WO2005120528A1 - Nouveaux extraits de plantes presentant des proprietes neuroprotectrices, neuroregeneratrices et antiphlogistiques - Google Patents

Nouveaux extraits de plantes presentant des proprietes neuroprotectrices, neuroregeneratrices et antiphlogistiques Download PDF

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Publication number
WO2005120528A1
WO2005120528A1 PCT/EP2005/006166 EP2005006166W WO2005120528A1 WO 2005120528 A1 WO2005120528 A1 WO 2005120528A1 EP 2005006166 W EP2005006166 W EP 2005006166W WO 2005120528 A1 WO2005120528 A1 WO 2005120528A1
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styrax
extract
crotonoides
extracts
knema
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PCT/EP2005/006166
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Oliver Ullrich
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Oliver Ullrich
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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)
    • 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
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

  • New plant extracts with neuroprotective, neuroregenerative and anti-inflammatory properties are provided.
  • the present invention relates to extracts from the plants Knema laurina and / or Styrax crotonoides, in particular the subspecies Styrax crotonoides subsp. fraserensis, Styrax benzo ⁇ n, and / or Styrax tonkinensis, process for the preparation of these extracts, pharmaceutical compositions containing these extracts, and the use of the extracts for the production of a pharmaceutical composition, in particular for the prevention and / or treatment of neurological diseases and / or inflammation-related diseases.
  • Plant extracts have been used in many different cultures for medical and other purposes for many years. New plant extracts are continually being extracted to examine their medicinal effects. Many plants, the benefits of which were previously unknown and which were considered exotic and insignificant, are widely used today, particularly in medicine. Natural product research therefore plays a central role in drug development. Without this discipline, entire therapeutic areas would be completely unused (Verdine G. Nature (1996) 384: 11-13).
  • An active ingredient that has been isolated from a natural source is now chemically and pharmaceutically optimized so that the side effects of the active ingredient are reduced and the specific main effect is improved.
  • An example of the successful search for medically relevant active ingredients from natural sources is the cytostatic taxol from the bark of a Pacific yew species (Taxus brevifolia). This discovery has brought the search for active substances in plants to the fore again.
  • this object according to the invention is achieved by providing an extract and / or extracts of the Styrax plant crotonoides, especially the subspecies Styrax crotonoides subsp. fraserensis, Styrax benzoin, and or Styrax tonkinensis from the Styracaceae family.
  • the Styrax crotonoides plant is a shrub or tree.
  • the term plant means both whole plants and parts of plants such as leaves, barks, flowers, fruits, seeds, roots etc. and mixtures thereof.
  • Another aspect of the invention relates to an extract from the plant Styrax crotonoides, in particular the subspecies Styrax crotonoides subsp. fraserensis, Styrax benzoin, and / or Styrax tonkinensis, solvents or mixtures of solvents which are selected from the group of distilled or undistilled water, low molecular weight alcohols, esters, ethers, polyols, chlorinated solvents, hydrocarbons, ketones being used as the extraction medium or halogenated hydrocarbons.
  • the extracts to be used according to the invention are produced by customary methods of extracting plants or parts of plants.
  • solvents with a certain polarity preferably organic solvents, water (distilled or not distilled) or mixtures of organic solvents and water, in particular low molecular weight alcohols, esters, hydrocarbons, ketones or halogen-containing hydrocarbons with more or less can be used as solvents for carrying out the extractions high water content can be used.
  • organic solvents water, alcohols, acids, primary and secondary amines
  • aprotic acetonitrile, dimethylformamide, dimethyl sulfoxide
  • Hexamethylphosphoric triamide, nitromethane, tert-amines) solvents Extraction with water, methanol, ethanol, pentane, hexane, heptane, acetone, chloroform, propylene glycols, polyethylene glycols, ethyl acetate, dichloromethane, trichloromethane and mixtures thereof is particularly preferred.
  • the extraction is generally carried out at 15 to 100 ° C., preferably at 20 to 45 ° C., more preferably approximately in the case of body heat. In one possible embodiment, the extraction is carried out under an inert gas atmosphere to avoid oxidation of the ingredients of the extract.
  • the extraction times are determined by the person skilled in the art depending on the starting material, the extraction process, the extraction temperature, the ratio of solvent to raw material, etc. set.
  • the crude extracts obtained can optionally be subjected to further customary steps, such as purification, concentration and / or decolorization. If desired, the extracts produced in this way can, for example, be subjected to a selective separation of individual undesirable ingredients.
  • the present invention includes the knowledge that the extraction conditions and the yields of the final extracts can be selected depending on the desired field of use. If desired, the extracts can then be subjected to spray or freeze drying, for example.
  • extract according to the present invention is accordingly understood to mean a substance and / or mixture of substances which is obtained from a plant by one or more extraction and / or other process steps.
  • the Styrax crotonoides in particular the Styrax crotonoides subsp.
  • Plant parts fraserensis, Styrax benzoin, and / or Styrax tonkinensis used to be the roots, barks (the root, the stem and the stem), the leaves and leafed stems, the fruits, grains (seeds) and / or flowers.
  • Styrax crotonoides in particular and preferably that, is used for the preparation of the extract or extracts of the plant Subspecies Styrax crotonoides subsp. fraserensis, Styrax benzoin, and / or Styrax tonkinensis, preferably above-ground plant material, particularly preferably the leaf material used, under "above-ground plant material" from Styrax crotonoides, in particular the subspecies Styrax crotonoides subsp.
  • fraserensis, Styrax benzoin, and / or Styrax tonkinensis is understood for the invention to be fresh or dried material from the leaves and / or stems, which can be harvested from plants without significantly impairing the viability, even if it is to a considerable extent, for example up to 90 % and typically around 50% is harvested.
  • "Above-ground plant material” has the advantage over material from the roots that crops of this perennial can be harvested repeatedly without new planting.
  • the present invention further relates to a pharmaceutical composition, the pharmaceutical composition comprising at least one Styrax crotonoides according to the invention, in particular subspecies Styrax crotonoides subsp. contains fraserensis, Styrax benzoin, and / or Styrax tonkinensis extract and / or an extract of the Knema laurina plant.
  • Knema laurina from the Myrisicaceae family has already been described in publications, inter alia in connection with the isolation of novel alkyl and alkenylphenols (Kijjoa A. et al. Planta Mediaca (1991) 57.6: 75-577 .; Gonzalez M. et al Phytochemistry (1996) 43.6: 1333-1337), Kijjoa A. et al. the isolation and identification of five compounds from the bark of Knema laurina and Knema tenuinervia subsp. setosa. Extracts of the latter have been used to treat cancer in traditional Thai medicine. Knema laurina is a shrub or tree that can grow up to 30m high. The strain of Knema oblongifolia Wall is known for use as a blood tonic. Knema angustifolia is used against stomatitis (inflammation of the oral cavity).
  • compositions or compositions of the invention are produced using conventional solid or liquid carriers or diluents and customary pharmaceutical and technical auxiliaries in accordance with the desired type of application with a suitable dosage in a manner known per se.
  • Preferred preparations exist in a dosage form which is oral, enteral or parenteral, for example ip (intraperitoneal), iv (intravenous), im (intramuscular) or percutaneous, application is suitable.
  • dosage forms are, for example, tablets, film-coated tablets, dragees, pills, capsules, powders, creams, ointments, lotions, liquids, such as syrups, gels, injectable liquids, for example for ip, iv, in or percutaneous injection, etc.
  • depot forms such as implantable preparations and suppositories are suitable.
  • the individual preparations release the extracts according to the invention gradually, depending on their type, or the entire amount in a short time to the body.
  • capsules, pills, tablets, coated tablets and liquids or other known oral dosage forms can be used as pharmaceutical preparations.
  • the medicaments can be formulated in such a way that they either release the active substances in a short time and release them to the body or have a depot effect, so that a longer-lasting, slow supply of active substance to the body is achieved.
  • the dosage units can contain one or more pharmaceutically acceptable carriers, for example substances for adjusting the rheology of the medicament, surface-active substances, solubilizers, microcapsules, microparticles, granules, diluents, binders such as starch, sugar, sorbitol and gelatin Fillers such as silica and talc.
  • pharmaceutically acceptable carriers for example substances for adjusting the rheology of the medicament, surface-active substances, solubilizers, microcapsules, microparticles, granules, diluents, binders such as starch, sugar, sorbitol and gelatin Fillers such as silica and talc.
  • Lubricants, dyes, fragrances and other substances for example substances for adjusting the rheology of the medicament, surface-active substances, solubilizers, microcapsules, microparticles, granules, diluents, binders such as starch, sugar, sorbito
  • Corresponding tablets can be obtained, for example, by mixing the extract according to the invention with known auxiliaries, for example inert diluents such as dextrose, sugar, sorbitol, mannitol, polyvinylpyrrolidone, disintegrants such as corn starch or alginic acid, binders such as starch or gelatin, lubricants such as carboxypolymethylene, carboxymethyl cellulose, cellulose acetate phthalate, or poly become.
  • auxiliaries for example inert diluents such as dextrose, sugar, sorbitol, mannitol, polyvinylpyrrolidone, disintegrants such as corn starch or alginic acid, binders such as starch or gelatin, lubricants such as carboxypolymethylene, carboxymethyl cellulose, cellulose acetate phthalate, or poly become.
  • auxiliaries for example inert diluents such as dextrose, sugar
  • Coated tablets can accordingly be produced by coating cores produced analogously to tablets with agents conventionally used in tablet coatings, for example polyvinylpyrrolidone or shellac, gum arabic, talc, titanium oxide or sugar.
  • the coated tablet can also consist of several layers, wherein the auxiliaries mentioned above for the tablets can be used.
  • Active ingredients can also be formulated in the form of a solution which is intended for oral administration and which, in addition to an active plant extract according to the invention, comprises a pharmaceutically acceptable oil and / or a pharmaceutically acceptable component contains compatible lipophilic, surface-active substance and / or a pharmaceutically acceptable hydrophilic, surface-active substance and or a pharmaceutically acceptable water-miscible solvent.
  • compositions which can be used externally are to be used, they must be such that the extracts according to the invention are supplied to the body in sufficient quantities.
  • dosage forms contain auxiliaries, for example substances for adjusting the rheology of the pharmaceuticals, surface-active agents, preservatives, solubilizers, thinners, substances for increasing the permeability for the extracts according to the invention through the skin, dyes, fragrances and skin protection agents, such as conditioners and moisture regulators.
  • other active substances can also be contained in the medicament (Ulimanns Enzyklopadie der Technische Chemie, Volume 4 (1953), pp. 1-39; J. Pharm. Sei.
  • the extracts according to the invention can also be used in suitable solutions, such as, for example, physiological saline, as a reflection or injection solution.
  • suitable solutions such as, for example, physiological saline, as a reflection or injection solution.
  • the active ingredients can be dissolved or suspended in a physiologically compatible diluent.
  • Particularly suitable diluents are oily solutions, such as solutions in sesame oil, castor oil and cottonseed oil.
  • solubilizers such as benzyl benzoate or benzyl alcohol, can be added.
  • any liquid carrier in which the extracts according to the invention are dissolved or emulsified can be used to formulate an injectable preparation.
  • These liquids often also contain substances for regulating the viscosity, surface-active substances, preservatives, solubilizers, thinners and other additives with which the solution is adjusted iosotonic.
  • Such preparations can be formulated so that a delayed release of the active ingredient is made possible.
  • Known techniques can be used for this purpose, for example depots that dissolve or work with a membrane.
  • Implants can be used as inert materials, for example contain biodegradable polymers or synthetic silicones, for example silicone rubber, the dosage of the extracts can vary depending on the type of application, age and weight of the patient, type and severity of the disease to be treated and similar factors.
  • the daily dose can be given as a single dose to be administered once or divided into two or more daily doses.
  • Neurodegeneration as a consequence of one or more pathological events primarily destroys the brain parenchyma and then also neighboring neurons through invading microglial cells / macrophages.
  • the latter consequential damage is essentially responsible for the loss of brain functions, which e.g. occur after a stroke (Mabuchi et al. (2000) Stroke 31 (7): 1735-1743); Stoll et al. (1998) Glia 22 (4): 329-337).
  • the death of neurons is mainly caused by toxic cytokines, nitrogen radicals and oxygen radicals, which are formed in large quantities by the invading microglial cells and macrophages.
  • compositions based on the plant Styrax fraserensis, Styrax benzoin, and / or Styrax tonkinensis and / or Knema laurina now show a surprisingly good neuroprotective, neuroregenerative and / or anti-inflammatory effect with regard to the fact that they inhibit the production and release of the pro-inflammatory cytokines, as well as the radicals by the microglial cells and the macrophages.
  • a further aspect of the present invention provides a pharmaceutical composition according to the invention which contains a plant extract according to the invention or plant extracts according to the invention in amounts between 0.001% and 25%, based on the final preparation, with the proviso that the amounts indicated are in contact with water and possibly other auxiliary substances. and add additives to 100% by weight.
  • the amount of plant extracts used in the preparations mentioned depends on the concentration of the individual ingredients and on the type of Use of the extracts .. As a rule, 0.001 to 25% by weight, in particular 0.03 to 10, and in particular 0.1 to 5% by weight of the plant extract, based on the final preparation of the pharmaceutical composition.
  • the total proportion of auxiliaries and additives can be 1 to 50, preferably 5 to 40,% by weight, based on the final preparation of the pharmaceutical composition.
  • the preparation can be produced by customary cold or hot processes
  • the pharmaceutical composition according to the invention generally contains at least one ingredient from the group consisting of saponins, flavone derivatives, tannins, sterols, proteins, carbohydrates, phenolic acids, xanthone derivatives, carotenoids and triterpenes.
  • Another object of the invention is a method for producing an extract of the plant Styrax crotonoides, in particular the subspecies Styrax crotonoides subsp. fraserensis, Styrax benzoin, and / or Styrax tonkinensis, solvents or mixtures of solvents being used for the extraction, which are selected from the group formed by distilled or undistilled water, low molecular weight alcohols, esters, hydrocarbons, ketones or halogen-containing hydrocarbons , Proteins, carbohydrates, phenolic acids, xanthone derivatives, carotenoids and triterpenes.
  • Another object of the present invention is the use of at least one extract of the Knema laurina plant and / or Styrax crotonoides, in particular the subspecies Styrax crotonoides subsp. fraserensis, Styrax benzoin, and / or Styrax tonkinensis, alone or in combination with at least one other active ingredient, for the production of a pharmaceutical composition for the prevention and / or treatment of neurological diseases and / or inflammation-related diseases.
  • Neuroinflammation is a characteristic of various neurodegenerative diseases and is characterized by the accumulation of a large number of activated microglial cells and asterocytes and a small number of T cells in the affected brain regions.
  • the extracts according to the invention can be used neuroprotectively (i.e. preventively) in order to prevent damage or killing of neuronal cells.
  • the invention also includes the use of the extracts for the treatment of diseases according to the invention in order, for. B. localize inflammatory reactions and prevent further damage to neighboring cells.
  • neurological diseases are therefore diseases in which primary or secondary inflammatory processes play a role and in which neuronal damage or neuronal cell death occurs.
  • neurological diseases in the sense of the invention denotes diseases of the central and peripheral nervous system such as. B.
  • Senile dementia multi-infarct dementia
  • Restless leg syndrome epilepsy, cell damage caused by hypolgyemia, hypoxia and ischemia; neuronal damage caused by uncontrolled movements; Asph xia as well as psychoses, schizophrenia, anxiety, pain, migraines and vomiting; functional disorders such as memory disorders (amnesia), disorders of the learning process, vigilance symptoms and withdrawal symptoms after chronic use of addictive substances such as benzodiazepines, hallucinogens, alcohol, cocaine or opiates, as well as AIDS-induced encephalopathy and other infection-related encephalopathies caused by rubella viruses, herpes viruses , Borrelia and caused by unknown pathogens; Creutzfeld-Jakob disease and neurodegenerative diseases of the peripheral nervous system such as polyneuropathies and polyneuritides.
  • Inflammation-related diseases in the sense of the invention generally refer to diseases which arise as a result of uncontrolled reactions of the immune system. Inflammatory diseases can occur throughout the body. For example, mucous membrane events can occur, such as: B. inflammation of the upper respiratory tract and stomatitis apthosa. Inflammation can also occur anywhere in the airways as well as in the gastrointestinal tract. Some inflammation of the Mucous membranes are not directly mediated by an infectious agent, but are caused by overreactions of the immune system in response to a microbial infection. Exemplary diseases are acute obstructive bronchitis and asthma-aggravated respiratory infections. An inflammation harmful to health can occur in places other than the mucous membranes.
  • rheumatic arthritis systemic juvenile rheumatoid arthritis and psoriasis arthritis
  • Reiters syndrome ankylosing spondylitis, Crohn's and M's disease
  • Whipple's disease with arthritis ulcerative colitis and systemic lupus erythematosus.
  • inflammation-related diseases such as B. allergies, autoimmune diseases, arteriosclerosis or transplant rejection reactions.
  • TNF- ⁇ Tumor necrosis factor alpha
  • IL-6 interleukin 6
  • TNF, nitrogen and oxygen radicals and IL-6 play a central role Many immune effector functions and cellular interactions necessary to elicit an effective host response during inflammation and immune response, but uncontrolled generation of inflammatory cytokines is detrimental to the host.
  • the extracts according to the invention are able to prevent or decisively minimize the release of the inflammatory cytokines by the macrophages or microglial cells.
  • Further studies of the intracellular mechanism of inhibition or the minimizing of the release of the inflammatory cytokines have shown that the extracts from Knema laurina inhibit the phosphorylation of ERK-1/2 by phosphorylated MEK and thereby the translocation of the dimerized P-ERK-1/2 in the cell nucleus and the activation of the "downstream" kinases such as. B. reduce p70 S6K in the macrophages or microglial cells.
  • Knema laurina extracts almost completely translocate NF-kappa- into the cell nucleus and then induce the expression of iNOS, which is responsible for the release of nitric oxide.
  • iNOS which is responsible for the release of nitric oxide.
  • Styrax crotonoides especially with the subspecies Styrax crotonoides subsp. fraserensis, Styrax benzoin, and or Styrax tonkinensis do not appear to inhibit NF-kappa-j ⁇ translocation.
  • Styrax crotonoides especially the subspecies Styrax crotonoides subsp.
  • the present invention further relates to the use according to the invention of the plant extracts from Knema laurina, solvents used as the extraction medium for the plant extracts, which are selected from the group formed by distilled or undistilled water, low molecular weight alcohols, esters, ethers, Polyols, chlorinated solvents, hydrocarbons, ketones or halogenated hydrocarbons.
  • solvents used as the extraction medium for the plant extracts which are selected from the group formed by distilled or undistilled water, low molecular weight alcohols, esters, ethers, Polyols, chlorinated solvents, hydrocarbons, ketones or halogenated hydrocarbons.
  • the same conditions apply to the extraction of Knema laurina extracts as those for Styrax crotonoides, in particular the subspecies Styrax crotonoides subsp. fraserensis, Styrax benzoin, and / or Styrax tonkinensis have been described in detail above.
  • Another object of the present invention relates to the use according to the invention of the plant extracts from Knema laurina, the plant parts used for the preparation of the Knema laurina extract or the Knema laurina extracts the roots, bark, leaves and leafed stems, the fruits, grains and / or Flowers are. It is particularly preferred to use the leaf material to prepare the Kema laurina extracts.
  • Yet another object of the present invention relates to the use according to the invention of the plant extracts as mentioned above in connection with at least one further neuroprotective and / or anti-inflammatory medicinally active agent / active substance.
  • these are agents that are currently used in the therapy of infiammatory damage in the CNS or as neuroprotective agents either in clinical studies or in experimental stage: NMDA receptor antagonists; Calcium channel antagonists; Antioxidants and radical scavengers; Antibodies against cell surface molecules; Cytokines and chemokines; Antibodies to cytokines and chemokines; Immunosuppressive drugs; statins; Phosphodiesterase inhibitors and antibiotics.
  • Fig. 5 Reduced inflammatory neuronal damage by extract from Knema laurina (ALM 5540): Propidium iodide staining of initially NMDA-damaged living Brain tissue from organotypic hippocampal cut cultures 3d after transfer of BV-2 microglial cells in the presence of various dilutions (ALM 5540) or absence of the extract from Knema laurina.
  • Fig. 6 Reduced inflammatory neuronal damage by extract from Styrax crotonoides subsp. fraserensis (ALM 5552): Propidium iodide staining of initially NMDA-damaged living brain tissue from organotypic hippocampal cut cultures 3d after transfer of BV-2 microglial cells in the presence of various dilutions (ALM 5552) or absence of the extract from Styrax fraserensis.
  • ALM 5552 Propidium iodide staining of initially NMDA-damaged living brain tissue from organotypic hippocampal cut cultures 3d after transfer of BV-2 microglial cells in the presence of various dilutions (ALM 5552) or absence of the extract from Styrax fraserensis.
  • Fig. 10 Altered signal transduction by extract from Knema laurina in microglial cells
  • I Effect of extract from Knema laurina on the phosphorylation of MEK (p-MEK, Fig. 10a), of ERK-1/2 ( ⁇ -ERK-1 / 2, Fig. 10b) and von raf and elk (p-raf, p-elk, Fig. 10c) and the induction of iNOS (Fig. 10c) 0-24h after stimulation with LPS with or without the presence of extract from Knema laurina (ALM 5540) in BV-2 microglial cells.
  • Fig. 11 Modified signal transduction by extract from Knema laurina in microglial cells
  • II Effect of extract from Knema laurina on the nuclear re translocation of the phosphorylated ERK-1/2 (p-ERK-1/2, Fig. 11a) and of p65 (NF-kappa ß, Fig. 11b) and on the phosphorylation of IkB (Fig. 11c) 0-24h after stimulation with LPS with or without the presence of extract from Knema laurina (ALM 5540) in BV-2 microglial cells.
  • Fig. 13 Proapoptotic effect through extracts from Knema laurina and Styrax crotonoides subsp. fraserensis in activated microglial cells.
  • Annexin-5 expression in relation to the unstimulated control without LPS (con, -LPS) 24h after stimulation with LPS (+ LPS) in the presence or absence of extracts from Knema laurina (ALM 5540, Fig. 13a) or Styrax crotonoides subsp. fraserensis (ALM 5552, Fig. 13b).
  • Fig. 14 Induction of neurogenesis by extract of Knema laurina in living brain section cultures according to OGD.
  • Living organotypic hippocampal brain section cultures OHSC were treated for 3 days with Knema laurina extract (1: 5,000, corresponding to 30 ⁇ g / ml ww) under basal conditions (without OGD) or directly after OGD.
  • Control treatment with solvent only (1: 5000 ethanol in PBS).
  • BrdU was added to the medium for the first 2 days. After 6 days the cultures were fixed and double stained with BrdU / DCX. Newly formed neurons are double positive BrdU / DCX.
  • Plant components (leaves, bark, roots) from Styrax crotonoides or Knema laurina were collected from the Malaysian peninsula or Sabah (collection areas: Fräsers Hill, West Malaysia; Danum Valley, Sabah; Keläbit Highlands, Sarawak).
  • the pulp was removed by filtration and the solvent was removed by rotovaporation (Buchi, Switzerland) to obtain a first crude methanol extract (F0001).
  • the methanol extract was then partitioned with a 1: 1 mixture of chloroform and water.
  • the undissolved material was then removed by filtration (F0002) and the solvent was separated by a separating funnel.
  • the aqueous extract was freeze-dried (F0004).
  • the extract F0003 was obtained by evaporation of the chloroform.
  • the chloroform extract was then further partitioned with a 1: 1 mixture of hexane and 20% aqueous methanol.
  • a hexane extract F0005
  • an aqueous methanol extract F0006
  • the methanolic extracts (F0006) were transferred from the National University of Malaysia, Bangi, Kuala Lumpur, Malaysia to the Institute for Cell and Neurobiology, Center for Anatomy, University Medicine Berlin. There the extracts were blown in a nitrogen stream at room temperature to maximum dryness and the respective extract weights were determined. The blown extracts were then taken up in 5 ml of ethanol, various dilutions were made in phosphate-buffered physiological saline (PBS) and these were frozen at -80 ° C.
  • PBS phosphate-buffered physiological saline
  • Knema Laurina extract in dilutions above 10 "4 inhibits the CD14 / TLR4-stimulated release of NO by BV-2 microglial cells by 50% (Fig. La), while the vitality of the cells up to a dilution of 2X10 "4 was not impaired (Fig. lbc). Above a dilution of 5 x 10 "4 , the number of vital cells also decreased (Fig. Lbc).
  • the extracts were examined in dilution ranges from 10 " to 10 in 8 independent experiments for their toxicity against a neuronal cell line, as well as with regard to a possible protective effect in different neuronal damage models.
  • HT22 cells an NMDA receptor-deficient primary cell line
  • the damage models used include oxidative damage by hydrogen peroxide, nitrosative damage by the NO donor sodium nitroprusside and the oxidative NMDA receptor-independent glutamate damage by incubation with glutamate. These scenarios are related to brain tissue damaged by inflammation Damaging mechanisms based on invading inflammatory cells, and the NMDA receptor-independent glutamate damage involves activation of the MAPK cascade in neurons, a central activating signaling pathway in microglial cells Coincidence of a deactivating effect on microglial cells with a protective effect in the case of NMDA receptor-independent glutamate damage in neurons indicates an intervention of the extract in the MAPK signaling pathway, which could actually
  • the Styrax crotonoides subsp. fraserensis extract developed no toxic effects on HT22 neurons in dilutions of 10 " to 10 " 3 (FIG. 4a). From a dilution of 10 "4 it was protective against oxidative glutamate damage (FIG. 4c), protective effects against hydrogen peroxide (FIG. 4b) or NO-mediated damage (FIG. 4d) could not be found. 1.5. Effect on inflammatory neuronal damage in living organotypic brain tissue
  • the model of the organotypic hippocampal cut culture serves this purpose, in which after 9 days in culture in the middle of the tissue there are organotypic conditions (no glia activation, normal metabolism, neuronal activity).
  • organotypic conditions no glia activation, normal metabolism, neuronal activity.
  • the Knema laurina extract reduced the release of the B-cell-stimulating cytokine IL-6 by approximately 50% in living brain tissue at a dilution of 5X10 "4 and in cultured BV-2 microglial cells by approximately 50% at a dilution of 2X10 "4 (Fig. 8).
  • the release of TNF- ⁇ was reduced by this extract in cultured BV-2 microglial cells at a dilution of 5x10 "4 by about 50%, but not in living him tissue (Fig. 8). Therefore, the dilution at 10 "4 Measured neuroprotective effects in living him tissue are unlikely to be due to a reduction in the pro-inflammatory cytokine TNF- ⁇ , while the reduction of IL-6 may be involved.
  • the extract of Styrax crotonoides subsp. fraserensis reduced the release of the cytokine IL-6 in living rasp tissue by about 50% at a dilution of 10 " and in cultivated BV-2 microglial cells by less than 20% at this dilution (FIG. 9).
  • the release of TNF- ⁇ was only reduced by this extract in cultured BV-2 microglial cells at a dilution of 2X10 "4 by about 10%, but by about 50% in living him tissue (FIG. 9). Therefore, the complete neuroprotective effect in living rasping tissue measured at a 10 " dilution is unlikely to be a reduction in the pro-inflammatory cytokine IL-6 to be led back.
  • TNF- ⁇ reduction can be involved in the measured neuroprotective effect.
  • Annexin-5 The exposure of Annexin-5 to the outside of the outer cell membrane is an early and specific event of apoptosis. While stimulation of BV-2 microglial cells with LPS via the CD14 / TLR4 receptor does not lead to an increase in the proportion of apoptotic cells, simultaneous incubation with the extract of Knema laurina and Styrax fraserensis leads to an increase of 10 "4 Detection of Annexin-5 on the surface of BV-2 microglial cells (Fig. 13) Unstimulated BV-2 microglial cells showed no increased proportion of apoptotic cells after incubation with the extracts (Fig. 13) However, the total number of cells is too low to be able to detect a reduced number of vital cells there (FIGS. 1c, 3c). 3. Summary of results
  • Extracts of the Knema laurina plant contain active ingredients, individually or in their entirety:
  • NO nitrogen monoxide
  • pro-inflammatory cytokines such as Can decrease or cancel IL-6 from activated immune cells
  • NO nitrogen monoxide
  • pro-inflammatory cytokines such as Reduce or cancel TNF- ⁇ from activated immune cells
  • the extracts were transported as methanolic suspensions in glass vessels from the National University Malaysia to Germany and stored immediately at -80 ° C on arrival. Before use, the methanol was blown under a stream of nitrogen, the dry extract weighed and resuspended in 5 ml of absolute ethanol. Thereby 147.9 mg / ml for Knema Laurina and for Styrax crotonoides subsp. fraserensis 5552 84 mg / ml reached (w / w.). The suspensions thus produced were diluted 1:10 2 , 1:10 3 , 1:10 4 , 1:10 5 in sterile PBS and these dilutions were used as stock solutions.
  • BV-2 microglial cells have morphological, phenotypic and functional properties of freshly isolated primary microglia and represent an adequate model system to test the activation and deactivation of microglial cells in vitro.
  • the cells were cultured in medium consisting of DMEM, heat-inactivated fetal calf serum (FCS) [10%], glutamine [10%] and penicillin / streptomycin [5%]. Cultivation in the incubator was carried out at a CO 2 content of 5% and a temperature of 37 ° C. The cells were passed every 2-3 days.
  • the cells were activated by CD 14 / TLR4 activation via lipopolysaccharide (LPS) [10 ⁇ g / ml].
  • LPS lipopolysaccharide
  • the microglia cells were seeded in 96-well microtiter plates with 100 ⁇ l / well in a concentration of 2 ⁇ 10 5 cells / ml and left for 24 hours.
  • the supernatant was removed and new medium with the respective substrates was added (LPS, plant extract, ethanol, PBS).
  • LPS plant extract, ethanol, PBS
  • the experiment was ended and the cells or supernatant were fed to the respective assays.
  • HT-22 Hippocampal Neurons (murine): HT-22 cells are an established murine hippocampal neuron cell line, which were developed as a subclone from immortalized HT-4 cells and do not carry an ionotropic glutamate receptor (Mäher et al. "The role of monoamine metabolism in oxidative glutamate toxicity "(1996) J. of Neuroscience 16 (20), 6394-6401). The cells are cultured in DMEM medium containing FCS [10%], glutamine [10%], penicillin / streptomycin [ 5%] and glucose is added. The passage took place every 2-3 days. Model neuronal damage was treated with glutamate [20 mM] (excitotoxic damage), HO 2 [150 ⁇ M] (oxidative damage) and the NO donor sodium nitroprasside [ 250 ⁇ M] (nitrosative damage).
  • MEM ice-cold preparation medium
  • the coronary sections with a layer thickness of 350 ⁇ m obtained after cutting with the vibratome are placed on Millipore filter membranes (pore size 0.1 ⁇ m) in the wells of a 6-hole culture dish and with culture medium (MEM: HBSS 2: 1, 25% normal horse serum, 2% L-glutamine, 2.64 mg / ml glucose and penicillin / streptomycin) at 35 ° C, 5% CO 2 and 95% ambient air in a humidified atmosphere.
  • MEM HBSS 2: 1, 25% normal horse serum, 2% L-glutamine, 2.64 mg / ml glucose and penicillin / streptomycin
  • the medium only has direct contact with the hippocampal tissue from below.
  • BV-2 microglial cells incubated with 20 ⁇ g / ml rhodarnine-labeled dextran MiniRuby for 24 h are washed several times with PBS, resuspended in a small volume of culture medium (see above) and placed on the surface of the hippocampal cut cultures. 1, 2 and 3 days after the NMDA lesion, the migration of the microglial cells to the site of the neurodegeneration is first documented in an intact section using fluorescence and light microscopy.
  • the sections are then cut after fixation (4% formaldehyde, 0.1% glutaraldehyde, 15% picric acid) and transfer to 0.8 M sucrose solution in a cryostat, mounted on gelatinized slides and capped with hnmunomount.
  • fixation 4% formaldehyde, 0.1% glutaraldehyde, 15% picric acid
  • transfer 0.8 M sucrose solution in a cryostat
  • the site-specific migration into the area of neurodegeneration is then statistically evaluated.
  • the number and density of damaged neurons is determined by propidium-iodide staining in unfixed cut tissue.
  • the supernatant was then carefully removed and an amount of HCl / isopropanol (1: 100) equal to the medium supernatant was added to the wells for cell lysis.
  • the dye formed in the cells was then able to dissolve on a shaker at room temperature for 30 minutes.
  • the absorbance was measured on a plate reader at 562 nm (measuring filter) against 630 nm (reference filter).
  • the aim of these investigations was 1.) to characterize the effects of the plant extracts on surface protein expression (CD 11a, CD 11b, CD 18, ICAM-1 expression) of the BV-2 cells and 2.) together with the non-specific detection of the Cell vitality using the MTT assay, the quantification of the proportion of apoptotic cells using annexin V determination.
  • Method After the end of the test, the BV-2 cells were fixed in a 75 cm2 bottle with formaldehyde [4%] by scraping before harvesting for the surface protein measurement. There was no fixation for the apoptosis measurement.
  • the cells were counted and in an amount of 2-5 x 105 cells in FACS buffer (FCS 2%, NaN3 0.1% in PBS) or annexin buffer (HEPES 0.1 M in NaOH, NaCl 1.4 M, CaCl 2 25 mM, pH 7.4) distributed over the FACS tubes.
  • FACS buffer FCS 2%, NaN3 0.1% in PBS
  • annexin buffer HPES 0.1 M in NaOH, NaCl 1.4 M, CaCl 2 25 mM, pH 7.4
  • the antibodies were added to the cells according to the instructions.
  • the measurement was carried out by the FACS device and evaluated using the FLOWJO program.
  • the isotypes of the respective antibodies recommended by the manufacturer were used to control the surface proteins.
  • the cells were stained twice with propidium iodide (PT) to check the cells that had died from necrosis.
  • Annexin-positive PI-negative cells were assigned to the stage of the onset of apoptosis, A-neg./PI-neg. were classified as vital, A.-pos / PI-pos. count as "total death rate” and A.-neg./PI-pos. Cells were discarded as an artifact.
  • Source Longobardi-Givan, A. "Flow Cytometry - First Principles” Wiley Liss, New York 1992 Nebe, CT. "Flow cytometric measurement technology and data evaluation" Infusionsther Transfusionsmed 23: 111 - 113 (1996)
  • Immunoblot analyzes were carried out with the aim of characterizing the effects of the plant extracts on CD14 / TLR4-induced signal transduction in BV-2 microglial cells.
  • the specific phosphorylation of p38, CREB, MEK, ERK-1/2, raf, p70, AKT and I-kappaB, the expression of egr-1 was examined.
  • OHSC living organotypic hippocampal cut cultures
  • OGD model oxygen and glucose
  • the cell culture inserts with the OHSCs were transferred to 1 ml glucose-free medium (GFM) in sterile 6-well culture dishes (TPP).
  • GFM medium was previously saturated with 5% CO 2 /95% N 2 for 10 min.
  • the 6-well culture dishes were then transferred to a hypoxia chamber (Billups-Rothenberg). After 40 min under ODG conditions in the temperature-controlled hypoxia chamber in GFM medium and N 2 / CO 2 atmosphere, the cultures were again cultivated in a normal, glucose-containing medium under normoxia conditions.
  • the OHSCs were analyzed after 24h or 48h after OGD. (see Fig. 14)

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Abstract

L'invention concerne des extraits des plantes suivantes Knema laurina et/ou Styrax crotonoides, notamment de la sous-espèce Styrax crotonoides subsp. fraserensis, Styrax benzoin, et/ou Styrax tonkinensis. L'invention concerne également un procédé pour produire ces extraits, des compositions pharmaceutiques les contenant, ainsi que l'utilisation de ces extraits pour produire une composition pharmaceutique servant notamment à la prévention et/ou au traitement de maladies neurologiques et/ou inflammatoires.
PCT/EP2005/006166 2004-06-08 2005-06-08 Nouveaux extraits de plantes presentant des proprietes neuroprotectrices, neuroregeneratrices et antiphlogistiques WO2005120528A1 (fr)

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EP3259991A1 (fr) 2016-06-21 2017-12-27 Universität Basel Compositions fongicides

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EP3451836B1 (fr) * 2016-05-02 2021-08-25 Y&B Mother's Choice Ltd. Composés comprenant des saponins et des extraits de plant
CN109316540A (zh) * 2017-07-31 2019-02-12 吴冰 一种健脾和胃穴位贴及其制备方法

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Publication number Priority date Publication date Assignee Title
JPH10306019A (ja) * 1997-05-08 1998-11-17 Takasago Internatl Corp 口腔用組成物

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Publication number Priority date Publication date Assignee Title
JPH10306019A (ja) * 1997-05-08 1998-11-17 Takasago Internatl Corp 口腔用組成物

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Title
DATABASE WPI Section Ch Week 199905, Derwent World Patents Index; Class B04, AN 1999-054264, XP002347732 *
KIJJOA A ET AL: "CONSTITUENTS OF KNEMA-LAURINA AND KNEMA-TENUINERVIA-SSP-SETOSA", 1991, PLANTA MEDICA, VOL. 57, NR. 6, PAGE(S) 575-577, ISSN: 0032-0943, XP009054573 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3259991A1 (fr) 2016-06-21 2017-12-27 Universität Basel Compositions fongicides
WO2017220565A1 (fr) 2016-06-21 2017-12-28 Universität Basel Compositions fongicides

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