lignin derivative in the present invention can be used as a cytoprotective agent against cell death of such optic nerve cells, and can also be used as a prophylactic / therapeutic agent for eye diseases that can be caused by such optic nerve cell death.

  The optic nerve cells in which the lignin derivative of the present invention is effective include retinal ganglion cells, amacrine cells (axon cells), bipolar cells, horizontal cells, photoreceptor cells (photoreceptor cells), interplexifilm cells, and Mueller cells (retinal glia). Cell). The lignin derivative in the present invention is effective for animals including humans, but is particularly effective for mammals including humans.

  The eye disease targeted by the lignin derivative in the present invention is preferably an eye disease of mammals including humans. Examples of eye diseases in which lignin is effective in the present invention include retinal vascular occlusion, diabetic retinopathy, ischemic optic neuropathy, macular degeneration, retinitis pigmentosa, label disease, and optic neuropathies such as retinal diseases and glaucoma. Eye diseases associated with

  The lignin derivative in the present invention can be formulated using a technique widely used as a single preparation or a combined preparation by adding a pharmaceutically acceptable additive as necessary.

  The prophylactic / therapeutic agent and cytoprotective agent for eye diseases of the present invention can be administered parenterally or orally. Examples of parenteral dosage forms include eye drops, injections, nasal drops, and oral dosage forms include tablets, capsules, fine granules, granules, powders, etc. It can be formulated using technology.

  For example, in the case of eye drops, isotonic agents, buffers, pH adjusters, solubilizers, thickeners, stabilizers, preservatives and the like can be appropriately added as additives. In addition, a stable eye drop can be obtained by suspending the drug by adding a pH adjuster, a thickener, a dispersant and the like. Examples of isotonic agents include glycerin, propylene glycol, sodium chloride, potassium chloride, sorbitol, mannitol and the like. Examples of the buffer include phosphoric acid, phosphate, citric acid, acetic acid, and ε-aminocaproic acid. Examples of the pH adjuster include hydrochloric acid, citric acid, phosphoric acid, acetic acid, sodium hydroxide, potassium hydroxide, boric acid, borax, sodium carbonate, sodium hydrogen carbonate and the like. Examples of the solubilizer include polysorbate 80, polyoxyethylene hydrogenated castor oil 60, macrogol 4000, and the like. Examples of thickeners and dispersants include cellulose polymers such as hydroxypropylmethylcellulose and hydroxypropylcellulose, polyvinyl alcohol, polyvinylpyrrolidone and the like. Examples of stabilizers include edetic acid and sodium edetate. be able to. Examples of preservatives (preservatives) include general-purpose sorbic acid, potassium sorbate, benzalkonium chloride, benzethonium chloride, methyl paraoxybenzoate, propyl paraoxybenzoate, and chlorobutanol. These preservatives are combined. Can also be used.

  The pH of the eye drop may be within the range acceptable for ophthalmic preparations, but is preferably in the range of 4.0 to 8.5, and the osmotic pressure ratio is preferably set to around 1.0. The injection includes solutions, suspensions, emulsions, and solid injections that are used by dissolving or suspending in the solution at the time of use. For example, the drug is dissolved, suspended, or emulsified in the solution. Used.

  The injection can be prepared using sterile purified water and sodium chloride for isotonicity.

  Tablets are excipients such as lactose, glucose, D-mannitol, anhydrous calcium hydrogen phosphate, starch, sucrose; carboxymethylcellulose, carboxymethylcellulose calcium, croscarmellose sodium, crospovidone, starch, partially pregelatinized starch , Disintegrating agents such as low-substituted hydroxypropylcellulose; binders such as hydroxypropylcellulose, ethylcellulose, gum arabic, starch, partially pregelatinized starch, polyvinylpyrrolidone, polyvinyl alcohol; magnesium stearate, calcium stearate, talc, hydrous silicon dioxide , Lubricants such as hydrogenated oil; refined sucrose, hydroxypropylmethylcellulose, hydroxypropylcellulose, methylcellulose, ethylcellulose, polyvinyl Coating agents such as pyrrolidone; citric acid, aspartame, ascorbic acid, may be formulated using appropriately selected and flavoring agents such as menthol.

  The dose can be appropriately selected depending on symptoms, age, dosage form, etc. In the case of eye drops, 0.001 to 10% (w / v) may be instilled once to several times a day. If so, it is usually sufficient to administer 0.1 mg to 250 mg daily or divided into several times. In the case of an oral preparation, 250 mg to 2.5 g per day can be usually administered once or divided into several times. If necessary, an amount outside the above range can be used.

  Since lignin derivatives have non-physiological cell death inhibitory activity and cytoprotective activity, these lignin derivatives can be used as reagents for non-physiological cell death inhibitor and cytoprotective agent. For example, direct administration to various cultured cell lines can protect cells against inappropriately induced apoptosis. Although the form of these reagents is not specifically limited, For example, solid agents, such as a powder, or the liquid agent melt | dissolved in the organic solvent or the water-containing organic solvent can be mentioned. Usually, the effective use concentration for exhibiting the non-physiological cell death inhibitory action and cytoprotective action using the above compound as a reagent is 10 to 50 μM, but the appropriate use amount is the kind of the cultured cell line. It depends on the purpose of use and can be appropriately selected by those skilled in the art. In addition, when administered to animals, the blood concentration is preferably 10 to 100 μM. Further, if necessary, an amount outside the above range can be used.

  Furthermore, this drug is also useful as a nutritional supplement (food) via the oral or intestinal tract. Various types of conventionally known forms can be adopted as the form of the food. It can also be used as a food additive. When ingested as a dietary supplement, the preferred intake is about 250 mg to 2.5 g per day, and an amount outside the above range can be used if necessary.

  In addition, since the lignin derivatives in the present invention have effective optic nerve cell protecting activity, these compounds are used as lead compounds to search for more useful compounds using non-physiological cell death inhibitory activity and cytoprotective activity as indicators. And can get.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, the scope of the present invention is not limited to the following Example.

(Preparation of carboxymethylated lignocresol and carboxymethylated lignovanillin)
2 mol of p-cresol or vanillin as a phenol compound was dissolved in acetone with respect to the basic unit (phenylpropane unit, C ₉ ) of lignin contained in bamboo (the stem part excluding leaves). After each acetone solution was immersed in a bamboo powder degreased sample for a day and night, acetone was completely distilled off and air-dried to sorb each phenol compound on the degreased sample. 68 wt% sulfuric acid (4 ml / g degreased sample) was added to these sorption samples and stirred at 30 ° C. for 1 hour. After completion of the stirring, the reaction system was poured into a large excess of water, and the sulfuric acid concentration of the reaction system was diluted to about 10%. The insoluble precipitate generated at this time was separated by centrifugation, sulfuric acid and unreacted phenol compound were removed, washed, and then dried under reduced pressure on phosphorus pentoxide. Acetone was added to the completely dried insoluble precipitate and stirred for a whole day and night. Thereafter, insoluble matters were removed by centrifugation. The extract was concentrated with an evaporator and then added dropwise to a large excess of diethyl ether, and the insoluble precipitate was centrifuged and washed. As a lignophenol sample, lignocresol derived from bamboo (weight average molecular weight; 7200, p -Cresol introduction amount; 28 wt%) and lignovanillin (weight average molecular weight; 5050, vanillin introduction amount; 29 wt%). The amount of phenol compound introduced was measured by NMR analysis.

  1 g of each obtained lignophenol sample (lignocresol and lignovanillin) was placed in a container and dispersed in 4 g of isopropyl alcohol. Thereafter, 6.25 g of a 40% aqueous sodium hydroxide solution was added to the dispersion and allowed to stand sufficiently. After standing, 12 g of isopropyl alcohol was further added and stirred to make the solution in the container a heterogeneous mixed dispersion solution. Maintaining the temperature of this dispersion at 50 ° C., a separately prepared monochloroacetic acid solution (a solution of monochloroacetic acid (about 1.3 mol per hydroxyl group in the lignophenol sample) dissolved in 4 g of isopropyl alcohol) was added over 1 hour. did. After the addition, the mixture was further stirred at 50 ° C. for 2 hours to be reacted. In the course of the reaction, a precipitate of carboxymethylated derivative containing sodium hydroxide was produced in the reaction system, and this precipitate was filtered off after the reaction was completed. The precipitate was dissolved in water and the solution was neutralized with 1N hydrochloric acid and further acidified to pH2. The precipitate generated at this time was separated and removed by centrifugation, and then the supernatant was desalted by dialysis. The desalted solution was freeze-dried and, as carboxymethylated derivatives, CM-derived lignocresol (sample 1, weight average molecular weight; 5330, carboxymethyl group amount; 11.6 wt%) and bamboo-derived lignovanillin (sample), respectively. 2, weight average molecular weight; 4500, carboxymethyl group amount; 20 wt%). The amount of carboxymethyl group was calculated by NMR analysis and non-aqueous potentiometric titration using tetra-n-butylammonium hydroxide.

  To examine the optic neuroprotective action of the lignophenol derivative thus obtained, (1) action against hypoxia / low glucose (OGD) -induced ischemia-like cell death using PC12 cells, (2) PC12 The effects on cell death induced by tunicamycin administration using cells, and (3) the effects on retinal injury model mice by NMDA administration in vivo were tested.

(Evaluation of effects on hypoxia / low glucose (OGD) -induced ischemia-like cell death)
Insufficient supply of oxygen and nutrients leads to death of retinal neurons and optic neuropathy, so the effect on hypoxia and low glucose (OGD) -induced ischemia-like cell death was tested.

(Evaluation methods)
PC12 cells were seeded on a collagen-coated 24-well plate at 2 × 10 ⁵ cells / well, and Dulbecco's modified Eagle basal medium containing 10% horse serum and 5% fetal calf serum for 2 days. Cultured. After 2 days of culture, hypoxia / low glucose (OGD) treatment was performed to induce ischemia-like cell death. For low glucose treatment, the medium was replaced with Dulbecco's modified Eagle basal medium (solvent) containing no glucose, or the medium was added with CMized lignophenol sample 1 or sample 2 (1 μM, 3 μM, 10 μM, 30 μM). The medium was replaced with Dulbecco's modified Eagle basal medium containing no glucose, and then the atmosphere was changed to 94% N ₂ , 5% CO ₂ , 1% O ₂ conditions as a hypoxic treatment and cultured for 4 hours. After the OGD treatment, glucose and fetal calf serum were added to the medium so as to be 1 g / 1 glucose and 1% fetal calf serum, and cultured for 18 hours (37 ° C., 5% CO ₂ ). Thereafter, cell viability was calculated by the resazurin reduction method using CellQuanti-Blue ^™ Cell Viability Assay Kits (BioAssaySystems). That is, resazurin solution was added to the medium and incubated for 3 hours, and the fluorescence intensity of each group was measured (excitation wavelength 530 nm, fluorescence wavelength 590 nm). Since dark blue resazurin is reduced by viable cells and converted to resorufin that emits pink fluorescence, the fluorescence intensity correlates with the number of viable cells. Therefore, the fluorescence intensity of the control group not subjected to OGD treatment at all was determined as the control group. As the cell viability (100%), the survival rate of each group relative to the control group was calculated from the difference from the fluorescence intensity of each group. The group which performed only OGD processing was made into the "solvent group", and the group which added OGD processing and each CM-ized lignophenol sample was compared as "sample 1 group" and "sample 2 group", respectively. The result is shown in the graph of FIG. In addition, the bar | burr in a graph shows the average value of each group, and an error line shows a standard error.

  From the results shown in FIG. 1, it can be seen that, at the cellular level, the lignin derivative has a concentration-dependently suppressing effect on cell death that is deeply involved in retinal diseases and eye diseases accompanied by optic neuropathy.

(Evaluation of effects of tunicamycin administration on endoplasmic reticulum stress-induced cell death)
Tunicamycin is a nucleoside antibiotic and has a structure similar to N-acetylglucosamine. Therefore, tunicamycin binds from the sugar donor uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) to N-acetylglucosamine transferase (polypeptide transferase or O-GlcNAc transferase (OGT)) and UDP-GlcNAc. And inhibits enzyme activity. As a result, abnormal proteins are excessively accumulated in the endoplasmic reticulum of the cells, causing endoplasmic reticulum stress and inducing cell death. Since it is known that retinal damage is also caused by endoplasmic reticulum stress induction, the effect on cell death induced by tunicamycin administration was examined. (In addition, various antioxidants, calcium antagonists, etc. have no effect on this tunicamycin-induced cell death (2nd Gifu Brain Science Society, Abstracts of Speech, p25).)

(Evaluation methods)
PC12 cells were seeded onto a collagen-coated 96-well plate at 2 × 10 ⁵ cells / well, and Dulbecco's modified Eagle basal medium containing 10% horse serum and 5% fetal calf serum was used for 2 days. Cultured. After 2 days of culture, the medium was replaced with Dulbecco's modified Eagle basal medium (solvent) containing 1% fetal calf serum, or the medium was CMized lignophenol sample 1 or sample 2 (1 μM, 3 μM, 10 μM, 30 μM). Was replaced with Dulbecco's modified Eagle basal medium containing 1% fetal calf serum supplemented with One hour after the medium change, tunicamycin was administered while diluting with a phosphate buffer (containing 0.1% dimethyl sulfoxide) so that the tunicamycin concentration in the medium was 2 μg / ml, and cultured for 24 hours (37 ° C., 5 ° C. % CO ₂ ) and tunicamycin treatment was performed. Thereafter, cell viability was determined by the resazurin reduction method using CellQuanti-Blue ^™ Cell Viability Assay Kits (BioAssay Systems). That is, a resazurin solution was added to the medium and incubated for 3 hours, and the fluorescence intensity of each group was measured (excitation wavelength 530 nm, fluorescence wavelength 590 nm). Since dark blue resazurin is reduced by viable cells and converted to resorufin that emits pink fluorescence, the fluorescence intensity correlates with the number of viable cells. From this, the fluorescence intensity of the control group that was not treated with tunicamycin was taken as the cell survival rate (100%) of the control group, and the survival rate of each group relative to the control group was calculated from the difference with the fluorescence intensity of each group. The group which performed only the tunicamycin treatment was compared as a “solvent group”, and the group where the tunicamycin treatment and the addition of each CM lignophenol sample were compared as “sample 1 group” and “sample 2 group”, respectively. The result is shown in the graph of FIG. In addition, the bar | burr in a graph shows the average value of each group, and an error line shows a standard error.

  From the results shown in FIG. 2, it can be seen that, at the cellular level, the lignin derivative has a concentration-dependently suppressing effect on cell death that is deeply involved in retinal diseases and eye diseases accompanied by optic neuropathy.

(Evaluation of effects on endoplasmic reticulum stress-induced cell death by in vivo NMDA administration)
N-methyl-D-aspartic acid (NMDA) increases intracellular Ca ²⁺ concentration and induces cell death by binding to NMDA receptor which is one of receptors for glutamate which is a neurotransmitter. NMDA receptor is involved downstream in the mechanism of normal-tension glaucoma, and NMDA antagonist memantine is in the clinical trial stage of normal-tension glaucoma in the United States (2nd Gifu Brain Science Institute, Abstract) Collection, p15). From these facts, NMDA was directly administered to the vitreous body of mice to prepare in vivo retinopathy model mice, and their effects on retinal cells were tested.

(Evaluation methods)
An anesthesia machine for small animals (70% laughing gas, 30% oxygen gas) in a ddy male mouse (weight: 36-39 g, Japan SLC Co., Ltd.) and anesthesia with isoflurane (at the time of induction of anesthesia; 2% Isoflurane, at the time of maintenance of anesthesia; 1% isoflurane). Thereafter, physiological saline (NMDA solvent) containing 10 mM N-methyl-D-aspartic acid (NMDA) was administered into the mouse vitreous with an injection needle (NMDA dose; 2 nmol / eye), or NMDA administration At the same time, physiological saline containing the CMized lignophenol sample 1 was administered into the mouse vitreous (NMDA dose: 2 nmol / eye, CMized lignophenol sample dose; 0.01 nmol / eye or 0.1 nmol / eye), Retinopathy model mice were prepared. In addition, levofloxacin ophthalmic solution (Santen Pharmaceutical Co., Ltd.) was instilled in retinal disorder model mice immediately after NMDA for the purpose of anti-inflammation. Seven days after NMDA administration, the eyes of mice were removed after cervical dislocation for histological analysis. After excision, a 4% paraformaldehyde solution as a fixative solution was intravitreally administered to each eye and immersed in the solution at 4 ° C. for at least 24 hours. Paraffin-embedded, sliced, and 6 histopathological sections (4 μm thick) stained with hematoxylin-eosin were prepared. As samples for evaluating retinal neuropathy, 3 sections were randomly selected from 6 sections collected so that the optic disc was inserted per eye, and used for histological analysis. The selected section is photographed from the optic disc to the retina between 375 μm and 625 μm, the number of retinal ganglion cells (RGC) in the retina and the thickness of the inner plexiform layer (IPL) are measured, and the histological analysis is performed. went. A group in which NMDA solvent was administered to induce retinal neuropathy, and a group in which CMDA lignophenol sample 1 was administered simultaneously with NMDA solvent to induce retinal neuropathy were referred to as “NMDA solvent group” and “sample 1 administration group”, respectively. " These results were compared with the corresponding measurements for the “control group” that had not been treated at all. The results of photography are shown in FIG. 3, and the measurement results of the number of RGCs and the thickness of IPL in the retina are shown in the graphs of FIGS. 4 and 5, respectively. In addition, the bar | burr in a graph shows the average value of each group, and an error line shows a standard error.

  As is clear from the results shown in FIGS. 3, 4, and 5, the lignin derivative is excellent in the effect of suppressing the decrease in the number of ganglion cells and the thinning of the inner plexiform layer in the retina of the living body. I understand.

Graph showing cell viability of each group induced by hypoxia / low glucose induction Graph showing cell viability of each group by tunicamycin administration Micrograph showing cross section of mouse retinal in each group by NMDA administration The graph which shows the number of ganglion cells (RGC) of the mouse | mouth retina of each group by NMDA administration Graph showing inner reticulated layer (IPL) thickness of mouse retina by NMDA administration

Claims (15)

(A) a water-soluble lignin secondary derivative obtained by subjecting a primary lignin derivative obtained by adding and mixing an acid to a lignin-containing material solvated with a phenol compound to an alkali;
(B) A lignin secondary derivative carboxyalkylated with a carboxyalkyl group having a lower alkyl group having 1 to 5 carbon atoms with respect to the lignin primary derivative described in (a), and (c) a lignin solvated with a phenol compound Carboxy having a lower alkyl group having 1 to 5 carbon atoms with respect to a water-soluble and / or water-insoluble lignin secondary derivative obtained by alkali treatment of a primary derivative of lignin obtained by adding an acid to the contained material and mixing. A lignin tertiary derivative obtained by carboxyalkylation with an alkyl group,
A prophylactic / therapeutic agent for eye diseases comprising one or more lignin derivatives selected from the group consisting of as active ingredients.   The preventive / therapeutic agent according to claim 1, wherein the phenol compound is a monovalent phenol compound.   The preventive / therapeutic agent according to claim 1 or 2, wherein the phenol compound is cresol or vanillin.   The preventive / therapeutic agent according to any one of claims 1 to 3, comprising the lignin tertiary derivative according to (c) as an active ingredient.   The preventive / therapeutic agent according to claim 4, wherein the phenol compound is cresol or vanillin.   The prophylactic / therapeutic agent according to any one of claims 1 to 5, wherein the lignin-containing material is bamboo.   The prophylactic / therapeutic agent according to any one of claims 1 to 6, wherein the eye disease is an optic nerve cell death-related eye disease caused by hypoxia and / or low glucose.   The prophylactic / therapeutic agent according to any one of claims 1 to 7, wherein the eye disease is an optic nerve cell death-related eye disease caused by endoplasmic reticulum stress. The prophylactic / therapeutic agent according to any one of claims 1 to 8, wherein the eye disease is an optic nerve cell death-related eye disease caused by an increase in intracellular Ca ²⁺ concentration.   The prophylactic / therapeutic agent according to any one of claims 1 to 9, wherein the eye disease is a retinal disease.   The prophylactic / therapeutic agent according to any one of claims 1 to 9, wherein the eye disease is glaucomatous optic neuropathy. (A) a water-soluble lignin secondary derivative obtained by subjecting a primary lignin derivative obtained by adding and mixing an acid to a lignin-containing material solvated with a phenol compound to an alkali;
(B) A lignin secondary derivative carboxyalkylated with a carboxyalkyl group having a lower alkyl group having 1 to 5 carbon atoms with respect to the lignin primary derivative described in (a), and (c) a lignin solvated with a phenol compound Carboxy having a lower alkyl group having 1 to 5 carbon atoms with respect to a water-soluble and / or water-insoluble lignin secondary derivative obtained by alkali treatment of a primary derivative of lignin obtained by adding an acid to the contained material and mixing. A lignin tertiary derivative obtained by carboxyalkylation with an alkyl group,
An optic neuroprotective agent comprising one or more lignin derivatives selected from the group consisting of as active ingredients.   The protective agent according to claim 12, wherein the optic nerve includes retinal ganglion cells.   A nutritional supplement containing the optic neuroprotective agent according to claim 12 or 13.   A prophylactic / therapeutic agent for eye diseases, comprising the optic neuroprotective agent according to claim 12 or 13.