CN107708717B - Application of rhinacanthin quinone C as nerve cell apoptosis inhibitor - Google Patents
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Abstract
The invention provides a nerve cell apoptosis inhibitor, an anti-Alzheimer disease agent, an anti-cerebral hypofunction agent and a medicine with anti-Alzheimer disease or anti-cerebral hypofunction, which contains the following components as effective components: "a predetermined fraction partitioned in dichloromethane when the ethanol extract of rhinacanthus nasutus is subjected to liquid-liquid partition with hexane and methanol and the fraction partitioned in methanol is subjected to liquid-liquid partition with dichloromethane and water", "a predetermined fraction as a fraction eluted from a silica gel column by an elution solvent when the predetermined fraction is subjected to silica gel column chromatography using the elution solvent composed of n-hexane, ethyl acetate and anhydrous methanol" or rhinacanthin C. The present invention provides a neuronal apoptosis inhibitor, an anti-alzheimer agent, an anti-cerebral function-reducing agent, and a pharmaceutical agent having an anti-alzheimer effect or an anti-cerebral function-reducing effect, each of which has an excellent inhibitory effect on neuronal apoptosis.
Description
Technical Field
The present invention relates to a neuronal apoptosis inhibitor, an anti-alzheimer agent, an anti-cerebral function-lowering agent, a pharmaceutical or food having an anti-alzheimer effect or an anti-cerebral function-lowering effect, and a method for producing the neuronal apoptosis inhibitor.
Background
With the rapid advancement of japan in recent years, the number of patients with dementia (dementia) has increased. It is expected that the number of patients with dementia will continue to increase in the future unless an epoch-making method for preventing and treating dementia is established. Therefore, a method for preventing and treating dementia is demanded not only from the medical point of view but also from the administrative point of view.
However, there are various diseases causing dementia, and three types of Alzheimer's disease, dementia with Lewy bodies and vascular dementia (vascular dementia) are considered to account for the majority of the diseases. Among them, since the number of patients with alzheimer's disease is large and fundamental prevention and treatment methods have not yet been established, various studies on alzheimer's disease have been conducted.
As a mechanism of alzheimer's disease, the amyloid cascade hypothesis (amyloid cascade hypothesis) is widely supported. The amyloid cascade hypothesis refers to the following hypothesis: amyloid β (amyloid β) is accumulated as amyloid protein (amyloid protein) in the brain, and as a result, Alzheimer's neurofibrillary changes, and death or shedding of nerve cells due to direct or indirect toxicity occurs, and various symptoms of dementia occur along with death or shedding of nerve cells. This hypothesis is widely supported at present and is a guideline in the development and study of a prophylactic method and a therapeutic method (see, for example, non-patent document 1).
Donepezil (donepezil), galantamine (galanthamine), rivastigmine (rivastigmine), and the like have been conventionally used for the treatment of alzheimer's disease (see, for example, non-patent document 2). According to the anti-Alzheimer's disease agent containing the above-mentioned substances as active ingredients, the development of dementia symptoms can be suppressed.
In addition, Rhinacanthus nasutus (L.) Kurz, also known as Rhinacanthus nasutus, is an evergreen dwarf shrub belonging to the acanthaceae family of Rhinacanthus genus, native to south german Plateau (Deccan Plateau) in india. The whole plant of rhinacanthus nasutus is known to have an insect-repellent, anti-inflammatory and antifungal activity against dermatophytes (see, for example, non-patent document 3), and is mainly used as a traditional Chinese medicine or food in china (taiwan), and recently also used as a traditional Chinese medicine or food in japan. In addition, prior applications filed by the present applicant disclose: rhinacanthus nasutus has an ability to remove active oxygen (for example, refer to patent document 1); has an effect of promoting excretion (see, for example, patent document 2); and an antiallergic effect (see, for example, patent document 3) and an antitumor effect (see, for example, patent document 4).
Patent document 1: japanese laid-open patent publication No. 9-143091
Patent document 2: japanese laid-open patent publication No. 9-169662
Patent document 3: japanese laid-open patent publication No. 2001-10964
Patent document 4: japanese patent laid-open publication No. 2002-53481
Non-patent document 1: experimental medicine, Vol.26No.16, No. 10 (2008), 2550, pp.10, published by Yangtze company, 10 (2008) and 1 (2008)
Non-patent document 2: the latest MEDICAL treatment of dementia, Vol.4No.2, No. 4/2014, pages 58-63, published by FUJI medicine, 4/25/2014
Non-patent document 3: herbal atlas of Mediterranean and Han dynasty in Probiotics, 492, Beilongwan, 1988
Disclosure of Invention
However, it is not known that a predetermined fraction, a predetermined fraction (fraction) or rhinacanthin C contained in rhinacanthin, which is obtained by a predetermined method from an extract of rhinacanthin, has a neuronal apoptosis inhibitory effect on the toxicity against amyloid β.
Accordingly, the technical object to be solved by the present invention is to provide a neuronal apoptosis inhibitor, an anti-alzheimer agent, an anti-cerebral function lowering agent, and a pharmaceutical or food having an anti-alzheimer agent or an anti-cerebral function lowering agent each having an excellent inhibitory activity on neuronal apoptosis. Further, it is an object of the present invention to provide a method for producing an apoptosis inhibitor for nerve cells, which can produce an apoptosis inhibitor for nerve cells having an excellent apoptosis inhibitory effect.
The present inventors have conducted intensive studies on a substance having an inhibitory effect on apoptosis against amyloid β, which is one of substances useful for the prevention or treatment of alzheimer's disease, and as a result, have found that a predetermined distribution obtained by a predetermined method from an extract of rhinacanthus nasutus, a predetermined fraction, and rhinacanthin C contained in rhinacanthin have an inhibitory effect on apoptosis, and have completed the present invention. The present invention is constituted by the following matters.
In the following description, unless otherwise specified, the solvent ratio is expressed by volume.
[1] An apoptosis inhibitor for nerve cells, characterized by comprising as active ingredients only the following:
performing liquid-liquid distribution of the ethanol extract of Rhinacanthus nasutus with hexane and methanol, performing liquid-liquid distribution of the distribution in methanol with dichloromethane and water,
a predetermined distribution as a distribution distributed in the dichloromethane.
[2] An apoptosis inhibitor for nerve cells, characterized by comprising as active ingredients only the following:
subjecting the ethanol extract of Rhinacanthus nasutus to liquid-liquid partition with hexane and methanol, subjecting the partition distributed in the methanol to liquid-liquid partition with dichloromethane and water, subjecting the partition distributed in the dichloromethane to silica gel column chromatography using an eluting solvent composed of n-hexane, ethyl acetate and anhydrous methanol,
as a prescribed component of the component eluted from the silica gel column by the eluting solvent.
[3] The neuronal apoptosis inhibitor according to the above [2], wherein the eluting solvent is a solvent consisting of n-hexane: ethyl acetate: anhydrous methanol ═ 3: 4: 2, in the solvent used for elution.
[4] The neuronal apoptosis inhibitor according to the above [3], wherein the predetermined component is a component eluted when the elution solvent is used in a volume 0.1 to 4 times the volume of the silica gel used for the silica gel column chromatography.
[5] The neuronal apoptosis inhibitor according to the above [4], wherein the predetermined component is a component that is eluted when the elution solvent is used in a volume of 0.2 to 2.5 times the volume of the silica gel used in the silica gel column chromatography.
[6] A neuronal apoptosis inhibitor characterized by containing only Rhinacanthus nasuta quinone C as an active ingredient.
[7] An anti-Alzheimer's disease agent characterized by containing the neuronal apoptosis inhibitor according to any one of the above-mentioned [1] to [6] as an active ingredient.
[8] An anti-cerebral hypofunction agent comprising the neuronal apoptosis inhibitor according to any one of the above [1] to [6] as an active ingredient.
[9] A pharmaceutical or food comprising the anti-Alzheimer's disease agent according to [7] or the anti-cerebral hypofunction agent according to [8], which has an anti-Alzheimer's disease activity or an anti-cerebral hypofunction activity.
[10] A method for producing an inhibitor of apoptosis of nerve cells, comprising the following steps in order:
1, extracting the rhinacanthus nasutus by using ethanol to obtain an ethanol extract;
a step 2 of subjecting the ethanol extract to liquid-liquid partition with hexane and methanol to obtain a partition distributed in the methanol; and
a 3 rd step of subjecting the partition material partitioned into methanol to liquid-liquid partition with methylene chloride and water to obtain a predetermined partition material of the partition material partitioned into methylene chloride,
and the neuronal apoptosis inhibitor contains only the prescribed distribution as an active ingredient.
[11] A method for producing an inhibitor of apoptosis of nerve cells, comprising the following steps in order:
1, extracting the rhinacanthus nasutus by using ethanol to obtain an ethanol extract;
a step 2 of subjecting the ethanol extract to liquid-liquid partition with hexane and methanol to obtain a partition distributed in the methanol;
a 3 rd step of subjecting the partition distributed in the methanol to liquid-liquid distribution using dichloromethane and water to obtain a partition distributed in the dichloromethane; and
a 4 th step of subjecting the partition material partitioned with the dichloromethane to silica gel column chromatography using an elution solvent composed of n-hexane, ethyl acetate and anhydrous methanol to obtain a predetermined fraction as a fraction eluted from the silica gel column by the elution solvent,
and the neuronal apoptosis inhibitor contains only the prescribed component as an active ingredient.
According to the present invention, it is also apparent from test examples described below that a neuronal apoptosis inhibitor, an anti-alzheimer agent, an anti-cerebral function lowering agent, and a pharmaceutical or food having an anti-alzheimer effect or an anti-cerebral function lowering effect, each having an excellent neuronal apoptosis inhibitory effect, can be provided.
Further, the present invention provides a method for producing a neuronal apoptosis inhibitor having an excellent inhibitory effect on neuronal apoptosis.
Further, regarding [1] above, for example, the following can be expressed: "use of an active ingredient as the predetermined partition for producing a neuroapoptosis inhibitor containing only the following predetermined partition as an active ingredient, wherein the predetermined partition is a partition distributed in dichloromethane when an ethanol extract of Rhinacanthus nasutus is subjected to liquid-liquid distribution using hexane and methanol and a partition distributed in the methanol is subjected to liquid-liquid distribution using dichloromethane and water. "
Further, the above [2] can be expressed as follows, for example: "use of an active ingredient as a predetermined component for producing a neuronal apoptosis inhibitor containing only the following predetermined component as an active ingredient, wherein an ethanol extract of Rhinacanthus nasutus is subjected to liquid-liquid partition with hexane and methanol, a partition distributed in the methanol is subjected to liquid-liquid partition with dichloromethane and water, and the partition distributed in the dichloromethane is subjected to silica gel column chromatography using an elution solvent composed of n-hexane, ethyl acetate and anhydrous methanol, as the predetermined component eluted from the silica gel column by the elution solvent. "
Further, the above [6] can be expressed as follows, for example: "use of the extract as an active ingredient of Rhinacanthus nasuta quinone C for producing a nerve cell apoptosis inhibitor containing only Rhinacanthus nasuta quinone C as an active ingredient. "
Further, the above [7] can be expressed as follows, for example: "use of the inhibitor of neuronal apoptosis according to any one of the above [1] to [6] as an active ingredient for producing an anti-Alzheimer's disease agent. "
Further, the above [8] can be expressed as follows, for example: "use of the inhibitor of neuronal apoptosis according to any one of the above [1] to [6] as an active ingredient for producing an anti-cerebral hypofunction agent. "
Further, the above [9] can be expressed as follows, for example: "use of an active ingredient of the anti-Alzheimer's disease agent of the above [7] or the anti-cerebral hypofunction agent of the above [8] for producing a pharmaceutical or food having an anti-Alzheimer's disease effect or an anti-cerebral hypofunction effect. "
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FIG. 1 is a flow chart showing the sequence of dispensing and fractionating a predetermined dispenser and a predetermined component from Rhinacanthus nasutus.
FIG. 2 is a flow chart showing the sequence of isolation of Rhinacanthus nasutus quinone C from Rhinacanthus nasutus.
FIG. 3 is a table showing the results of the test examples.
Detailed Description
The following describes a neuronal apoptosis inhibitor, an anti-alzheimer agent, an anti-cerebral hypofunction agent, a pharmaceutical or food having an anti-alzheimer effect or an anti-cerebral hypofunction, and a method for producing the neuronal apoptosis inhibitor of the present invention.
The invention relates to a nerve cell apoptosis inhibitor, which only contains the following components as effective components: the present invention provides a method for producing an apoptosis inhibitor for nerve cells, which comprises the steps of, in the case of liquid-liquid partition of an ethanol extract of rhinacanthus nasutus with hexane and methanol and liquid-liquid partition of a partition distributed in methanol with dichloromethane and water, the steps of: 1, extracting the rhinacanthus nasutus with ethanol to obtain an ethanol extract; a 2 nd step of subjecting the ethanol extract to liquid-liquid partition with hexane and methanol to obtain a partition distributed in the methanol; and (3) performing liquid-liquid partition of the partition distributed in methanol with dichloromethane and water to obtain a predetermined partition distributed in the dichloromethane, wherein the neurocyte apoptosis inhibitor is obtained by containing only the predetermined partition as an active ingredient.
The distribution defined in the present invention can be obtained under various conditions as long as the described conditions are satisfied. Hereinafter, usable methods and the like will be described.
In addition, in the present specification, "a distribution" means a purified product and can be obtained by liquid-liquid distribution.
In the present specification, "containing only a predetermined distribution as an active ingredient" means that the predetermined distribution is contained only as an active ingredient having an apoptosis inhibitor action, and does not deny the component (various solvents, auxiliary additives, or the like) other than the active ingredient.
The rhinacanthus nasutus can be used as an extraction raw material by collecting leaves, stems, roots, flowers, and the like of rhinacanthus nasutus as a raw material at an appropriate time and then subjecting the collected leaves, stems, roots, flowers, and the like to a drying step such as ordinary air drying. The Rhinacanthus nasutus as the extraction material is preferably pulverized or chopped.
Extraction with ethanol can be performed by a known extraction method generally used in industry and experiments (for example, refer to "FIGHT MEDICINE, vol. 16, pages 929 to 934, 2009"). The ethanol extract of Rhinacanthus nasutus can be obtained by the extraction.
In the present specification, the term "ethanol" refers to a "solvent containing ethanol as a main component (containing 50% or more of ethanol)". That is, "ethanol" includes not only so-called anhydrous ethanol but also a mixed solvent of ethanol and other solvents. In the case of extraction from rhinacanthus nasutus, a mixed solvent of ethanol and water (hydrous ethanol) is preferably used, and particularly 90% ethanol is more preferably used.
The extraction temperature is usually 0 to 100 ℃, preferably 5 to 50 ℃. When the extraction is performed under the condition that the solvent is boiling, the extraction is preferably performed while refluxing the solvent. The extraction time is about 1 hour to 10 days, and the solvent amount is 1 to 30 times of the weight of the raw material per time of unit drying, preferably 5 to 10 times of the weight. The extraction may be performed by stirring or by dipping alone. The extraction operation may be repeated as many times as necessary. Moreover, extraction operations under different conditions may also be combined.
Further, it is preferable to perform an operation of removing insoluble residues (insoluble residues) from the crude extract obtained in the above-described operation by filtration or centrifugation.
The liquid-liquid distribution can be performed by a known method generally used in industry and experiments.
Liquid-liquid partitioning is a method also called two-phase partitioning, two-phase solvent partitioning method, liquid-liquid extraction, etc., and is a method of recovering a target compound by a difference in solubility using 2 types of solvents different in polarity.
Preferably, after liquid-liquid partitioning, the solvent is removed to obtain a dry solid state of the partition. However, the case where the solvent is not required to be removed for the purpose of use is not limited thereto.
In the present specification, the term "methanol" refers to a "solvent containing methanol as a main component (containing 50% or more of methanol)". That is, "methanol" includes not only so-called anhydrous methanol but also a mixed solvent of methanol and other solvents. In the liquid-liquid distribution, a mixed solvent of methanol and water is preferably used, and 90% methanol is particularly preferred. On the other hand, the term "anhydrous methanol" as used herein means anhydrous methanol and does not include a mixed solvent containing methanol.
Can be obtained by a "method for producing an inhibitor of apoptosis of nerve cells comprising only a predetermined distribution as an active ingredient".
The "a neuronal apoptosis inhibitor" of the present invention contains only the following components as active ingredients: a process for producing a neuronal apoptosis inhibitor, which comprises the steps of subjecting an ethanol extract of Rhinacanthus nasutus to liquid-liquid partition with hexane and methanol, subjecting a partition material partitioned with methanol to liquid-liquid partition with dichloromethane and water, and subjecting the partition material partitioned with dichloromethane to silica gel column chromatography using an eluting solvent comprising n-hexane, ethyl acetate and anhydrous methanol, wherein a predetermined fraction of the fraction eluted from the silica gel column by the eluting solvent is allowed to pass through the column chromatography, the process comprising the following steps: 1, extracting the rhinacanthus nasutus with ethanol to obtain an ethanol extract; a 2 nd step of subjecting the ethanol extract to liquid-liquid partition with hexane and methanol to obtain a partition distributed in the methanol; a 3 rd step of subjecting the partition distributed in the methanol to liquid-liquid distribution using methylene chloride and water to obtain a partition distributed in the methylene chloride; subjecting the partition partitioned with dichloromethane to silica gel column chromatography using an elution solvent composed of n-hexane, ethyl acetate and anhydrous methanol to obtain a predetermined fraction of the fraction eluted from the silica gel column by the elution solvent, and the neuronal apoptosis inhibitor is obtained by containing the predetermined fraction as an active ingredient.
The components specified in the present invention can be obtained under various conditions as long as the conditions described are satisfied. The following describes methods and the like that can be used.
In addition, in the present specification, "fraction" refers to a purified substance and a substance obtained by fractionation by column chromatography (column chromatography).
In the present specification, "containing only a predetermined component as an active ingredient" means that only a predetermined component is contained as an active ingredient having an apoptosis inhibitor action, and that a component other than the active ingredient (various solvents, auxiliary additives, and the like) is not negatively contained.
Since the description has already been made with respect to the extraction and the liquid-liquid distribution, the description thereof will be omitted.
Preferably, after silica gel column chromatography, the eluting solvent is removed to give the components in a dry solid state. However, the case where the solvent is not required to be removed for the purpose of use is not limited thereto.
The silica gel column chromatography can be carried out by a known method generally used in industrial and experimental fields.
The elution solvent is preferably a mixture of n-hexane: ethyl acetate: anhydrous methanol ═ 3: 4: 2 (see experimental examples described later). In this case, the predetermined component is preferably a component eluted when the elution solvent is used in an amount of 0.1 to 4 times the volume of the silica gel used for the silica gel column chromatography, and more preferably a component eluted when the elution solvent is used in an amount of 0.2 to 2.5 times the volume of the silica gel used for the silica gel column chromatography (see test examples described later).
The predetermined distribution and the predetermined components may be obtained by further performing a purification method other than the above-described method, as long as the technical problem to be solved by the present invention is not impaired.
It is considered that the solvent and the mixed solvent used for obtaining the predetermined distribution and the predetermined component may be replaced by a solvent or a mixed solvent having the same polarity. In this case, if the solvent is used, a predetermined distribution and a predetermined component can be obtained. Examples of the solvent include: water, various alcohols, esters such as ethyl acetate, various linear, branched, and cyclic hydrocarbons, methylene chloride (dichloromethane), chloroform (chloroform), acetone (acetone), various ethers, and the like, and a solvent suitable for the method and purpose can be selected from among usable solvents and used.
In addition, it is considered that the distribution and the predetermined components defined in the present invention contain rhinacanthin C as 1 component, when considered from the results of the experimental examples described later.
The rhinacanthin C in the "neuronal apoptosis inhibitor containing rhinacanthin C as an active ingredient" of the present invention may be obtained by extracting and purifying rhinacanthin C from a raw material (see experimental examples described later), or may be obtained from other plants. Furthermore, the rhinacanthin C can also be obtained by synthesis.
Rhinacanthin C is a compound represented by the following chemical formula (1).
The rhinacanthin C of the present invention is not limited as long as it has an action mechanism in vivo, and may be in the form of a pharmaceutically acceptable salt before administration.
The predetermined distribution, the predetermined components and the rhinacanthin C have the effect of inhibiting apoptosis of nerve cells caused by toxicity of amyloid β as shown in experimental examples described later. Therefore, it is considered that the neuronal apoptosis inhibitor of the present invention containing a predetermined distribution, a predetermined composition and rhinacanthin C as active ingredients has an effect as an anti-alzheimer agent and an anti-cerebral hypofunction agent.
The term "anti-alzheimer agent" as used herein means a substance that can be used for at least one of prevention and treatment of alzheimer. The "anti-cerebral hypofunction agent" is a substance that can be used for at least one of prevention and treatment of cerebral hypofunction.
The administration route of the neuronal apoptosis inhibitor, the anti-brain function lowering agent, the anti-alzheimer agent and the pharmaceutical (here, the pharmaceutical for internal use or oral administration) of the present invention is not particularly limited, and examples thereof include: gastrointestinal tract administration such as oral administration and rectal administration, mucosal administration such as nasal administration, and injection administration such as intravenous administration and subcutaneous administration. The dosage form may be any suitable dosage form for any administration method, and examples thereof include: solid dosage forms such as tablet, powder, fine granule, capsule, powder, pill, buccal tablet, etc.; liquid dosage forms of solutions, suspensions, emulsions, syrups, injections, and the like; gel-like preparations, and the like. The neuronal apoptosis inhibitor, the anti-brain function lowering agent, the anti-alzheimer agent and the pharmaceutical agent of the present invention may be administered as they are, or may be administered together with a pharmacologically acceptable excipient (excipient). As the excipient, any of monosaccharides (monosachhoride), disaccharides (disacchoride), polysaccharides (polysaccharide), inorganic salts, oils and fats, distilled water, and the like can be used as the excipient which can be generally used for a preparation. When formulated, additives such as binders, lubricants, dispersants, suspending agents, emulsifiers, diluents, buffers, antioxidants, bacteriostats, and the like may be used.
The effective dose of the neuronal apoptosis inhibitor, the anti-brain function lowering agent, the anti-alzheimer agent and the pharmaceutical product of the present invention varies depending on the administration route, the dosage form, the disease symptoms, the age of the subject, and the like, and it is considered that the prescribed distribution, the prescribed fraction or the rhinacanthin C is usually 0.1 to 1000mg, preferably 0.5 to 300mg, and more preferably 1 to 100mg per day for an adult. The content of the predetermined fraction, the predetermined component or the rhinacanthin C can be set to an effective component content in the preparation most suitable for each administration form, based on the form of the preparation, the effective dose, and data as the dose of the preparation.
The pharmaceutical composition of the present invention may be an external pharmaceutical composition. The form of the external pharmaceutical preparation is not particularly limited.
Examples of the form of the external pharmaceutical preparations include ointments, creams, pastes (cataplasms), patches, and external preparations. The external pharmaceutical preparation of the present invention may contain various pharmaceutical ingredients as required in a predetermined distribution, a predetermined composition, or rhinacanthin C. Further, additives such as binders, dispersants, suspending agents, emulsifiers, diluents, buffers, antioxidants, bacteriostats, and the like may also be used.
Examples of the food of the present invention include foods in the form of tea containing an active ingredient and foods in the form of processed foods containing an active ingredient.
The tea is preferably used by mixing with dried leaves, stems or roots of Rhinacanthus nasutus, or with other tea materials. As the tea raw material, any tea raw material can be used as long as it is a substance that can be generally used as tea, such as green tea, oolong tea, puer tea, black tea, decocted tea, brown rice tea, eucommia tea, persimmon leaf tea, mulberry leaf tea, and the like. In addition, dried leaves, stems or roots of rhinacanthus nasutus can be used by baking the same as other tea materials.
The form of the food containing the active ingredient may be any form as long as it is a form that can be usually provided as a food, such as a beverage, jelly, biscuit, tablet, pill, soft capsule, hard capsule, powder, fine granule, or granule. Additives such as excipients, binders, lubricants, dispersants, suspending agents, emulsifiers, diluents, buffers, antioxidants, and bacteriostatic agents can also be used as auxiliary materials for foods.
The effective intake amount of the food of the present invention varies depending on the form of intake, the health condition of the subject, the age of the subject, and the like, and it is considered that the predetermined distribution, the predetermined component, or the rhinacanthin C is generally 0.1 to 1000mg, preferably 0.5 to 300mg, and more preferably 1 to 100mg per day for an adult.
The content of the prescribed distribution, the prescribed ingredient or the rhinacanthin C in the food of the present invention is generally 0.0001 to 1 wt%, preferably 0.001 to 0.5 wt%, more preferably 0.01 to 0.1 wt%, depending on the form of the food.
Examples
The present invention will be described in further detail below with reference to examples of distribution, fractionation and isolation of a predetermined distribution, a predetermined fraction, and rhinacanthin C, or test examples relating to an apoptosis inhibitory effect. The following experimental examples are specific examples, but the present invention is not limited to the following experimental examples.
1. Partition/fractionation/isolation of partition of the present invention, component of the present invention, and example of partition/fractionation/isolation of Rhinacanthus nasutus quinone C
The partitioning and fractionation of the partition of the present invention and the components of the present invention are carried out according to the flow shown in FIG. 1.
First, 2kg of dried root of Rhinacanthus nasutus (L.) Kurz was prepared as a raw material, and pulverized with a general-purpose grinder (grind).
Subsequently, extraction was performed with 90% ethanol. The extraction is carried out by immersing Rhinacanthus nasutus in 20L of 90% ethanol for 3 days, and then refluxing the solvent for 1 hour. The extract was separated from the extraction residue by filter paper. The same raw materials were subjected to the extraction 3 times in total, and the solvent was removed from the extract under reduced pressure to obtain 77.82g of a dry solid ethanol extract.
After removing 4g of the ethanol extract as a sample for analysis, 73.82g of the total amount of the remaining ethanol extract was subjected to liquid-liquid partition using hexane and 90% methanol. First, 500mL of 90% methanol was added to the ethanol extract, followed by 500mL of hexane and shaking for 1 minute. Wait about 10 minutes until the solvent separates and collect the 90% methanol phase. The same operation was further repeated twice, and the obtained 90% methanol phases were combined, and then the solvent was removed under reduced pressure to obtain 54.32g of a dry solid partition.
After 4g of the above fraction was removed as a sample for analysis, 50.32g of the total amount of the remaining fraction was subjected to liquid-liquid partition using methylene chloride and water. First, 500mL of water (purified water) was added to the partition, followed by 500mL of methylene chloride and shaking for 1 minute. Wait about 10 minutes until the solvent separates and collect the dichloromethane phase. The same operation was further repeated twice, and the obtained methylene chloride phases were combined, and then the solvent was removed under reduced pressure to obtain 27.55g of a predetermined partition material. In the following description, the dispenser obtained here is referred to as "predetermined dispenser a".
Then, 20.00g of the fraction A thus obtained was subjected to silica gel column chromatography.
First, 20.00g of a prescribed distribution A was mixed with 60g of silica gel (general purpose articles of 0.063 to 0.2 mm). Thereafter, a column (open column) filled with 500g of silica gel (0.040 to 0.063mm common product) was prepared, the column was equilibrated with an elution solvent (n-hexane: ethyl acetate: anhydrous methanol: 3: 4: 2), and a mixture of a predetermined fraction a and silica gel was loaded and eluted with the elution solvent. After 1500mL of an elution solvent was used, 500mL of acetone and 500mL of absolute ethanol were passed through a column in this order to elute the silica gel-adsorbed material.
In silica gel column chromatography, each about 20mL was collected in test tubes, and the pattern (pattern) eluted by TLC (thin-layer chromatography) was observed. According to the rough elution pattern, the substance obtained by passing through 1 st to 6 th test tubes is used as a component 1-1 (the solvent amount is about 0 to 120mL), the substance obtained by passing through 7 th to 24 th test tubes is used as a component 1-2 (the solvent amount is about 120 to 480mL), the substance obtained by passing through 25 th to 39 th test tubes is used as a component 1-3 (the solvent amount is about 480 to 780mL), the substance obtained by passing through 40 th to 70 th test tubes is used as a component 1-4 (the solvent amount is about 780 to 1400mL), and the substance obtained by passing through the rest of the elution solvent, acetone and absolute ethyl alcohol is used as a component 1-5.
Further, the above components 1-1 to 1-4 are specified components.
Isolation of rhinacanthin C was carried out according to the scheme shown in FIG. 2. Since the method for obtaining the predetermined fraction a is different from the method for silica gel column chromatography, only the differences from the above-mentioned methods will be described.
First, 2.50g of a prescribed distribution A was mixed with 10g of silica gel (general-purpose articles of 0.063 to 0.2 mm). Thereafter, a column (hollow column having a diameter of 2cm, a length of 30cm, and a volume of about 60 mL) filled with 200g of silica gel (general-purpose product of 0.040 to 0.063 mm) was prepared, and the column was equilibrated with an elution solvent (n-hexane: ethyl acetate: 4: 1), and then a predetermined mixture of the fraction a and silica gel was loaded and eluted with the elution solvent. Further, the amount of the elution solvent was 500 mL.
In silica gel column chromatography, the fractions were collected in test tubes each about 10mL, and the pattern eluted by TLC was observed. According to the rough elution pattern, the fraction obtained in the 1 st test tube was designated as fraction 2-1 (about 0 to 10mL of solvent), the fraction obtained in the 2 nd to 3 rd test tubes was designated as fraction 2-2 (about 10 to 30mL of solvent), and the fraction obtained in the 4 th to 10 th test tubes was designated as fraction 2-3 (about 30 to 100mL of solvent).
Then, regarding the components 2-1 to 2-3, generalWill for a long time1HNMR and13the data (data) of CNMR was compared with literature values (Journal of Natural Products, volume 59, pages 808-811, 1996) and structural analysis was performed, confirming that component 2-2 was Rhinacanthus nasutus quinone C. The obtained rhinacanthin C content is 444.2 mg.
Further, a JEOL JNM-GSX500 type nuclear magnetic resonance spectrometer (nuclear magnetic resonance spectrometer) (manufactured by Japan Electron Ltd.) was used as the nuclear magnetic resonance spectrometer.
2. Specified distribution, specified composition and test example relating to inhibitory action of Agrimonoquinone C on apoptosis of nerve cells
In this test example, experiments were conducted on cytotoxicity caused by amyloid β and nerve cell apoptosis inhibitory effects of a predetermined distribution, a predetermined composition and rhinacanthin C. Amyloid β (25 to 35), which is known as the center of action of neurotoxicity of amyloid β, was used in the evaluation of the neurotoxicity of amyloid β.
In this test example, PC12 cells (derived from rat adrenal pheochromocytoma cell line) widely used in tests for evaluating effects (toxicity or drug efficacy) on nerve cells were used. The PC12 cells used were obtained from BCRC (Bioresource Collection and Research Center: Center for biological resources preservation and Research, Taiwan).
The obtained PC12 cells were cultured at 37 ℃ and saturation humidity in the presence of 5% carbonic acid gas using RPMI-1640 medium (each obtained from Invitrogen Gibco) supplemented with 5% fetal bovine serum, 10% horse serum, 100units/ml penicillin (penicillin) and 100mg/ml streptomycin (streptomycin).
For the evaluation of the cytotoxicity of amyloid β (25-35), a 96-well microplate (microplate) coated with poly-L-lysine was used. Each well of the microplate was seeded (diseminate) 2X 104Mu.l of PC12 cells were cultured in the presence of 5% carbon dioxide gas at 37 ℃ under saturated humidity.
After 24 hours of incubation, 0.1% DMSO (j.t. baker) was added to the control (control), each sample was added to the evaluation zone (see below), and 10 μ M galantamine hydrobromide (galantamine hydrate), which is an anti-alzheimer agent and known to have neuroprotective effect, was added to the positive control (positive control), obtained from Sigma-Aldrich).
For all test zones, tests were performed with addition of amyloid β (25-35) (obtained by Sigma-Aldrich) at 15. mu.M and without addition of both (described later). The cell viability in each test area was calculated by measuring the absorbance at 570nm (absorbance) for 72 hours after the start of the culture by a predetermined method using MTT (Molecular Probes).
In this test example, the cell viability of the control, the positive control and each sample was first calculated in a state where amyloid β was not added. Next, the cell viability of each sample under the same conditions except that amyloid β was added was calculated. Then, the following calculation is performed: the cell survival rate in the control was subtracted from the value obtained by dividing the value of the cell survival rate in the case where the amyloid β was added by the value of the cell survival rate in the case where the amyloid β was not added by multiplying the value calculated by the calculation by 100 times in% as a correction rate for cell survival.
The cell survival correction rate is an index indicating that the cell survival rate was restored by several% as compared with the control in a state in which the influence of the sample on the cell survival rate was corrected.
A prescribed partition a (dichloromethane partition), component 1-1 as a prescribed component, component 1-2 as a prescribed component, component 1-3 as a prescribed component, component 1-4 as a prescribed component, and rhinacanthin C (component 2-2) were used as samples. In addition, a screening (screening) or the like relating to cytotoxicity is performed in advance with respect to the concentration of each sample to determine an appropriate concentration.
In addition, since the experiment relating to the positive control was performed separately from the experiment relating to each sample of the present invention, the numerical value of the cell survival rate in each control was different as shown in fig. 3.
The results of the test examples are shown in FIG. 3. Fig. 3(a) is a table showing test results relating to the positive control, and fig. 3(b) is a table showing test results relating to each sample of the present invention.
As shown in fig. 3, it was first found that amyloid β has high toxicity to PC12 cells (see the item of the control in fig. 3). Furthermore, it was found that the prescribed fraction A, the prescribed fraction (fractions 1-1 to 1-4) and the Rhinacanthus nasutus quinone C exhibited a high cell survival correction rate.
The cell survival correction rates achieved by components 1-1 and 1-2 are comparable to galanthamine hydrobromide.
Moreover, although the concentration was as low as 1. mu.M, the correction rate of cell survival achieved by Rhinacanthus nasutus quinone C was 30.2%. Since the correction rate for cell survival achieved by 10 μ M galanthamine hydrobromide was 17.2%, the fact that rhinacanthin C showed a particularly high correction rate for cell survival was found.
The above test examples confirm that the neuronal apoptosis inhibitor of the present invention has a good effect of inhibiting neuronal apoptosis caused by the toxicity of amyloid β, i.e., an excellent inhibitory effect of neuronal apoptosis. Therefore, the neuronal apoptosis inhibitor of the present invention containing the above-mentioned substance as an active ingredient is considered to have an effect as an anti-alzheimer agent and an anti-cerebral hypofunction agent.
[ examples ]
The following examples describe the preparation of pharmaceutical products and foods containing rhinacanthin C, which is an active ingredient of the neuronal apoptosis inhibitor of the present invention and is also an active ingredient of the anti-alzheimer agent or anti-cerebral hypofunction agent of the present invention. It is needless to say that the item of the rhinacanthin C may be changed to a predetermined distribution or a predetermined composition.
(1) Tablet formulation
Tablets were prepared using rhinacanthin C according to the following formulation.
(preparation method)
To lactose (95.8g), rhinacanthin C (0.2g), dried corn starch (2g), talc (1.8g), and calcium stearate (0.2g) were added and mixed. Next, tablets were made by a usual method using a single stroke tablet press (single stroke tablet press).
(2) Hard capsule
Hard capsules (360 mg per 1 capsule) were prepared using rhinacanthin C according to the following formulation.
(preparation method)
Lactose (220g) and corn starch (110g) were added to and mixed with rhinacanthin C (5g), and an aqueous solution of hydroxypropyl cellulose (25g) was added thereto and kneaded (kneading). Subsequently, pellets were produced by a usual method using an extrusion granulator. A hard capsule was prepared by filling the granules into a gelatin hard capsule (gelatin hard capsule).
(3) Soft capsule
Soft capsules (170 mg per 1 capsule) were prepared using Rhinacanthus nasutus quinone C according to the following formulation.
Rhinacanthus nasutus Quel C0.5 mg
Soybean oil 169.5mg
(preparation method)
To soybean oil (169.5g), rhinacanthin C (0.5g) was added and mixed. Next, soft capsules were manufactured by filling into soft capsules according to a conventional method using a rotary mold automatic molding machine.
(4) Pill preparation
A pill (100 mg per 1 capsule) was prepared using Rhinacanthus nasutus quinone C according to the following formulation.
(preparation method)
The raw materials were mixed and mixed as described above, and an appropriate amount of water was added to the mixture, and then a homogeneous kneaded mass was produced by a kneader, and the obtained kneaded mass was rolled, and then pelletized by a pelletizer (pelletizer) and dried to produce pellets.
(5) Powder preparation
Powder (1000 mg per 1 pack) was prepared from rhinacanthin C by the following method.
Rhinacanthus nasutus Quinq C1 mg
Lactose 799mg
Corn starch 200mg
(6) Jelly and its production process
Jelly (100g) was prepared from rhinacanthin quinone C by the following method according to the conventional recipe.
(preparation method)
The above ingredients were mixed and heated to 90 ℃. After confirming that the gelatin was dissolved, the container was filled with the gelatin and cooled. Jelly is made by solidifying gelatin.
(7) Ointment
Ointment (100g) was prepared from rhinacanthin C by the following general method.
(oil phase component)
(aqueous phase component)
(preparation method)
Heating the oil phase component and the water phase component to 80 deg.C respectively to make them uniform, adding the water phase into the oil phase while stirring, emulsifying, and cooling to obtain ointment.
(8) Patch preparation
Patch (100g) was prepared from rhinacanthin quinone C by the following method according to a conventional method.
(adhesive solvent)
(medicinal effective ingredient)
Rhinacanthus nasutus Quinq C0.1 g
Ethanol 5.0g
(percutaneous absorption enhancer)
Oleyl alcohol (oleyl alcohol) 0.8g
The total amount is 100g
(preparation method)
The adhesive solvent and the active ingredient are homogenized, and the active ingredient and the percutaneous absorption enhancer are added to the adhesive solvent and stirred at room temperature to prepare the composition. This composition was spread on a polyester film (polyester film) subjected to silicon treatment (silicone treatment), dried at 120 ℃ and cooled, and then the adhesive layer was transferred to a polyethylene film (polyethylene film) to prepare an adhesive patch.
Claims (3)
1. Use of a substance containing only agrimonoquinone C as an active ingredient for the preparation of a medicament having a neuronal apoptosis inhibitory effect on direct toxicity against amyloid β.
2. Use of a substance containing a neuronal apoptosis inhibitor as an active ingredient for the preparation of a medicament as an anti-alzheimer's disease agent, wherein the neuronal apoptosis inhibitor contains only agrimonoquinone C as an active ingredient and has a neuronal apoptosis inhibitory effect on direct toxicity to amyloid β.
3. Use of a substance containing a neuronal apoptosis inhibitor as an active ingredient for the preparation of a medicament as an anti-cerebral hypofunction agent, wherein the neuronal apoptosis inhibitor contains only agrimonoquinone C as an active ingredient and has a neuronal apoptosis inhibitory effect against direct toxicity to amyloid β.
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| PCT/JP2015/069363 WO2017006407A1 (en) | 2015-07-04 | 2015-07-04 | Nerve cell death inhibitor, anti-alzheimer's disease agent, brain hypofunction inhibitor, drug or food having anti-alzheimer's disease effect or brain hypofunction-inhibiting effect, and method for producing nerve cell death inhibitor |
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| CN (1) | CN107708717B (en) |
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| CN111671089A (en) * | 2020-06-24 | 2020-09-18 | 天津市泉又今生物科技有限公司 | Application of biological enhanced cell nutrient in preparation of anti-beta-amyloid protein cytotoxicity functional food |
| CN115385795B (en) * | 2022-10-11 | 2023-11-17 | 广西中医药大学赛恩斯新医药学院 | Preparation method and application of Rhinacanthus nasuta (L.) kurz naphthoquinone monomer Rhinacanthin-C |
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| CN103467293B (en) * | 2010-02-19 | 2015-06-10 | 株式会社安露莎 | Novel compound and application thereof in pharmaceuticals, food or cosmetics with anti-tumor function |
| CN102391277A (en) * | 2011-10-10 | 2012-03-28 | 南京泽朗医药科技有限公司 | Method for preparing high-purity rhinacanthin C |
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