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WO2003006038A1 - Composition servant a proteger et a regenerer les cellules nerveuses et contenant un extrait de coptidis rhizoma - Google Patents

Composition servant a proteger et a regenerer les cellules nerveuses et contenant un extrait de coptidis rhizoma Download PDF

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Publication number
WO2003006038A1
WO2003006038A1 PCT/KR2002/001306 KR0201306W WO03006038A1 WO 2003006038 A1 WO2003006038 A1 WO 2003006038A1 KR 0201306 W KR0201306 W KR 0201306W WO 03006038 A1 WO03006038 A1 WO 03006038A1
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Prior art keywords
cells
nerve
coptidis rhizoma
rhizoma extract
composition
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English (en)
Inventor
Byung-Kil Choi
Yun-Hee Kim
Soo-Kyung Kim
Jung-Su Lim
Hyo-Sup Kim
Dae-Sung Park
Chi-Young Chang
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Regen Biotech Inc
EUGENBIO Inc
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Regen Biotech Inc
EUGENBIO Inc
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Priority claimed from KR1020020040110A external-priority patent/KR20030007105A/ko
Publication of WO2003006038A1 publication Critical patent/WO2003006038A1/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/71—Ranunculaceae (Buttercup family), e.g. larkspur, hepatica, hydrastis, columbine or goldenseal
    • A61K36/718—Coptis (goldthread)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives

Definitions

  • the present invention relates to a composition for protecting nerve cells, promoting nerve cell growth and regenerating nerve cells comprising a Coptidis
  • Rhizoma extract Further, the present invention relates to a composition for drugs and functional foods useful in the prevention and treatment of nervous diseases or nerve injuries comprising a Coptidis Rhizoma extract.
  • composition according to the present invention can be used as therapeutic agents for the prevention and treatment of neurodegenerative diseases, ischemic nervous diseases or brain injuries, and for the improvement of learning capability.
  • Synapses are the connection points between nerve cells, and one nerve cell connects to 1000-5000 other nerve cells on average. It is estimated that since at least lO 1 1 nerve cells exist in the human brain, there are at least 10 14 synapses in the human brain. All complex and various brain functions, for example thoughts, sensations, memory, learning and actions, cannot be understood without consideration of these neural networks. Synaptic connections are essential to nerve cell survival. Special functions according to the connections between nerve cells make it possible to express high-level brain functions intrinsic to humans. In particular, it is known that once the central nervous system is damaged, its regeneration is very difficult. Many ideas and attempts for treating damaged nerve tissues or chronic degenerative diseases have been made in various ways.
  • nerve cells die. There are two nerve cell death pathways, like all other cells: necrosis and apoptosis. Necrosis and apoptosis have different morphological and molecular biological characteristics. When an axon is cut (axotomy), a part attached to the cell body and a terminal forming a synapse are separated each other. Such axotomy leads to not only synaptic denaturation due to cut off of supply of protein factors from target cell body, but also synaptic detachment. That is, regeneration is a key to nerve cell survival.
  • Dead nerve cells are replaced with glial cells in the peripheral nervous system, and astrocytes or microglias in the central nervous system, in a process called "synaptic stripping".
  • immune system cells such as monocytes, macrophages, etc., can replace the dead nerve cells, depending on the extent of damages.
  • monocytes, macrophages, etc. can replace the dead nerve cells, depending on the extent of damages.
  • Many theories explaining mechanisms of physical injuries to nerve cells, acute neurotoxicity, acute and chronic nervous disorders, dementia, epileptic, etc. have been introduced, but these theories all have a common point. That is, these diseases affect nerve cells and supporting tissue cells thereof. These cells extend horizontally and perpendicularly to form many dendrites and axons, which form many neural networks.
  • the glutamatergic neural net responding to glutamate, an excitatory neurotransmitter, is a neural net to which has drawn attention in terms of development of acute and chronic cranial nervous diseases.
  • All mammalian brains develop a systematic neural network through a series of division, differentiation, survival and apoptosis of neuronal stem cells, and synaptic formation, thereby performing complex brain functions.
  • cranial nerve cells produce many substances necessary for nerve growth to make their axons and dendrites grow. Therefore, as new learning and memories are introduced, synaptic connections and neural networks are continuously remodeled.
  • Neuronal stem cells exist in the adult brain.
  • the development and differentiation of the stem cells in the adult brain lead to the regeneration of nerve cells (Johansson, C. B., Momma S., Clarke D. L., Risling M., Lendahl U., and Frisen J. (1999) Identification of a neural stem cell in the adult mammalian central nervous system, Cell 96, 25-34).
  • Neuronal stem cells are mainly found in the subventricular zones of striatum adjacent to lateral ventricles.
  • Neural stem cells in the subgranular zones at dentate gyrus of the hippocampus divide to form granule cells (van Praag et al., Nature 415, 1031-1034 (2002)). Therefore, increased development and differentiation of neuronal stem cells can promote nerve regeneration.
  • transplanted cells In order to survive in the nervous system, transplanted cells must differentiate into their compatible nerve cells to form synapses together with target cells, and participate in electrical signal transmission to continuously receive survival factors from the target cells.
  • Neuronal stem cells divide into other stem cells or cells to be differentiated. At this time, cells suffering from false cell division and unnecessary cells experience cell death. Surviving cells are classified according to types of cells they are differentiated into. Neuronal precursors or neuroblasts, which are differentiated into nerve cells, are differentiated into cells secreting suitable neurotransmitters. Glial precursors, which are differentiated into glial cells, are differentiated into astrocytes and oligodendrocytes. These are cells assisting nerve cells. Astrocytes mechanically and metabolically support nerve cells, and comprise 70-80% of adult brain cells. Oligodendrocytes insulate axons and produce myelin to increase the rate of transmission of signals. Neuronal stem cells in the central nervous systems of fetus and adult can be differentiated into three types of brain cells, depending on environment of brain tissues and type of signals transmitted to neuronal stem cells.
  • stem cells there are three types of cells as stem cells in the central nervous system. These cells all exist in the adult rodent brain, and it is believed that the cells exist in the adult human brain. One area containing these cells exists in the brain tissues adjacent to ventricles known as ventricular zones and subventricular zones. Ventricle is spaces through which cerebrospinal fluid can flow. During fetal neurogenesis, rapid cell division takes place in the tissues around the ventricles. In the adult, stem cells around ventricles can exist, but the tissues are very small. The second area in which stem cells exist is not found in humans. The area is rostral migratory stream connecting lateral ventricles and olfactory bulbs in rodents. The third area is the hippocampus, which is associated with memory formation, and exists in both the adult rodent and human brains.
  • Stem cells in the hippocampus exist in the subgranular zones of dentate gyms.
  • BrdU bromodeoxyuridine
  • about half of the labeled cells are differentiated into granule cells of dentate gyrus, and 15% are differentiated into glial cells, and the rest do not have particular phenotypes.
  • BrdU-labeled cells in dentate gyrus of human and rat express nerve cell markers such as NeuN, neuron-specific enolase, calbindin.. etc. These nerve-like cells are similar to granule cells of dentate gyrus in terms of morphology.
  • the other BrdU-labeled cells express GFAP, which is an astrocyte marker.
  • FGFs Fibroblast growth factors
  • aFGF acidic FGFs
  • bFGF basic FGFs
  • Membrane- associated proteoglycans bind to low-affinity binding sites of FGF receptors, and are essential to FGF's binding with a high-affinity binding site.
  • FGF receptors express 4 genes into various transcripts by alternative splicing. The receptors can bind with at least one FGF family member, and their ligand binding specificities are determined by thei types and splicing forms. FGFs have mitogen activity and induce cell differentiation. The treatment of pheochromocytomas (PC 12) with FGF causes their differentiation into cells having neuronal phenotype. Little is known about the signal transmission system of FGF receptors.
  • MAP kinase mitogen-activated protein kinases
  • mRNAs of FGF receptors are mainly found in the adult rat brain, in particular in primary cultured nerve cells of developing rat brain and hippocampus. Furthermore, it is known that FGF increases the survival of retinal optic nerves during the development of Xenopus retinal optic nerve cells, and in particular the expression of FGF is drastically increased in a short period of time.
  • the present inventors found that the treatment of the hippocampal primitive nerve cell line (HiB5) with PDGF and FGF inhibits apoptosis of cells and influences the differentiation into nerve cells or glial cells (Kwon, Y. Kim (1997) Expression of brain-derived neutrophic factor mRNA stimulated by basic fibroblast grwoth factor and platelet-derived growth factor in rat hippocampal cell line, Mol. Cells 7, 320-325.).
  • Nerve growth factors initiate the division of nerve stem cells, regulate the number of divided cells into apoptosis, initiate the differentiation of divided cells, induce the survival of cells orthodromically moving toward target-derived growth factors and the apoptosis of cells moving in a false direction to regulate the survival of presynaptic nerve cells, and regulate synaptic formation and synaptic remodeling.
  • Coptidis Rhizoma root of a plant belonging to Coptis japonica, Makino
  • Coptidis Rhizoma has been traditionally used as a stomachic, a calmative, an antiphlogistic, antimicrobial, an antipyretic, etc., and thus used in the treatment of dyspepsia, gastritis, enteritis, colic. vomiting, dysentery, schizophrenia, melena, burn, etc.. in Oriental medicine.
  • Rhizoma mainly refers to pharmacological effects such as antiinflammatory, antiphlogistic, antidiabetic, antineoplastic, immunostimulant, dysrhythmia-modulatory, nephroprotective, etc (Birdsall & Kelly, 1977).
  • U.S. Pat. No. 5,916,555 discloses a natural drug composition for the treatment of diabetes comprising a Coptidis Rhizoma extract.
  • Coptidis Rhizoma have been undertaken, but there has been no study explaining the use of a Coptidis Rhizoma extract for treating nervous diseases. Furthermore, effect of a Coptidis Rhizoma extract on regeneration and protection of neurites and effect of a Coptidis Rhizoma extract on promotion of neurite growth have not hitherto been reported.
  • a drug and food composition for protecting nerve cells, promoting the differentiation of nerve cells and regenerating nerve cells comprising a Coptidis Rhizoma extract.
  • composition according to the present invention is useful for preventing and treating physical injuries to nervous systems, degenerative and ischemic cranial nerve injuries, and peripheral nerve injuries.
  • the present inventors identified protective and differentiative effects of the Coptidis Rhizoma extract on neuronal stem cells and nerve cell lines cultured in vitro in stress models (serum deprivation-induced stress and oxidative stress).
  • stress models serum deprivation-induced stress and oxidative stress.
  • the present inventors identified the neuroprotective effect of the Coptidis Rhizoma extract in nerve apoptosis-induced animal models and dementia-induced animal models, by treating with a neurotoxin. Further, they identified the effect of the Coptidis Rhizoma extract on regeneration of injured peripheral nerves in peripheral nerve-injured animal models.
  • the present inventors examined the inhibitory effect of the Coptidis Rhizoma extract against apoptosis induced by serum deprivation-induced stress and oxidative stress, and the effect of the Coptidis Rhizoma extract on differentiation and regeneration of nerve stem cells and nerve cells, by treating human neuroblastomas (SH-SY5Y), rat neuronal stem cells (HiB5), and PC 12 cells with the Coptidis Rhizoma extract.
  • the Coptidis Rhizoma extract will be useful for preventing and treating nervous system disorders, degenerative brain diseases including dementia, nervous system diseases, and nerve injuries by traffic accidents, etc.
  • a Coptidis Rhizoma extract can be prepared in accordance with conventional processes. For example, after dried roots of Coptidis Rhizoma were homogenized to 10-20 mesh and charged into a round-bottomed flask equipped with a reflux condenser, the homogenized roots of Coptidis Rhizoma were extracted using hot water or ethanol respectively to prepare the Coptidis Rhizoma extracts.
  • the cells were cultured under serum deprivation. After the culture was treated with the Coptidis Rhizoma extract, cell survival was evaluated by performing the MTT assay to identify the protective effect against cell injuries. Retinoic acid, which increases cell survival as a cell differentiation factor, was used as a positive control. 3 hours before depriving serum, the cells were treated with the Coptidis Rhizoma extract, and cultured in chemically defined media, N2, for 2 days to induce nerve regeneration.
  • the cell survival rate in the group treated with the Coptidis Rhizoma extract was 1.7 times higher than in the control group.
  • the Coptidis Rhizoma extract has an excellent protective effect against apoptosis, a positive effect on cell survival and a neuroprotective effect.
  • the Coptidis Rhizoma extract has an excellent neuroprotective effect in PC 12 cells.
  • Apoptosis of nerve cells is a cause of degenerative brain diseases.
  • a common cause of apoptosis is oxidative stress. Since H 2 0 2 is a strong oxidant and produces oxidative stress in cells, it is used to prepare a cell model for degenerative brain diseases such as stroke and dementia.
  • SH-SY5Y cells were treated with H 2 0 2 to induce cell injuries. 3 hours before the treatment, the
  • Coptidis Rhizoma extract was added to cell culture solution to examine its protective effect against oxidative stress.
  • the cell protection effect against apoptosis in the group treated with the Coptidis Rhizoma extract was more than two times higher than in the control group. Therefore, it can be seen that the Coptidis Rhizoma extract has an excellent protective effect against apoptosis of neuroblastomas and a positive effect on cell survival under condition of oxidative stress.
  • neuronal stem cells (HiB5) were cultured under conditions for initiation of differentiation for 1 day. After the culture was treated with the Coptidis Rhizoma extract prepared above and further cultured for 2 days, neurite growth was observed. A positive' control group was treated with bFGF to induce the differentiation into nerve cells.
  • the group treated with the Coptidis Rhizoma extract and the positive group all were differentiated into nerve cells. It was also observed that cell bodies were dwindled, and neurites were extended to be two times longer than their cell bodies. Therefore, it can be seen that the Coptidis Rhizoma extract has an excellent effect of promoting differentiation of neuronal stem cells into nerve cells.
  • Rhizoma extract in PC 12 cells the group treated with the Coptidis Rhizoma extract showed high NGF mRNA expression, compared with the untreated group. This suggests that the Coptidis Rhizoma extract functions as a nerve growth factor in PC 12 cells.
  • the mechanisms by which nerve cell growth factors inhibit apoptosis of nerve cells are as follows: 1) inhibition of death effector gene expression, and 2) promotion of cell survival promoting genes (e.g., bcl-2, bcl-xL, etc) expression (Helmreich, 2001). Therefore, it is assumed that the Coptidis Rhizoma extract functions as a nerve growth factor, and the Coptidis Rhizoma extract increases the production of Bcl-2, a representative anti-apoptosis protein, thereby efficiently inhibiting apoptosis of nerve cells.
  • a common characteristic of Alzheimer's disease in the early stages is memory loss.
  • the hippocampus is involved in the formation of short-term and long-term memories.
  • Degeneration in the hippocampus and forebrain are most commonly found in the brain of Alzheimer's patients, and senile plaques are most commonly found in the hippocampus and forebrain.
  • degeneration of nerve cells in the CA1 and entorhinal cortex of the hippocampus is fastest. Since survival of cholinergic neurons projecting from basal forebrain depends on NGF and BDNF, which are target- derived neurotrophic factors, cholinergic neurons are rapidly degenerated in patients suffering from Alzheimer's disease.
  • Alzheimer's disease Many etiological studies on initiating factors of Alzheimer's disease have been carried out. Among them, many experiments have noted that abnormal phosphorylation of ⁇ -amyloid, which is a main component of senile plaques, or tau proteins found in the neurofibrillary tangles of dying nerve cells, is associated with apoE4, etc. However, there exist too many genes related with Alzheimer's disease, and no initiating factors and gene mutations commonly found in all patients have been found. Currently used therapeutic agents of Alzheimer's disease include acetylcholine esterase inhibitors for enhancing activities in the cholinergic signal transmission system, acetylcholine esterase precursors, and a drug for improving energy metabolism of nerve cells.
  • neurotrophins or nerve cell stimulants capable of increasing nerve cell survival in order to reduce Alzheimer's disease progress and etiologically treat Alzheimer's disease.
  • nerve growth factors have drawn attention. So far, clinic trials with NGF have shown some effects in cholinergic neurons (Knusel B. and Gao H., Life Sci. 58:2019-2027(1996); Lapchak P. A., Exp Neurol 124:16-20(1993)), but did not exhibit satisfactory effects (Neve et al., Brain Res Mol Res. 1996. 39(1-2) : 185-197(1996)). Therefore, it is necessary to select substances capable of protecting against apoptosis of nerve cells, increasing survival and regeneration of nerve cells, and increasing the survival and differentiation of neuronal stem cells.
  • the present inventors identified the protective effect of the Coptidis Rhizoma extract against apoptosis, and a neuroregenerative effect of the Coptidis Rhizoma extract in an animal model for dementia.
  • Schwann cells play an important role in the generation and regeneration of the peripheral nervous system.
  • Schwann cells derived from the neural crest previously divide at the sites occupied by axons. That is, axonal growth in the peripheral nervous system depends on Schwann cells.
  • Schwann cells produce trophic factors to regulate nerve survival and neurite growth.
  • Axons in nerve cells secrete neuregulin to increase Schwann cell survival and to regulate the ratio between axons and Schwann cells.
  • Schwann cells receiving no influence from axons die.
  • Schwann cells produce myelin sheaths to insulate axons and the differentiation of Schwann cells is completed.
  • peripheral nerves When peripheral nerves are injured in adults suffering from neurogenesis, they undergo Wallerian degeneration at the distal stumps toward nerve endings from the injured sites. However, the proximal stumps toward cell bodies from the injured sites start to regrow. At the distal stumps toward nerve endings from the injured sites, the degenerated axons and myelin sheaths are removed. On the other hand, at the proximal stumps toward cell bodies from the injured sites, the environment is modified to promote axonal regrowth (Kwon, Y. Kim, Bhattacharyya, W.V., Cheon, K., Stiles, CD., and Pomeroy, S.L. (1997) Activation of ErbB2 during Wallerian degeneration of sciatic nerve, J.
  • Schwann cells Immediately after nerves are damaged, Schwann cells rapidly divide. Such Schwann cell division is believed to be due to the fact that Schwann cells fail to make contact with axons, or the division is promoted by growth factors secreted from axons. During axonal regrowth, contact of Schwann cells with axons promotes axonal differentiation and regenerates myelin sheaths. Further, Schwann cells can influence axonal regeneration from a distance. For example, though nerves are cut and separated by a gap of 1cm, axons regenerate toward the distal stumps. Such orthodromic movement of axons is possible only when living Schwann cells exist in the distal stump.
  • Regeneration in the peripheral nervous system occurs in accordance with the following processes: first, Schwann cells are separated from cut axons to obtain division potential (dedifferentiation), axons of nerve cells regrow from injured sites, Schwann cells insulate the regrown axons with myelin sheaths (redifferentiation), and axons grow enough to reach muscles and form neuromuscular junctions at muscle cells.
  • the present inventors examined whether the Coptidis .Rhizoma extract promotes axonal regrowth, the regeneration of myelin sheaths, and the formation of neuromuscular junctions in muscle cells, in the regeneration process of sciatic nerves through which most nerve fibers pass in the peripheral nervous system.
  • the present inventors observed the degree of nerve regeneration 1 week, 2 weeks and 4 weeks after intraperitoneally injecting PBS (phosphate-buffered saline) or the Coptidis Rhizoma extract into sciatic nerves of a rat.
  • PBS phosphate-buffered saline
  • Coptidis Rhizoma extract into sciatic nerves of a rat.
  • the present inventors separated hindlimb muscle connected to sciatic nerve. As a result, it was observed in the control group that nerve endings were stained, but did not spread to muscle fibers and thus did not form neuromuscular junctions. In the group administered with the Coptidis Rhizoma extract, the nerve endings spread to all muscle fibers. Therefore, it is believed that the Coptidis Rhizoma extract promotes axonal growth, the regeneration of myelin sheaths and the regeneration of nerve endings to form neuromuscular junctions during regeneration of peripheral nerves.
  • Nerve growth factors initiate the division of neuronal stem cells, regulate the divided cells into apoptosis, induce the survival of cells orthodromically moving toward target-derived growth factors and apoptosis of cells moving in a false direction to regulate the survival of presynaptic nerve cells, and regulate new synaptic formation and remodeling. Since the Coptidis Rhizoma extract induces the differentiation of neuronal stem cells, inhibits apoptosis and promotes neurite differentiation, it is expected that the Coptidis Rhizoma extract will perform functions of nerve growth factors.
  • the dosage for the Coptidis Rhizoma extract can be varied depending upon known factors, such as age, sex, body weight, disease severity and health condition of the recipient.
  • the daily dosage is commonly in the range of 100 to 150mg/60kg of body weight in two or three installments.
  • the Coptidi Rhizoma extract may be mixed with an appropriate carrier or excipient, or may be diluted in an appropriate diluent.
  • composition according to the present invention can further comprise fillers, anti-coagulating agents, lubricants, wetting agents, flavors, emulsifying agents, preservatives, etc.
  • the composition according to the present invention can be formulated in accordance with well-known processes.
  • the formulation may be in dosage form such as tablets, powders, pills, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile water for injection, sterilized powders, etc.
  • the composition according to the present invention may be administered through a suitable route such as oral, transdermal, subcutaneous, intravenous or intramuscular route.
  • the Coptidis Rhizoma extract may be formulated into pharmaceutical preparations for preventing and treating nervous system diseases, or may be added to foods or beverages.
  • composition according to the present invention may be used as drugs or foods to treat degenerative brain diseases such as dementia, chronic epilepsy, palsy, ischemic brain diseases, Parkinson's disease and Alzheimer's disease.
  • degenerative brain diseases such as dementia, chronic epilepsy, palsy, ischemic brain diseases, Parkinson's disease and Alzheimer's disease.
  • foods include beverages, gums, teas, vitamin complexes, health care products, etc.
  • Fig. 1 is a graph showing the protective effect of the Coptidis Rhizoma extract against apoptosis induced by serum deprivation-induced stress in human neuroblastoma SH-SY5Y, which is a differentiated nerve cell line;
  • Fig. 2 is a graph showing the protective effect of the Coptidis Rhizoma extract against apoptosis under condition of serum deprivation-induced stress in pheochromocytomas (PC 12) derived from white rat neural crest;
  • Fig. 3 is a graph showing the protective effect of the Coptidis Rhizoma extract against apoptosis induced by oxidative stress in human neuroblastoma SH- SY5Y, which is a differentiated nerve cell line;
  • Fig. 4 is confocal microscopic images showing the effect of the Coptidis Rhizoma extract on inducing differentiation of white rat-derived neuronal stem cells (HiB5).
  • bFGF+ represents bFGF (basic fibroblast growth factor)-treated cells, and bFGF- represents bFGF-untreated cells;
  • Fig. 5 is confocal microscopic images showing the effect of the Coptidis
  • Rhizoma extract on neurite regeneration in human neuroblastoma SH-SY5Y which is a differentiated nerve cell line.
  • Retinoic acid is a positive control group which causes the neurite differentiation of SH-SY5 Y;
  • Figs. 6a to 6c are photographs showing morphological changes of PC 12 cells after administering 20, 60, 200 ⁇ g/ml of the Coptidis Rhizoma extract, respectively, for 18 days. These figures show distinct formation of neurites;
  • Fig. 7 is a photograph showing the neurite growth in PC 12 cells 3 days after administering NGF (Nerve Growth Factor. 50ng/ml).
  • Figs. 8a and 8b are bar graphs showing the length of neurites after treating the Coptidis Rhizoma extract (120 ⁇ g/ml) in cultures of PC 12 cells over various time intervals (Fig. 8a: a group treated for 4 days, and Fig. 8b: a group treated for 10 days)
  • Control a control group treated with physiological saline
  • NGF a group treated with
  • NGF 50ng/ml
  • CC a group treated with the Coptidis Rhizoma extract (120 ⁇ g/ml));
  • Fig. 9a is a photograph showing the expression of NGF mRNA after treating the Coptidis Rhizoma extract in PC 12 cells. A group treated with the Coptidis Rhizoma extract exhibits far higher expression of NGF than control group.
  • Fig. 9b shows the expression of GAPDH mRNA as a control group in the quantification of mRNA (M: 100 bp DNA marker, N: control group, 1 : a group treated with the Coptidis Rhizoma extract (120 ⁇ g/ml) for 5 days, 2: a group treated with the Coptidis Rhizoma extract (120 ⁇ g/ml) for 14 days, and 3: a group treated with NGF (50ng/kg) for 14 days);
  • Figs. 10a and 10b show normal cerebral slices of 7-day old white rats stained by the TUNEL method.
  • Fig. 10b is a magnified view (x400) of the open square indicated in Fig. 10a;
  • Figs. 11 a and l ib are photographs showing apoptosis of nerve cells in cerebral slices, 1 day after intraperitoneally injecting MK-801 (0.5mg/kg) into 7-day old white rats.
  • Fig. 11 a shows total cerebral coronal slice (Black points represent cells positive to the TUNEL method, which is an apoptosis searching method capable of staining only cells exhibiting nuclear DNA-fragmentation).
  • Fig. l ib is a magnified view (X400) of the open square represented in Fig. 1 la;
  • Figs. 12a and 12b are photographs of the cerebral slices taken 3 days after intraperitoneally injecting the Coptidis Rhizoma extract (20mg/kg) alone into 7-day old white rats.
  • FIG. 12b is a magnified view (X400) of the open square indicated in Fig. 12a;
  • Figs. 13a and 13b are representative photographs of the cerebral slices taken after pretreating the peritoneal cavity of 7-day old white rats with the Coptidis Rhizoma extract (20mg/kg) alone for 3 days, followed by intraperitoneally injecting MK-801 (0.5mg/kg) into the rat.
  • MK-801 0.5mg/kg
  • Figs. 14a and 14b are photographs of the cerebral slices taken after intraperitoneally injecting MK-801 (0.5mg/kg) into 7-day old white rats to induce nerve cell apoptosis, followed by intraperitoneally injecting the Coptidis Rhizoma extract (20mg/kg) for 6 days. These figures reveal that the Coptidis Rhizoma extract inhibits nerve cell apoptosis induced by MK-801 (0.5mg/kg).
  • Fig. 14b is a magnified view (X400) of the open square indicated in Fig. 14a;
  • Fig. 15 is a graph quantitatively showing the extent to which the Coptidis Rhizoma extract inhibits nerve cell apoptosis induced by MK-801 (0.5mg/kg) in the cerebral slice of young rat [1 : a group administered with MK-801 (0.5mg/kg) alone, 2: a group administered with the Coptidis Rhizoma extract (20mg/kg) alone for 6 days, 3 : a group pretreated with the Coptidis Rhizoma extract (20mg/kg) for 3 days and then administered with MK-801 (0.5mg/kg)], and 4: a group administered with MK-801 (0.5mg/kg) and then administered with the Coptidis Rhizoma extract (20mg/kg) for 6 days;
  • Fig. 16a is a result of RT-PCR showing the expression of bcl-2 mRNA, an anti-apoptosis gene expressed in cerebral tissues of 7-day old white rats, after intraperitoneally injecting various doses of the Coptidis Rhizoma extract for 1 day (lane 1, 2, 3) or 3 days (lane 4, 5, 6). This figure reveals that the expression of bcl-2 mRNA is higher than in the control group.
  • FIG. 16b shows the expression of GAPDH mRNA (M: 100 bp DNA ladder, N: control group, 2: a group administered with the Coptidis Rhizoma extract (50mg/kg), 3: a group administered with the Coptidis Rhizoma extract (25mg/kg), 4: a group administered with the Coptidis Rhizoma extract (l OOmg/kg), 5: a group administered with the Coptidis Rhizoma extract (50mg/kg), and 6: a group administered with the Coptidis Rhizoma extract (25mg/kg));
  • Fig. 17 is microscopic images showing the neuroregenerative effect of the
  • Fig. 18 is confocal microscopic images showing the neuroregenerative effect of the Coptidis Rhizoma extract, after fluorescence-staining the brain tissues of dementia-induced white rats with calbindin antibody, which is a nerve-specific marker;
  • Fig. 19 is confocal microscopic images (X200). showing the neuroregenerative effect of the Coptidis Rhizoma extract, 1 week after intraperitoneally injecting into sciatic nerve-damaged white rats.
  • White lines indicate axons longer than 300 ⁇ m stained with beta-tubulin isotypelll (red), and arrowheads indicate degenerated myelin sheaths stained with MBP (myelin binding protein, green) antibody;
  • Fig. 20 is confocal microscopic images (X200) showing the neuroregenerative effect of the Coptidis Rhizoma extract, 2 week after intraperitoneally injecting the extract into sciatic nerve-damaged white rats.
  • White lines indicate axons longer than 300 ⁇ m stained with beta-tubulin isotypelll (red), and arrows indicate regenerated myelin sheaths of Schwann cells longer than 200 ⁇ m Gained with MBP (myelin binding protein, green) antibody,
  • Fig. 21 is confocal microscopic images (X200) showing the neuroregenerative effect of the Coptidis Rhizoma extract, 4 weeks after intraperitoneally injecting the extract into sciatic nerve-damaged white rats.
  • White lines indicate axons longer than 300 ⁇ m stained with beta-tubulin isotypelll (red), and arrowheads indicate myelin sheaths longer than 200 ⁇ m stained with MBP (myelin binding protein, green) antibody It was confirmed that the number of long and thick axons and myelin sheaths increased. When myelin sheaths were differentiated and then insulated regrowing axons, two antibody markers were overlapped to appear to be yellowish,
  • Fig 22 is magnified (x400) views of Fig. 19,
  • Fig 23 is magnified (x400) views of Fig. 20,
  • Fig 24 is magnified (x400) views of Fig 21 .
  • Fig 25 is photographs showing the neuroregenerative effect of the Coptidis Rhizoma extract during reformation of neuromuscular junctions.
  • nerve endings reached only one muscle fiber, but did not spread to other fibers
  • the nerve endings reached all muscle fibers to form neuromuscular junctions.
  • Example 1 General methods 1) Nerve cell line culture
  • HiB5 cells derived from white rat embryonic hippocampus were prepared by retroviral infection of temperature sensitive SV40 large T antigen, they divided at 34 ° C, but the cell division stopped at 39 ° C (body temperature of rat).
  • HiB5 cells When bFGF (20ng/ml) was added to the HiB5 cells, cell survival increased and HiB5 cells differentiated into nerve cells to express marker molecules of nerve cells.
  • Cell culture medium was prepared by adding a mixture of 10% FBS (fetal bovine serum), penicillin/streptomycin, glutamine and sodium pyruvate (0.11 g/L) to DMEM.
  • FBS fetal bovine serum
  • penicillin/streptomycin penicillin/streptomycin
  • glutamine sodium pyruvate
  • Another cell culture medium was prepared by adding pyruvate to a serum-free medium (N2, containing DMEM/F12, insulin, transferrin, Putreseine and BSA; Botten Stein & Sato., 1979).
  • PC 12 cells and SH-SY5Y cells were cultured in DMEM supplemented with 10% FBS.
  • NGF or retinoic acid was added to a serum-free medium.
  • MTT assay of nerve cell line Cell injuries under condition of serum deprivation-induced stress and oxidative stress were measured by MTT assay.
  • MTT assay Cells were seeded into N2 medium in an amount of 7.5* 10 3 cells, and then a neurotoxin or a nerve growth factor was added thereto to differentiate the cells and then 0.1 volume of 4mg/ml MTT was further added. The mixture was stored at 37 ° C for 3-4 hours. After 1 OO ⁇ l of solubilization buffer was added to the mixture and then stored for 24 hours, O.D. values were measured by ELISA assay.
  • Tissue sections were fixed with 4% paraformaldehyde and cryosected to a thickness of 40 ⁇ m.
  • the cryosected tissue sections were stained with nerve cell- or astrocyte-labeled antibody and FITC-labeled secondary antibody before examining under a confocal microscope.
  • cultured cells were fixed with 4% paraformaldehyde for 20 minutes, permeated in 0.5%> NP-40 for 5 minutes, and blocked using 1% BSA solution for 30 minutes.
  • proximal stumps and distal stumps were obtained over various time intervals (6 hours, 1 day, 3 day, 7 day, 14 day, 21 day and 28 day), respectively, before testing.
  • the right sciatic nerve was used as a control group.
  • Example 3 Protective effect of Coptidis Rhizoma extract against apoptosis induced by stress in nerve-related cells
  • the cells were cultured under serum deprivation. After the culture was treated with the Coptidis Rhizoma extract, cell survival was evaluated by performing the MTT assay, thereby identifying the protective effect against cell injuries. Retinoic acid, which induces the differentiation of SH-SY5Y as a cell differentiation factor, was used as a positive control. 3 hours before depriving serum, the cells were treated with the Coptidis Rhizoma extract (30 ⁇ 100 ⁇ g/ml). The cells were cultured in chemically defined media (N2) for 2 days to induce nerve regeneration (see, T ⁇ ble 1 and Fig. 1 ). [Table 1 ]
  • the cell survival rate in the group treated with the Coptidis Rhizoma extract was 1.5 times higher than in the control group, but when treated with the Coptidis Rhizoma extract (lOO ⁇ g/ml), the cell survival was somewhat decreased. Therefore, it can be seen that the Coptidis Rhizoma extract has an excellent protective effect against apoptosis, a positive effect on cell survival and a neuroprotective effect.
  • SH-SY5Y cells were treated with H 2 0 2 for 30 minutes to indupe cell injuries. 3 hours before the cells were treated with H 2 O , the Coptidis Rhizoma extract (30 ⁇ 100 ⁇ g/ml) was added to cell culture to examine its cell protection effect.
  • the cell protection effect against oxidative stress in the group treated with the Coptidis Rhizoma extract was more than two times higher than in the control group.
  • the Coptidis Rhizoma extract has an excellent protective effect against apoptosis of neuroblastomas and a positive effect on cell survival under condition of oxidative stress
  • HiB5 cells were cultured under conditions for initiation of differentiation for 1 day. Thereafter, the culture was treated with the Coptidis Rhizoma extract (lOO ⁇ g/ml) and further cultured for 2 days. The cultured cells were immunostained with nerve cell-specific labeled molecule, and then neurite growth was observed under a confocal microscope. A positive control was treated with bFGF (20ng/ml) under the same condition as described above to induce the differentiation into nerve cells.
  • the differentiation degree was measured by double- staining neurites with nerve cell-specific labeled moiecule (anti-neurofilament antibody) and FITC-labeled secondary antibody (green), followed by staining cell nuclei with propidium iodide (red).
  • Example 5 Quantitative comparison of neurites in PC12 cell line A group was treated with the Coptidis Rhizoma extract (120 ⁇ g/ml) alone, another group was treated with physiological saline alone, and last group was treated with NGF (50ng/m ), and then cultured in cultures of PC 12 cells, respectively, for more than 2 weeks. Subsequently, the length of neurites in each group was measured.
  • Differentiation index was scored as follows: no neurite expression (0), the length of expressed neurites was less than the diameter of cell bodies (1), the length of expressed neurites was similar to the diameter of cell bodies (2), the length of expressed neurites was less than two times as long as the diameter of cell bodies (3), and the length of expressed neurites was more than two times as long as the diameter of cell bodies, or the expressed neurites form synapses together with other nerve cells (4).
  • 200 differentiated cells from each microculture well were defined as one unit, and five units were statistically analyzed. The results are shown in Figs. 8a and 8b. As shown in Figs. 8a and 8b, the group treated with the Coptidis Rhizoma extract shows excellent neurite formation, compared with the group treated with physiological saline.
  • a group was treated with the Coptidis Rhizoma extract (120 ⁇ g/ml), another group was treated with physiological saline and final group treated with NGF
  • RNA isolation lml of TRI Reagent (Molecular Research Center Inc., USA) was added to 1 OOmg of tissue sections, and the mixture was homogenized and then left at room temperature for 10 minutes.
  • 0.1 ml of BCP (Sigma, USA) was added to l ml of the homogenized mixture, mixed with each other for 1 minute, and then left at 4 ° C for 10 minutes. After the mixture was centrifuged at 12,000rpm, 4 ° C for 15 minutes, the supernatant was added to cold isopropanol and left at a temperature of -20 ° C for 16 hours. Thereafter, the supernatant was centrifuged at 12,000 ⁇ m, 4 ° C for 15 minutes to obtain RNA precipitates.
  • RNA precipitates were washed with DEPC (diethylpyrocarbonate)-treated cold ethanol (75%), and dried using SpeedVac. The dried RNA was dissolved in DEPC-treated distilled water. After the concentration and purity of RNA were spectrophotometrically measured at 260nm, the isolated RNA was stored at a temperature of -20 °C before use.
  • DEPC diethylpyrocarbonate
  • RNA synthesis (Reverse Transcription: RT) 2 ⁇ g of total RNA obtained above was mixed wiih 4.0 ⁇ l of 5X RT buffer, l.O ⁇ l of oligo (dT16) (100 pmoles/ ⁇ l), 4 ⁇ l of 10 mM dNTPs (Promega, USA), 0.5 ⁇ l of RNasin (40 Units/ ⁇ l, Promega, USA) and l .O ⁇ l of MMLV reverse transcriptase (200units/ ⁇ l, Promega, USA), and DEPC-treated distilled water was added thereto until a total volume of the reaction solution was 30 ⁇ l. The reaction was performed in a DNA thermal cycler (Perkin Elmer 2400, USA) at 42 ° C for 1 hour to synthesize cDNA.
  • a DNA thermal cycler Perkin Elmer 2400, USA
  • PCR l ⁇ l of RT product was mixed with sense and antisense primers (each 10 pmoles), l ⁇ l of 10 mM dNTPs, 2 ⁇ l of 10X buffer (20mM Tris-Cl, 1.5mM MgCl 2 , 25mM KCl, 0.1 mg/ml gelatin, pH 8.4) and 1 unit of Taq DNA polymerase (Promege, USA), and then distilled water was added thereto until a total volume of the reaction solution was 25 ⁇ l. Polymerase chain reaction was perfoimed using a DNA thermal cycler (Perkin Elmer 2400, USA).
  • Figs. 9a and 9b The results are shown in Figs. 9a and 9b.
  • the group treated with the Coptidis Rhizoma extract shows high NGF expression, compared with the group treated with physiological saline.
  • Example 7 Regenerative and protective effects of Coptidis Rhizoma extract on cranial nerve cells using MK-801 model
  • MK-801 reaches maximal concentrations in plasma and brain within 10 to 30 minutes of injection with an elimination half-life of 1.9 hr (Vezzani et al, 1989). Olney et al. found that when MK-801 was administered to a young rat (7 ⁇ 8-days old) to inhibit NMDA receptors (for 2-3 hours), nerve cells highly sensitive to NMDA receptors died through apoptotic neurodegeneration. At this time, the number of dead nerve cells amounted to 12-26% of total nerve cells (Ikonomidou et al, 1999)
  • the protective effect of the Coptidis Rhizoma extract on nerve cells was evaluated using models for apoptosis of nerve cells induced by MK-801 in 7-day old white rats.
  • Young rats were divided into 5' groups: a) a group administered with physiological saline alone, b) a group administered with MK-801 (0.5mg kg) alone, c) a group administered with the Coptidis Rhizoma extract (20mg/kg) alone, d) a group pretreated with the Coptidis Rhizoma extract (20mg/kg) and then administered with MK-801 (0.5mg/kg), and e) a group pretreated with MK-801 (0.5mg/kg) and then administered with the Coptidis Rhizoma extract (20mg/kg). All groups were intraperitoneally injected.
  • Figs. 10a and 10b show TUNEL staining in normal cerebral sections of 7-day old white rats.
  • Figs. 1 1a and l ib show cerebral coronal slices.
  • Black cells represent cells positive to the TUNEL method, which stains only cells having segmented DNA in nuclei.
  • Rhizoma extract inhibits apoptosis of nerve cells induced by MK-801 (Figs. 13a and
  • Example 8 Quantitative comparison of nerve cell apoptosis in white rat cerebra
  • Example 9 Expression of bcl-2 mRNA and GAPDH mRNA in white rat cerebra
  • RT-PCR was performed to examine the expression of bcl-2 mRNA, which is an anti-apoptosis gene expressed in cerebral tissues.
  • bcl-2 mRNA is an anti-apoptosis gene expressed in cerebral tissues.
  • GAPDH mRNA was used as a control group. The expression of GAPDH mRNA was performed by RT-PCR method (Figs. 15a and 15b).
  • Example 10 Neuroregenerative effect of the Coptidis Rhizoma extract in animal model for dementia
  • ibotenic acid As an animal model for dementia, ibotenic acid, a kanate derivative, was microinjected into the entorhinal cortex of adult rat brain using a stereotaxic frame. Since injection of ibotenic acid into the entorhinal cortex exhibiting serious degeneration kills pyramidal cells in the CA1 region and granule cells in the dentate gyrus region, the animal model for dementia is also used a model for chemical lesion.
  • an adult rat was anesthetized, and its head was fixed with a stereotaxic frame. Subsequently, 1.5 ⁇ l (1 mg ml) of ibotenic acid was injected into the entorhinal cortex region to prepare a model for dementia, and then the Coptidis Rhizoma extract was injected into the same site. 2 weeks after operation, brain tissue sections were counter-stained with hematocylin to examine apoptosis and protective effect. After fluorescence-staining the cells, regions used for cell count were represented as squares (Fig. 17). In addition, the cells were fluorescence-stained with calbindin antibody, a nerve-specific marker, to examine nerve cell survival (Fig. 18). As shown in Fig.
  • the number of calbindin-positive neurons was reduced by 30-40% in the hippocampus and entorhinal cortex, and 4 weeks after injection, the cell numbers recovered somewhat. Cell numbers in the dentate gyrus regions were reduced more slowly than cell numbers in the CA1 region.
  • survival of calbindin-positive neurons increased, in particular, survival rates of pyramidal cells in the CA1 region had doubled, and those of granule cells in the dentate gyrus regions increased 1.5 times.
  • survival rate of calbindin-positive neurons in the entorhinal cortex were somewhat increased, but the increase was statistically significant. Therefore, it is believed that the Coptidis Rhizoma extract has a protective effect against apoptosis of nerve cells and a neuroregenerative effect in an animal model for dementia.
  • Example 11 Effect of the Coptidis Rhizoma extract on regeneration of sciatic nerves in the peripheral nervous system
  • cryosected sciatic nerves were double-stained using beta-tubulin isotypelll (cy3, red), which is an axon marker, and MBP (myelin binding protein, cy2, green) antibody, which is a differentiation (myelin) marker of Schwann cells. It was observed under a confocal microscope that axons were longer than 300 ⁇ m and myelin sheaths were longer than 200 ⁇ m (see, Figs. 19 to 24).
  • the Coptidis Rhizoma extract promotes axonal growth, the regeneration of myelin sheaths and the regeneration of nerve endings to form neuromuscular junctions during regeneration of peripheral nerves.
  • the composition according to the present invention regenerates axons and dendrites of nerve cells, thereby having a protective effect against nerve cell injuries, a positive effect on nerve cell growth and a regenerative effect on nerve cells.
  • the composition according to the present invention can be used as a therapeutic agent for the prevention and treatment of neurodegenerative diseases or nerve injuries, in particular, dementia, Parkinson's disease, Alzheimer's disease, epilepsy, palsy, ischemic brain diseases and peripheral nerve injuries.

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Abstract

L'invention concerne une composition servant à protéger les cellules nerveuses, à favoriser la croissance de ces cellules et à régénérer ces cellules nerveuses, ladite composition contenant un extrait de Coptidis Rhizoma. Cette composition exerce un excellent effet de protection contre l'apoptose des cellules nerveuses, un effet positif d'induction de la régénération de ces cellules nerveuses et de cellules souches neuronales, un effet régénérateur sur les neurites, un effet neurorégénérateur sur le système nerveux cérébral et périphérique, un effet reconstituant sur les jonctions neuromusculaires et un effet protecteur contre l'apoptose des cellules nerveuses chez les animaux atteints de démence ou d'ischémie cérébrale. On peut, de ce fait, utiliser cette composition en tant qu'agent thérapeutique pour la prévention et le traitement de maladies neurodégénératives, de maladies nerveuses ischémiques ou de lésions nerveuses, ainsi que pour améliorer les capacités intellectuelles.
PCT/KR2002/001306 2001-07-10 2002-07-10 Composition servant a proteger et a regenerer les cellules nerveuses et contenant un extrait de coptidis rhizoma Ceased WO2003006038A1 (fr)

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ES2302473A1 (es) * 2006-12-22 2008-07-01 Universidad De Barcelona Agente terapeutico para el tratamiento del desorden afectivo bipolar en mamiferos.
RU2348421C2 (ru) * 2007-03-13 2009-03-10 Государственное образовательное учреждение высшего профессионального образования "Нижегородская государственная медицинская академия Росздрава" (ГОУ ВПО "НИЖГМА РОСЗДРАВА") Средство для стимуляции роста мотонейронов коры головного мозга растущего организма млекопитающих и способ его применения
RU2355406C2 (ru) * 2007-03-28 2009-05-20 Государственное образовательное учреждение высшего профессионального образования "Нижегородская государственная медицинская академия Росздрава" (ГОУ ВПО "НИЖГМА РОСЗДРАВА") Средство для стимуляции роста мотонейронов спинного мозга растущего организма млекопитающих и способ его применения
GB2534228A (en) * 2015-01-19 2016-07-20 Hansemerkur Zentrum Fur Traditionelle Chinesische Medizin Am Univ Hamburg-Eppendorf Gemeinnutzige Gm Compound or mixture of compounds for the treatment of neurodegenerative diseases or oxidative stress injuries

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2302473A1 (es) * 2006-12-22 2008-07-01 Universidad De Barcelona Agente terapeutico para el tratamiento del desorden afectivo bipolar en mamiferos.
WO2008077978A1 (fr) * 2006-12-22 2008-07-03 Universidad De Barcelona Agent thérapeutique pour le traitement du trouble affectif bipolaire chez les mammifères
ES2302473B1 (es) * 2006-12-22 2009-06-12 Universidad De Barcelona Agente terapeutico para el tratamiento del desorden afectivo bipolar en mamiferos.
RU2348421C2 (ru) * 2007-03-13 2009-03-10 Государственное образовательное учреждение высшего профессионального образования "Нижегородская государственная медицинская академия Росздрава" (ГОУ ВПО "НИЖГМА РОСЗДРАВА") Средство для стимуляции роста мотонейронов коры головного мозга растущего организма млекопитающих и способ его применения
RU2355406C2 (ru) * 2007-03-28 2009-05-20 Государственное образовательное учреждение высшего профессионального образования "Нижегородская государственная медицинская академия Росздрава" (ГОУ ВПО "НИЖГМА РОСЗДРАВА") Средство для стимуляции роста мотонейронов спинного мозга растущего организма млекопитающих и способ его применения
GB2534228A (en) * 2015-01-19 2016-07-20 Hansemerkur Zentrum Fur Traditionelle Chinesische Medizin Am Univ Hamburg-Eppendorf Gemeinnutzige Gm Compound or mixture of compounds for the treatment of neurodegenerative diseases or oxidative stress injuries

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