WO2023072069A1 - 异野漆树苷及其衍生物用于促进神经修复的用途 - Google Patents

异野漆树苷及其衍生物用于促进神经修复的用途 Download PDF

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WO2023072069A1
WO2023072069A1 PCT/CN2022/127372 CN2022127372W WO2023072069A1 WO 2023072069 A1 WO2023072069 A1 WO 2023072069A1 CN 2022127372 W CN2022127372 W CN 2022127372W WO 2023072069 A1 WO2023072069 A1 WO 2023072069A1
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isorhoifolin
nerve
brain
use according
regeneration
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French (fr)
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陈令仪
陈裕仁
廖彣玲
王翊
李育棠
吕亭萱
李家维
陈方仪
焦传金
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陈令仪
陈裕仁
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Priority to CN202280071472.9A priority Critical patent/CN118215669A/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
    • C07H17/06—Benzopyran radicals

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  • the invention provides an application of isorhoifolin (Isorhoifolin) and its derivatives for promoting nerve repair.
  • Traumatic Brain Injury affect about 70 million people worldwide each year.
  • Common treatments include physical therapy, hyperbaric oxygen therapy, cranial magnetic stimulation, and cranial direct current stimulation. These non-invasive treatments can improve depression and cognitive function after TBI, but there are still no effective drugs that can promote nerve regeneration after brain injury.
  • Traumatic brain injury is an injury caused by external force hitting the brain. There are nearly 70 million confirmed cases worldwide every year. Traumatic brain injury can damage the cranial nerves, leading to loss of movement or cognitive function of the patient. Because the central nervous system is difficult to regenerate and restore after being damaged, there is currently no effective therapy for nerve regeneration in traumatic brain injury. Patients with traumatic brain injury often have brain lesions after a period of time and will cause brain degenerative diseases in the future. Therefore, the early use of drugs that promote brain nerve regeneration after brain injury is a therapeutic solution.
  • Isorhoifolin is a currently known compound, and the research on this compound as a drug for treatment is rare. Although it is seen that the composition containing this compound is used for the treatment of venous problems or hemorrhoids, but It is not intended for use in the treatment of nerve-related diseases. Therefore, further research in the present invention found that Isorhoifolin and its derivatives can effectively promote nerve repair and regeneration, and can penetrate the blood-brain barrier, which can be used to develop potential drugs for the treatment of traumatic brain injury.
  • the human nervous system is divided into central nervous system and peripheral nervous system, both of which are composed of neurons.
  • the trigeminal nerve is the cranial nerve in the peripheral nerves and connects to the pons (central nerve). Therefore, when Isorhoifolin and its derivatives of the present invention pass through the nasal mucosa, pass through the olfactory epithelial cells, enter the channels around the sense of smell and the trigeminal nerve, and enter the brain, they can also reach the systemic nerves through the peripheral nerves at the same time. Repair effect.
  • cranial nerves that are not easy to repair are used as experimental objects. In this way, both the central and peripheral nerves in the nervous system can be repaired.
  • the "central nervous system” consists of the brain and spinal cord.
  • the "central nervous system” described in the present invention includes but is not limited to the olfactory brain, amygdala, hippocampus, neocortex, lateral ventricle, superior optic thalamus, optic thalamus, hypothalamus, base optic thalamus, pituitary gland, pine Fruit body, third ventricle, midbrain tectum, peduncle, anterior part of the brain, cerebral aqueduct, pons, cerebellar medulla, spinal cord.
  • the "peripheral nervous system” consists of the somatic nervous system and the autonomic nervous system.
  • the "peripheral nerves” mentioned in the present invention include but are not limited to sensory nerves, motor nerves, cranial nerves, spinal nerves, sympathetic nerves, parasympathetic nerves, and enteric nervous system.
  • the invention provides an application of isorhoifolin (Isorhoifolin) and its derivatives for promoting nerve repair.
  • the isorhoifolin derivative is: rutin naringin (Narirutin).
  • the chemical structure of Isorhoifolin is as shown in chemical formula (1):
  • Isorhoifolin and its derivatives can promote 10-30% regeneration of injured hippocampal neurons (injured hippocampal neurons) and promote nerve regeneration in 3D brain tissue slices when used alone, proving The compound can effectively promote the regeneration of neurons and can be used to repair nerve damage.
  • isorhoifolin Isorhoifolin
  • its derivatives can penetrate the user's blood-brain barrier (blood-brain barrier) and enter the brain, so as to promote the repair of the damaged neurons.
  • isorhoifolin Isorhoifolin and its derivatives can penetrate the blood-brain barrier (blood-brain barrier) and enter the brain, so as to promote the repair and regeneration of cranial nerves or increase the number of nerves.
  • Isorhoifolin and its derivatives can promote the regeneration of post-injury cortical neurons, post-injury hippocampal neurons and post-injury retinal neurons.
  • Isorhoifolin has relatively low toxicity to Neuro2a cells.
  • the effective dosage concentration of isorhoifolin and its derivatives is 1 nM to 864 ⁇ M.
  • Isorhoifolin and its derivatives have significant effects on nerve repair, wherein the Isorhoifolin compound and its derivatives can penetrate the blood-brain barrier (blood-brain barrier) and enter the brain through nasal cavity administration , promote the repair of the damaged neurons; promote the repair and regeneration of cranial nerves or increase the number of nerves; and promote the nerve axis regeneration of cortical neurons and hippocampal neurons after injury.
  • blood-brain barrier blood-brain barrier
  • the Isorhoifolin compound and its derivatives can penetrate the blood-brain barrier (blood-brain barrier) and enter the brain through nasal cavity administration , promote the repair of the damaged neurons; promote the repair and regeneration of cranial nerves or increase the number of nerves; and promote the nerve axis regeneration of cortical neurons and hippocampal neurons after injury.
  • Fig. 1 is a flow chart of the present invention in vitro (in vitro) nerve repair test.
  • Fig. 2 is a reference schematic diagram for calculating the gap closure rate (gap closure rate) of the present invention.
  • 3A-3C are the experimental results of hippocampal nerve repair in vitro (in vitro) with isorhoifolin of the present invention.
  • Fig. 4 is the result of the hippocampal nerve regeneration experiment in vitro (in vitro) of Isorhoifolin derivative rutin naringin (Narirutin) of the present invention.
  • Fig. 5 is the result of cortical nerve regeneration experiment in vitro (in vitro) of Isorhoifolin derivative rutin naringin (Narirutin) of the present invention.
  • Fig. 6 is the experimental flow chart of the ex vivo brain tissue section of the present invention.
  • Fig. 7 is the result of the slice experiment of isorhoifolin (ex vivo) brain tissue of the present invention.
  • Fig. 8 is the result of an experiment on brain tissue slices of Isorhoifolin derivative rutin naringin (Narirutin) in vitro (ex vivo) of the present invention.
  • Fig. 9 is the result of the cytotoxicity test of the Isorhoifolin compound of the present invention.
  • Fig. 10 is a flow chart of the experiment of the present invention to control the model of cortical impact traumatic brain injury and the results of the experiment of Isorhoifolin compounds promoting the recovery of motor coordination after brain injury in mice.
  • Fig. 11 is a flow chart of the present invention promoting the repair of retinal nerve tissue in ex vivo.
  • Fig. 12 is the experimental result of Isorhoifolin of the present invention promoting the repair of retinal nerve tissue in ex vivo.
  • Embodiment 1 Isorhoifolin (Isorhoifolin) and its derivatives in vitro (in vitro) nerve repair test
  • the experimental procedure is shown in Figure 1.
  • the fetal rats of 18-day pregnant rats were taken out, and then the brains of the fetal rats were divided into cortex and hippocampus, and then the cortex and hippocampus were separated into cortical nerve cells. (cortical neurons) and hippocampal neurons (hippocampal neurons), the hippocampal neurons are planted in a 48-well plate, this day is called Day In Vitro 0 (DIV0), and Cytosine beta-D-arabinoside will be added at DIV2 (AraC) inhibits the proliferation of glia cells.
  • DIV0 Day In Vitro 0
  • Cytosine beta-D-arabinoside will be added at DIV2 (AraC) inhibits the proliferation of glia cells.
  • the nerve cells are scratched with a micropipette tip, and different concentrations of isorhoifolin (Isorhoifolin) are added. Seventy-two hours after the addition of isorhoifolin, nerve cells were labeled with TUJ1 antibody by immunofluorescence staining to observe the situation of nerve regeneration. This experiment was taken with a Zeiss Observer Z1 microscope.
  • Isorhoifolin isorhoifolin
  • the degree of nerve axis regeneration is quantified by the gap closure rate.
  • the white dotted line is the boundary created by the scratching of the nerve cells drawn by the tip of the micropipette, and the black part in the middle is the boundary produced by the micropipette. In the area scratched by the pipette tip, the regenerated neurites will grow from both sides of the dotted line to the middle.
  • the calculation method is to draw a line between the gaps (gap) every 50 ⁇ m in the injured area to calculate the width of the gap, and take the average after drawing a total of ten lines Value: the length of the gap between injured borders (Length of gap between injured borders, Lg), after a period of time in the same injury area, a line is drawn every 50 ⁇ m to connect the nerve axis extended by regeneration, the length is calculated, and the average value is taken after ten lines are drawn : The length of the gap between regenerated neurons (Ln), the gap closure rate is calculated by (Lg-Ln)/Lg, and the scale bar is 100 ⁇ m. This experiment was shot with Zeiss Observer Z1 microscope.
  • the white dotted line is the boundary generated by scratching the nerve cells with the tip of a micropipette, and the scale bar is 100 ⁇ m.
  • This experiment was taken with a Zeiss Observer Z1 microscope. From this experiment, it can be known that Isorhoifolin can promote the proliferation of neurite in the hippocampus.
  • 3A is the graph of the experimental results of Isorhoifolin from 3.5nM to 34.6nM
  • 3B is the graph of the experimental results of Isorhoifolin from 0.009 ⁇ M to 864 ⁇ M
  • 3C is the graph of the calculation results of the gap closure rate of different concentrations of Isorhoifolin.
  • Isorhoifolin Derivative Narirutin Promotes Nerve Regeneration in the Hippocampal Gyrus after Injury
  • the experimental procedure is also shown in Figure 1.
  • the fetuses of 18-day-pregnant rats were taken out, and then the brains of the fetuses were divided into cerebral cortex and hippocampus, and then separated into cortical nerve cells and hippocampal nerve cells. Nerve cells are planted in a 48-well dish. This day is called Day In Vitro 0 (DIV0).
  • DIV0 Day In Vitro 0
  • Cytosine beta-D-arabinoside (AraC) will be added to inhibit the proliferation of neuroglial cells.
  • a micropipette tip will be used Nerve cells were scratched, and 1nM and 10nM Narirutin were added respectively, and 0.1% DMSO was used as the solvent control group.
  • Isorhoifolin derivative rutin naringin (Narirutin) promotes post-injury cortical nerve regeneration in the cortex
  • the procedure of this experiment is also shown in Figure 1.
  • the fetuses of 18-day-pregnant rats were taken out, and then the brains of the fetuses were divided into cerebral cortex and hippocampus, and then separated into cortical nerve cells and hippocampal nerve cells.
  • the cells are planted in a 48-well plate, which is called Day In Vitro 0 (DIV0) on this day.
  • Cytosine beta-D-arabinoside (AraC) will be added to inhibit the proliferation of glial cells at DIV2, and the tip of a micropipette will be used at DIV8.
  • the nerve cells were scratched, and 10nM and 100nM Narirutin were added respectively, and 0.1% DMSO was used as the solvent control group. The results in the figure have been standardized with the 0.1% DMSO group. Seventy-two hours after adding Narirutin, nerve cells were labeled with TUJ1 antibody by immunofluorescence staining to observe the nerve regeneration. This experiment was taken with a Zeiss Observer Z1 microscope, and the scale bar is 100 ⁇ m.
  • Embodiment two Isorhoifolin and its derivatives ex vivo (ex vivo) brain tissue slice experiment
  • the present invention uses 3D brain tissue slice culture to observe the effect of compounds on nerves.
  • the experimental method is to take out the fetuses of 18-day-pregnant mother mice, then take out the brains of the fetuses and embed them in low-melting agarose gel, and then use Leica microtome VT100 slices to collect brains with a thickness of 350 ⁇ m. After slicing, use a scalpel to injure (injury), and plant it in an insert in a 6-well plate.
  • This culture method allows the brain slices to be in contact with medium and air at the same time.
  • brain slice culture is compared to in vitro Culture (in vitro culture) can more truly reflect the interaction and three-dimensional structure of nerve cells and other cells of the brain.
  • TUJ1 is antibody-labeled nerve cells
  • GFAP is antibody-labeled glial cells
  • DAPI is reagent-labeled cell nuclei.
  • the white dotted line marks the site of the scalpel injury
  • the left side of the dotted line is the brain slice tissue
  • the right side of the dotted line is the newborn nerve axis.
  • the effect of Isorhoifolin on promoting nerve regeneration can be quantified by calculating the length of the white dotted line and the area of the newborn nerve shaft on the right side of the white line, and dividing the area of the newborn nerve shaft by the length of the white dotted line to calculate the neurite length.
  • the scale bar is 100 ⁇ m
  • this experiment was taken with Zeiss LSM800confocal microscope.
  • the effect of Isorhoifolin on nerve regeneration was observed in ex vivo brain slices. From the experimental results, it can be known that Isorhoifolin has the effect of promoting nerve regeneration.
  • the experimental process of Isorhoifolin derivative rutin naringin is also shown in Figure 6.
  • the brains of fetal rats were taken out, and the brains were embedded in low-melting point agarose gel, and the Leica Microtome VT100 cut the brain into slices with a thickness of 350 ⁇ m, cultured after scratching the brain slices with a scalpel, and added ddH 2 O or Narirutin every day, and after 96 hours of culture, fluorescent immunostaining was used to label nerve cells with TUJ1 antibody , GFAP antibody labeled glial cells, DAPI reagent labeled cell nuclei, the scale bar of this experiment is 100 ⁇ m. This experiment was shot with a Zeiss LSM800 confocal microscope.
  • Embodiment three Isorhoifolin cytotoxicity test
  • CellTiter-Glo cell viability assay was used to detect the toxicity of Isorhoiofolin to Neuro2a cells.
  • CellTiter-Glo cell viability assay can detect the ATP content in cells, and then observe the survival rate of cells after adding compounds.
  • Embodiment four Isorhoifolin promotes the motor coordination recovery test after brain injury in mice
  • Pre-training was carried out at -5, -3, and -1Dpi (Day post injury), that is, 5, 3, and 1 days before the brain injury, and at 0Dpi, using controlled
  • the cortical impact model (control cortical impact traumatic brain injury model) performed brain injury on mice, and administered Isorhoifolin at 14 or 140 ⁇ g/kg by nasal administration at 0, 2, 4, 6, 8, 10, and 12 Dpi, and at 1 , 3, 6, 10, 13Dpi for horizontal bar experiments.
  • Embodiment five Isorhoifolin promotes the repair of isolated (ex vivo) retinal nerve tissue
  • Retinal explants were obtained from 8-day-old C57BL/6 mice, and the eyes were removed after sacrifice. Then, the eyeball was separated from the retina with forceps and micro scissors and removed. Remove the vitreous body, and finally cut the separated retina into four pieces, trim and scratch the edge of the tissue with micro scissors, culture each piece on a 18mm round glass slide, put it into a 12-well plate, and put it in the incubator , cultured at 5% CO 2 and 35°C for five days, and each piece of retinal tissue needed to be replaced with fresh culture medium every day and 9 ⁇ M or 90 ⁇ M Isorhoifolin was added.
  • the group given Isorhoifolin had a significant post-injury recovery effect compared with the group given only water (ddH 2 O).

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Abstract

一种异野漆树苷(Isorhoifolin)及其衍生物的新用途。该异野漆树苷(Isorhoifolin)及其衍生物可以用于促进受损脑神经元及视网膜神经的再生。

Description

异野漆树苷及其衍生物用于促进神经修复的用途 技术领域
本发明提供一种异野漆树苷(Isorhoifolin)及其衍生物用于促进神经修复的用途。
背景技术
目前神经损伤，像是创伤性脑损伤(TBI)，每年全球约有七千万人受此病症所扰。常见治疗方式为物理疗法、高压氧疗法、头颅磁刺激及头颅直流电刺激这种非侵入性疗法可以改善TBI后的忧郁和认知功能，但目前却仍没有有效的药物可以促进脑伤后神经再生。
创伤性脑损伤是因外力撞击脑部所产生的损伤，每年全球约有近7000万的确诊病例，创伤性脑损伤会损伤脑神经，进而导致患者行动或者认知上的功能缺失。由于中枢神经系统受损后很难再生复原，目前医学上对于创伤性脑损伤并无有效促进神经再生的疗法，脑外伤患者一段时间后常有脑部病变及日后造成脑神经退化疾病。因此，在脑伤后及早使用促进脑神经再生药物是治疗的解决办法。
不仅如此，在治疗药物的选择方面，由于大脑有血脑屏障，一般药物无法随意穿透血脑屏障达到有效剂量，因此于治疗脑部疾患时，有效药物投与便成为严重的问题。
有鉴于此，目前极需要研发出一种可治疗神经损伤的药物，除此 也极需一种可穿透血脑屏障的给药方式。
Isorhoifolin(异野漆树苷)为目前已知的化合物，该化合物做为药物进行治疗的研究并不多见，虽有看到含有该化合物的组合物用于治疗静脉问题或是痔疮的用途，但并无使用于治疗神经相关疾病的用途。因此本发明进一步研究发现Isorhoifolin及其衍生物可以有效的促进神经修复和再生，且能穿透血脑屏障，可用来开发用于治疗创伤性脑损伤的潜力药物。
发明内容
发明内容旨在提供本揭示内容的简化摘要，以使阅读者对本揭示内容具备基本的理解。此发明内容并非本揭示内容的完整概述，且其用意并非在指出本发明实施例的重要/关键组件或界定本发明的范围。
人体神经系统分为中枢神经和周边神经，两者均由神经元组成。其中三叉神经为周边神经中的脑神经且连接桥脑(中枢神经)。因此当本发明的异野漆树苷(Isorhoifolin)及其衍生物透过鼻腔黏膜，穿过嗅上皮细胞，进入嗅觉和三叉神经周围的通道，进入到大脑，亦可同时透过周边神经达到全身神经修复效果。
有鉴于此，在本发明中，以不易修复的脑神经作为实验对象。如此，以达到神经系统中的中枢神经和周边神经均可修复的效果。
“中枢神经”由脑和脊髓构成。本发明中所述的“中枢神经”是包含但不限于嗅脑、杏仁体、海马体、新皮质、侧脑室、上视丘、视丘、下视丘、底视丘、脑下垂体、松果体、第三脑室、中脑顶盖、大脑脚、脑盖前部、大脑导水管、桥脑、小脑延髓、脊髓。
“周边神经”由躯体神经系统和自主神经系统所组成。本发明中所述的“周边神经”是包含但不限于感觉神经、运动神经、脑神经脊神经、交感神经、副交感神经、肠神经系统。
本发明提供一种异野漆树苷(Isorhoifolin)及其衍生物用于促进神经修复的用途。
在一些实施例中，异野漆树苷(Isorhoifolin)衍生物为:芸香柚皮苷(Narirutin)。
在一些实施例中，异野漆树苷(Isorhoifolin)的化学结构如化学式(1)所示:
Figure PCTCN2022127372-appb-000001
在一些实施例中，异野漆树苷(Isorhoifolin)的衍生物芸香柚皮苷(Narirutin)的化学结构如化学式(2)所示:
Figure PCTCN2022127372-appb-000002
在一些实施例中，异野漆树苷(Isorhoifolin)及其衍生物在单独使用 下可以促进受伤的海马神经元(injured hippocampal neurons)10-30％的再生以及促进3D脑组织切片的神经再生，证明该化合物可有效的促进神经元的再生并且可用于修复神经损伤。
在一些实施例中，异野漆树苷(Isorhoifolin)及其衍生物可穿透该使用者的血脑屏障(blood-brain barrier)进入大脑，以促进该受损神经元之修复。
在一些实施例中，异野漆树苷(Isorhoifolin)及其衍生物可穿透血脑屏障(blood-brain barrier)进入大脑，以促进脑神经修复、再生或神经数量增加。
在一些实施例中，异野漆树苷(Isorhoifolin)及其衍生物可促进伤后皮质神经(cortical neurons)、伤后海马回神经(hippocampal neurons)以及伤后视网膜神经(retinal neurons)的再生。
在一些实施例中，异野漆树苷(Isorhoifolin)对于Neuro2a细胞毒性相当低。
在一些实施例中，异野漆树苷(Isorhoifolin)及其衍生物的有效剂量浓度为1nM至864μM。
在一些实施例中，异野漆树苷(Isorhoifolin)及其衍生物对于神经修复具有显著效果，其中该Isorhoifolin化合物及其衍生物经鼻腔给药可穿透血脑屏障(blood-brain barrier)进入大脑，促进该受损神经元的修复；促进脑神经修复、再生或神经数量增加；以及促进伤后皮质神经(cortical neurons)及海马回神经(hippocampal neurons)的神经轴再生。
在参阅下文实施方式后，本发明所属技术领域中具有通常知识者当可轻易了解本发明的基本精神及其他发明目的，以及本发明所采用的技术手段与实施态样。
附图说明
图1为本发明体外(in vitro)神经修复试验流程图。
图2为本发明gap closure rate(间隙闭合率)计算参考示意图。
图3A-3C为本发明异野漆树苷(Isorhoifolin)体外(in vitro)海马回神经修复实验结果。
图4为本发明Isorhoifolin衍生物芸香柚皮苷(Narirutin)体外(in vitro)海马回神经再生实验结果。
图5为本发明Isorhoifolin衍生物芸香柚皮苷(Narirutin)体外(in vitro)皮质神经再生实验结果。
图6为本发明离体(ex vivo)脑组织切片实验流程图。
图7为本发明异野漆树苷(Isorhoifolin)离体(ex vivo)脑组织切片实验结果。
图8为本发明Isorhoifolin衍生物芸香柚皮苷(Narirutin)离体(ex vivo)脑组织切片实验结果。
图9为本发明Isorhoifolin化合物细胞毒性实验结果。
图10为本发明控制皮质撞击脑外伤模型实验流程图及Isorhoifolin化合物促进小鼠脑伤后的运动协调性恢复实验结果。
图11为本发明促进离体(ex vivo)视网膜神经组织修复流程图。
图12为本发明Isorhoifolin促进离体(ex vivo)视网膜神经组织修复实验结果。
具体实施方式
为了使本揭示内容的叙述更加详尽与完备，下文针对了本发明的实施态样与具体实施例提出了说明性的描述；但这并非实施或运用本发明具体实施例的唯一形式。实施方式中涵盖了多个具体实施例的特征以及用以操作这些具体实施例的方法与用途，然而，亦可利用其他具体实施例来达成相同或均等的功效。应当理解，该些实施例系用于说明本发明，而非限制本发明的范围。
实施例一、:异野漆树苷(Isorhoifolin)及其衍生物体外(in vitro)神经修复试验
本实验流程如图1所示，将怀孕18天的大鼠的胎鼠取出，再将胎鼠的大脑分成大脑皮质(cortex)及海马回(hippocampus)，接着将cortex和hippocampus分离成皮质神经细胞(cortical neurons)以及海马回神经细胞(hippocampal neurons)后，将海马回神经细胞种在48孔盘中，这天称为Day In Vitro 0(DIV0)，在DIV2时会加入Cytosine beta-D-arabinoside(AraC)抑制神经胶细胞(glia cell)的增生，在DIV8时会用微量吸管尖对神经细胞进行划伤，并加入不同浓度之异野漆树苷(Isorhoifolin)。在加入Isorhoifolin的72小时后，利用免疫荧光染色法使用TUJ1抗体标记神经细胞进而去观察神经再生的情形。此实 验使用Zeiss Observer Z1 microscope进行拍摄。
如图2所示，通过间隙闭合率(gap closure rate)定量神经轴再生的程度，白色的虚线是微量吸管尖所划出的将神经细胞刮伤所产生的边界，中间黑色的部分是被微量吸管尖划伤的区域，再生的神经突会由虚线两侧向中间生长，计算方法为伤区每隔50μm在gap(间隙)之间拉一条线计算Gap的宽度，一共拉十条线之后取平均值:受伤边界间的间隙长度(Length of gap between injured borders,Lg)，一段时间后在同样的伤区每50μm拉一条线连接再生所延伸出的神经轴，计算长度，拉十条后取平均值:再生神经间的间隙长度(Length of gap between regenerated neurons,Ln)，将(Lg-Ln)/Lg后计算出gap closure rate，比例尺为100μm，此实验使用Zeiss Observer Z1 microscope进行拍摄。
如图3A-3C所示，白色虚线为利用微量吸管尖将神经细胞刮伤所产生的边界，比例尺为100μm，此实验使用Zeiss Observer Z1 microscope进行拍摄。由此实验可得知Isorhoifolin可促进海马回神经突增生。其中3A为3.5nM至34.6nM的Isorhoifolin实验结果图；3B为0.009μM至864μM的Isorhoifolin实验结果图；3C为不同浓度的Isorhoifolin间隙闭合率计算结果图。
异野漆树苷(Isorhoifolin)的衍生物芸香柚皮苷(Narirutin)促进伤后海马回神经再生实验
本实验流程同样如图1所示，将怀孕18天的大鼠的胎鼠取出，再将胎鼠的大脑分成大脑皮质及海马回，接着分离成皮质神经细胞以及 海马回神经细胞，将海马回神经细胞种在48孔盘中，这天称为Day In Vitro 0(DIV0)，在DIV2时会加入Cytosine beta-D-arabinoside(AraC)抑制神经胶细胞的增生，在DIV8时会用微量吸管尖对神经细胞进行划伤，并加入分别加入1nM和10nM Narirutin，0.1％DMSO为溶剂控制组，图中结果皆已与0.1％DMSO组别进行标准化。在加入Narirutin的72小时后，利用免疫荧光染色法使用TUJ1抗体标记神经细胞进而去观察神经再生的情形。此实验使用Zeiss Observer Z1 microscope进行拍摄，比例尺为100μm。
如图4所示，通过间隙闭合率(gap closure rate)定量神经轴再生的程度，此实验结果表示芸香柚皮苷(Narirutin)可有效的促进海马回神经突增生。
异野漆树苷(Isorhoifolin)的衍生物芸香柚皮苷(Narirutin)皮质促进伤后皮质神经再生实验
本实验流程同样如图1所示，将怀孕18天的大鼠的胎鼠取出，再将胎鼠的大脑分成大脑皮质及海马回，接着分离成皮质神经细胞以及海马回神经细胞，将皮质神经细胞种在48孔盘中，这天称为Day In Vitro 0(DIV0)，在DIV2时会加入Cytosine beta-D-arabinoside(AraC)抑制神经胶细胞的增生，在DIV8时会用微量吸管尖对神经细胞进行划伤，并加入分别加入10nM和100nM Narirutin，0.1％DMSO为溶剂控制组，图中结果皆已与0.1％DMSO组别进行标准化。在加入Narirutin的72小时后，利用免疫荧光染色法使用TUJ1抗体标记神经细胞进而去观察神经再生的情形。此实验使用Zeiss Observer Z1  microscope进行拍摄，比例尺为100μm。
如图5所示，通过间隙闭合率(gap closure rate)定量神经轴再生的程度，此实验结果表示芸香柚皮苷(Narirutin)可有效的促进皮质神经突增生。
实施例二:Isorhoifolin及其衍生物离体(ex vivo)脑组织切片实验
在in vitro中测试化合物对于神经再生的效果后，也试着在ex vivo(立体)的情况下观察化合物对于神经再生的效果。如图6所示，本发明使用了3D脑组织切片培养的方式观察化合物对于神经的效果。实验方法为将怀孕18天的母鼠的胎鼠取出，再将胎鼠的大脑取出后用低熔点琼脂糖胶(low-melting agarose gel)包埋，再用Leica microtome VT100切片收取厚度350μm的脑切片后用手术刀进行划伤(injury)，并将其种在6孔盘中的insert中，这个培养方式可以使脑切片同时接触介质(medium)和空气，同时脑切片培养相比于in vitro culture(体外培养)可以较为真实的反映神经细胞与大脑其他细胞的交互作用以及立体结构。每天加ddH 2O或加异野漆树苷(Isorhoifolin)，培养96小时后利用荧光免疫染色法使用TUJ1抗体标记神经细胞、GFAP抗体标记神经胶细胞、DAPI试剂标记细胞核。
如图7所示，TUJ1为抗体标记神经细胞、GFAP为抗体标记神经胶细胞、DAPI为试剂标记细胞核。白色虚线标示出手术刀injury的部位，虚线左侧为脑切片组织，虚线右侧则为新生的神经轴。可通过计算白色虚线的长度以及白线右侧新生神经轴的面积，将新生神经轴 面积除以白色虚线长度进而计算出神经突长度(neurite length)，以此来定量Isorhoifolin对于促进神经再生的效果，比例尺为100μm，此实验使用Zeiss LSM800confocal microscope进行拍摄。由ex vivo的脑切片中观察Isorhoifolin对于神经再生的效果，从实验结果可得知Isorhoifolin具有促进神经再生的效果。
异野漆树苷(Isorhoifolin)的衍生物芸香柚皮苷(Narirutin)实验流程同样如图6所示，将大鼠胎鼠的脑取出，并将脑包埋在低熔点琼脂糖胶中，利用Leica microtome VT100将脑切成厚度为350μm的切片组织，在以手术刀划伤脑切片后进行培养，并每天加ddH 2O或加Narirutin，培养96小时后利用荧光免疫染色法使用TUJ1抗体标记神经细胞、GFAP抗体标记神经胶细胞、DAPI试剂标记细胞核，此实验比例尺为100μm。此实验使用Zeiss LSM800 confocal microscope进行拍摄。
如图8所示，从ex vivo(离体)的脑切片中观察Narirutin对于神经再生的效果，从实验结果可得知Narirutin具有促进神经再生的效果。
实施例三:Isorhoifolin细胞毒性实验
利用CellTiter-Glo cell viability assay检测异野漆树苷(Isorhoiofolin)对于Neuro2a细胞的毒性。CellTiter-Glo cell viability assay可侦测细胞中ATP的含量，进而观察细胞加入化合物后的存活率。
如图9所示，结果可知Isorhoifolin和对于Neuro2a细胞的IC50大 约在11.616mM的范围，对于细胞的毒性相当低。
实施例四:Isorhoifolin促进小鼠脑伤后的运动协调性恢复试验
利用单杠(horizontal bar)实验，将小鼠放置在离地高49cm，长38cm，直径2mm、4mm、6mm的黄铜棒上，让小鼠用前爪抓住黄铜棒，通过计算小鼠在黄铜棒上待的时间以及是否到达黄铜棒末端的平台上，来评估小鼠运动协调性的能力。评估标准如下:1-5秒为1分，5-10秒为2分，10-20秒为3分，20-30秒为4分，30秒以上或到达平台为5分。
本实验实验模型如图10所示，分别在-5、-3、-1Dpi(Day post injury)，即脑伤前5,3,1天进行pre-training(预训练)，并在0Dpi，利用controlled cortical impact model(控制皮质撞击脑外伤模型)对小鼠进行脑伤，并在0、2、4、6、8、10、12Dpi利用鼻腔给药的方式给予14或140μg/kg的Isorhoifolin，并在1、3、6、10、13Dpi进行horizontal bar的实验。
由图10可看出，在脑伤后仅给予水(ddH 2O)的组别有运动协调性的丧失，而在给予Isorhoifolin的组别中，运动协调性与Sham(假手术组)组别相似，明显促进运动协调性的恢复。
实施例五:Isorhoifolin促进离体(ex vivo)视网膜神经组织修复
实验设计及流程如图11所示，视网膜组织(retinal explants)取自于出生8天之C57BL/6品系小鼠，牺牲后取下其眼睛，接着以镊子及 显微剪将眼球与视网膜分离并去除玻璃体，最后将分离后的视网膜剪成四瓣，并以显微剪将组织边缘进行修剪及划伤，每瓣培养于18mm圆形玻片，并放入12孔盘中，放入培养箱，以5％CO 2及35℃条件下培养五天，每片视网膜组织每天都需更换新鲜培养液并加入9μM或90μM的Isorhoifolin。视网膜组织培养至第五天时，以0.1％Glutaraldehyde solution及4％paraformaldehyde混和溶液于室温固定(Fix)一小时，并利用初级抗体axonal marker beta-III-tubulin(TUJ1)及DAPI进行染色，标记神经细胞和细胞核，再以超解析正立式共轭焦显微镜(LSM-800,Carl Zeiss)拍摄，利用图片分析程序ImageJ，圈选组织边界计算周长以及组织外之神经纤维面积，以神经纤维面积除以组织边界周长，进而得出单位周长神经纤维长度。
如图12所示，视网膜神经(retinal neurons)伤后，给予Isorhoifolin的组别相较于仅给予水(ddH 2O)的组别有着明显的伤后回复效果。
上述实验数据为在特定条件下所获得的初步实验结果，其仅用以易于了解或参考本发明的技术内容而已，其尚需进行其他相关实验。该实验数据及其结果并非用以限制本发明的权利范围。
前述较佳实施例仅举例说明本发明及其技术特征，该实施例的技术仍可适当进行各种实质等效修饰及/或替换方式予以实施；因此，本发明的权利范围权利要求书所界定的范围为准。

Claims (8)

  1. 一种异野漆树苷(Isorhoifolin)及其衍生物在用于制备治疗神经损伤的医药品中的用途。
  2. 如权利要求1所述的用途，其特征是，所述异野漆树苷(Isorhoifolin)衍生物为芸香柚皮苷(Narirutin)。
  3. 如权利要求1所述的用途，其特征是，所述神经损伤为中枢神经或周边神经损伤。
  4. 如权利要求1所述的用途，其特征是，所述神经损伤是指神经元损伤。
  5. 如权利要求1所述的用途，其特征是，所述神经损伤包含皮质神经(cortical neurons)、海马回神经(hippocampal neurons)或视网膜神经(retinal neurons)损伤。
  6. 如权利要求1所述的用途，其特征是，所述治疗是指神经再生、神经数量增加或神经修复。
  7. 如权利要求1所述的用途，其特征是，所述异野漆树苷(Isorhoifolin) 及其衍生物经鼻腔给药可穿透血脑屏障(blood-brain barrier)进入大脑。
  8. 如权利要求1所述的用途，其特征是，所述异野漆树苷(Isorhoifolin)及其衍生物有效剂量为1nM至864μM。
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