CN104288168A - Application of trillin in preparation of drug used for treating and/or preventing diseases mediated by microglial cells - Google Patents
Application of trillin in preparation of drug used for treating and/or preventing diseases mediated by microglial cells Download PDFInfo
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
Abstract
The invention relates to a new application of trillin in preparation of a drug used for treating and/or preventing diseases mediated by microglial cells, wherein the diseases includes stroke, Alzheimer disease, Parkinson's disease, cerebral trauma, multiple sclerosis, amyotrophic lateral sclerosis, AIDS relative dementia, bovine spongiform encephalopathy and the like. The traditional Chinese medicine monomer, the trillin, has following new functions of capable of significantly inhibiting generation of an inflammatory medium NO activating the microglial cells and inhibiting cell death activated and induced by the microglial cells. The trillin can be used for treating and preventing the cerebral immune inflammatory related diseases, such as the stroke, the Alzheimer disease, the Parkinson's disease, the cerebral trauma, the multiple sclerosis, the encephalitis and the like.
Description
Technical field
The present invention relates to medical art, relate to particularly cryptogenin for the preparation of prevent and/or treat microglia mediation relevant disease, especially nerve immunity diseases associated with inflammation medicine in purposes, especially relate to cryptogenin for the preparation of the purposes prevented and/or treated in the medicine of the immune correlated diseases such as apoplexy, Alzheimer, parkinson disease, cerebral trauma, multiple sclerosis, amyotrophic lateral sclerosis, aids related dementia, bovine spongiform encephalopathy.In addition, the invention still further relates to the medical composition and its use comprising cryptogenin.
Background technology
Microglia (microglia) is the one in the neurogliocyte of central nervous system, and its volume is minimum, and cell space is shaft-like or oval, and there are many spinous process on surface.The effect of microglia is main similar to macrophage, as participating in immunoreactive main effects cell in central nervous system, in the inflammation that nervous system primary disease or infection cause, participates in immunne response.Central nervous system disease is often with a large amount of necrosis and the apoptosis of neuronal cell, and around these necrotic lesions, assemble a large amount of microglia activated, and the cytokines such as its NO secreted, tumor necrosis factor (TNF-α), interleukin 6 (IL-6) can be detected, the effect of inflammatory reaction in these disease process of therefore its mediation is valued by the people gradually.
Some Autoimmune neuropathies systemic disease is relevant to microglia, and microglia makes nervous system injury by offering antigenic activation T cell.At present, the inflammatory reaction of this microglia mediation is considered to the mechanism of the generation of multiple nervous system disease or deterioration, thus causes the extensive concern of people.
Alzheimer is also called presenile dementia, and be a kind of neurodegenerative diseases, it is extensively dead that main pathology shows as cerebral cortex neurons, causes cerebral atrophy, but seldom involve motor neuron.Along with deepening continuously of research, it is found that: by the dissection to Alzheimer Died Patients cerebral tissue, except the neurofibril strand group of being twisted into and a large amount of downright bad neuronal cell widely can be observed, the microglia that is activated and expression product, such as TNF-α, IL-6 etc. can also be found in its vicinity.This shows that Alzheimer is relevant with the activation of microglia.
Parkinson disease main manifestations is the nigral cell Progressive symmetric erythrokeratodermia degeneration of Basal ganglia, belongs to chronic disease.The material such as microglia, inflammatory factor be activated in a large number is had in the brain that it is found that Parkinsonian, the inflammatory reaction of pointing out Parkinsonian process and microglia to mediate is about (inflammatory reaction that the microglia see people such as Xu Ling mediates and central nervous system disease, Immunology Today, 2011,31st volume, the 2nd phase: 160-163 page).
In recent years, along with deepening continuously of research, finding that the abnormal activation of microglia (microglia) all plays a significant role in the diseases such as apoplexy, Alzheimer, parkinson disease, cerebral trauma, multiple sclerosis, amyotrophic lateral sclerosis, aids related dementia, bovine spongiform encephalopathy, is one of Etiological causing nerve injury in above-mentioned disease pathology evolution.Microglia is central nervous system's immunocyte, and under the pathological state of the diseases such as apoplexy, Alzheimer, parkinson disease, cerebral trauma, the minor variations of microenvironment can activate microglia, makes it from the quiescent condition fast steering state of activation.Whole activation process presents water fall effect: differentiation, propagation, and immune molecule is expressed or up-regulated, migrates to damaging part, and form, immundominance and changing function etc. occur.Its form upper volume increases, cell space becomes and justifies and have distortion, phagocytosis; Functionally start great expression some with antigen recognition with offer relevant membrane surface molecule, as expressed MHC II (MHC) etc.; Overexpression stromatin enzyme 9 (MMP9), degraded blood brain barrier.The more important thing is, the secretion level of the many cytokines of remarkable rise, wherein small part such as GDNF promotes neuronal survival, and major part such as tumor necrosis factor α (TNF α), interleukin-11 β (IL-1 β), interleukin 6 (IL-6) etc. have Neuronal Damage effect, also has chemotactic factor as mediated leucocytes chemotactics such as MCP 1 (MCP-1), irregular chemotactic factor (Fractalkine), macrophage inflammatory proteins 1 (MIP-1); Also produce nitric oxide (NO), reactive oxygen species (ROS) and Eicosanoids (as PGE2) etc., cause subsequent brain damage.Microglia is not only natural immunity effector lymphocyte, participates in starting and regulating acquired immunity simultaneously, participates in multiple sclerosis morbidity.
Therefore, suppress the overactivity of microglia, balance the various proinflammatory factors of its secretion, can the effective disease such as prevention and therapy apoplexy, Alzheimer, parkinson disease, cerebral trauma, multiple sclerosis, amyotrophic lateral sclerosis, aids related dementia, bovine spongiform encephalopathy.Document in the past or patent also show, naloxone (naloxone, J Pharmacol Exp Ther2000, 293:607-617), dextromethorphan (dextromethorphan, J Pharmacol Exp Ther2003, 305:212-218), tetrahydro isoquinoline derivative (CN101553229A, Chinese Patent Application No. 200780045274.0, publication date on October 7th, 2009), tripchlorolide, T 4 (CN101332200A, Chinese Patent Application No. 200810071485.8, publication date December in 2008 31 days) etc. can by suppressing Activated Microglia control brain inflammation relevant disease.
Summary of the invention
The present inventor has found that cryptogenin can be used for preventing and/or treating the disease of Activated Microglia mediation, the nerve immunity inflammatory diseases caused by neurotoxic effect of particularly microglia mediation, and more particularly described disease is selected from: apoplexy, Alzheimer, parkinson disease, cerebral trauma, multiple sclerosis, amyotrophic lateral sclerosis, aids related dementia, bovine spongiform encephalopathy.Based on above-mentioned discovery, complete the present invention.Present invention also offers the pharmaceutical composition comprising cryptogenin, and comprising the purposes of pharmaceutical composition in the nerve immunity inflammatory diseases caused by the neurotoxic effect mediated for the preparation of the disease, the particularly microglia that prevent and/or treat Activated Microglia mediation of cryptogenin, more particularly described disease is selected from: apoplexy, Alzheimer, parkinson disease, cerebral trauma, multiple sclerosis, amyotrophic lateral sclerosis, aids related dementia, bovine spongiform encephalopathy.
A first aspect of the present invention provide the cryptogenin of following formula for the preparation of prevent and/or treat Activated Microglia mediation disease medicine in purposes,
In one embodiment, described disease is the nerve immunity inflammatory diseases caused by neurotoxic effect of microglia mediation.In another embodiment, described disease is selected from: apoplexy, Alzheimer, parkinson disease, cerebral trauma, multiple sclerosis, amyotrophic lateral sclerosis, aids related dementia, bovine spongiform encephalopathy.
In one embodiment, every daily dose that described medicine gives object in need counts 0.01-1000mg/kg body weight with cryptogenin, is preferably 0.1-100mg/kg body weight, is more preferably 1-100mg/kg body weight.
In another embodiment, cryptogenin provides with the form of cryptogenin monomer, or with the form of the plant extract comprising cryptogenin, preferably provide with the form of the Fructus Tribuli crude saponin comprising cryptogenin.Preferably, described plant includes but not limited to: Fructus Tribuli (Tribulus terrestris), Trillium tschonoskii Maxim (Trilliumtschonoskii maxim).More preferably, described plant extract, the preferably Fructus Tribuli crude saponin cryptogenin that comprises more than 30% by weight, be preferably more than 60%, be more preferably more than 90%.
A second aspect of the present invention provides a kind of pharmaceutical composition of the disease for preventing and/or treating microglia mediation, it contains the cryptogenin preventing and/or treating effective dose or the plant extract comprising cryptogenin, preferably comprises Fructus Tribuli crude saponin, optional other drug and the optional pharmaceutically acceptable carrier of cryptogenin.Preferably, the nerve immunity inflammatory diseases caused by neurotoxic effect that mediates for microglia of the disease of described microglia mediation.Especially, the disease of described microglia mediation is selected from: apoplexy, Alzheimer, parkinson disease, cerebral trauma, multiple sclerosis, amyotrophic lateral sclerosis, aids related dementia, bovine spongiform encephalopathy.
In one embodiment, based on the gross weight of active component in described pharmaceutical composition, the content of described cryptogenin is 1-99%; Be preferably 20-80%; Be more preferably 40-60%; The content of described other drug is 99-1%; Be preferably 80-20%; Be more preferably 60-40%.
In a preferred embodiment, described other drug is selected from: levodopa, carbidopa, Selegiline, benzhexol, benzatropine, amantadine, bromocriptine, pergolide, tacrine, huperzine A, galantamine, xanomeline, donepezil hydrochloride, rivastigmine, memantine, piracetam, Dihydroergotoxine Mesylate, pentoxifylline, nicergoline, aspirin, melatonin, curcumin, desferrioxamine, idebenone, Tirilazad Mesylate and vitamin C.
In one embodiment, pharmaceutically acceptable carrier is selected from solvent, diluent, dispersant, suspending agent, surfactant, isotonic agent, thickening agent, emulsifying agent, antiseptic, binding agent, lubricant, stabilizing agent, hydrating agents, emulsifying accelerator, buffer agent, absorbent, coloring agent, flavouring agent, sweeting agent, ion-exchanger, releasing agent, smears, correctives and antioxidant.
Preferably, described pharmaceutical composition is the dosage form of unit dosage, the cryptogenin comprise 0.001mg-5000mg in the dosage form of this unit dose, being preferably 0.1mg-1000mg, being more preferably 1mg-100mg.
Especially, the dosage form of described pharmaceutical composition includes but not limited to tablet, capsule, powder, granule, lozenge, pill, solution, suspensoid, Emulsion, syrup, powder, granula subtilis, pilule, elixir, injection, medicinal drops, ointment, lotion, gel, emulsifiable paste, spray, suppository, patch.
Cryptogenin of the present invention generally uses with the form of pharmaceutical composition, and this compositions is containing the cryptogenin as active component and the pharmaceutically acceptable auxiliaries for the treatment of effective dose.Pharmaceutical composition containing cryptogenin, by oral, injection or mucosa delivery, can make the dosage form such as tablet, capsule, powder, granule, lozenge for clinical practice.The medicine of above-mentioned various dosage form all can be prepared according to the conventional method of pharmaceutical field.
This application provides Chinese medicine monomer cryptogenin and there is following functions: the generation reducing the cytotoxicity inflammatory mediators such as activated microglia NO, TNF-α.Thus the control being applied to the brain immune inflammation relevant diseases such as apoplexy, Alzheimer, parkinson disease, cerebral trauma, multiple sclerosis for cryptogenin provides directly strong experimental basis and theoretical basis, existing very strong science and novelty, have again very high development and application values.
As used herein, term " cryptogenin " is also referred to as disogluside or Trillum glycoside.The molecular weight of cryptogenin is 576.76, and molecular formula is C
33h
52o
8, No. CAS is 14144-06-0.The present invention's cryptogenin used is prepared according to such as Chinese patent application CN201110187744.5, or also can by commercial acquisition.According to the present invention, cryptogenin used can also be the plant extract comprising cryptogenin form, preferably provide with the form of the Fructus Tribuli crude saponin comprising cryptogenin.
As used herein, term " disease of microglia mediation " comprises the nerve immunity inflammatory diseases caused by neurotoxic effect of microglia mediation.Especially, the disease of microglia mediation is selected from: apoplexy, Alzheimer, parkinson disease, cerebral trauma, multiple sclerosis, amyotrophic lateral sclerosis, aids related dementia, bovine spongiform encephalopathy.
As used herein, term " apoplexy " also referred to as apoplexy, is a kind of cerebral blood circulation obstacle disease of unexpected onset in the traditional Chinese medical science.
As used herein, term " Alzheimer " is also called presenile dementia, is a kind of neurodegenerative diseases, and main clinical manifestation is hidden forgetful and short term memory loss that attack, slowly progress, activity of daily living Progressive symmetric erythrokeratodermia goes down, and has various neuropsychic symptom and behavior disorder.
As used herein, term " parkinson disease " is the common dyskinetic disorder of a kind of middle-aged and elderly people, also known as Parkinsonism, its main pathology changes into the formation of Lewy body in the necrosis of substantia nigra of midbrain degeneration of dopaminergic neurons and brain, and clinical main manifestations is the symptoms such as static tremor, myotonia, bradykinesia.
As used herein, term " cerebral trauma " refers to the traumatic brain injury that external force causes.
As used herein, term " multiple sclerosis " is a kind of inflammation of the central nervous system demyelinating disease by autoimmune caused by abnormal, alleviate in the course of disease-recurrence is important feature.
As used herein, term " amyotrophic lateral sclerosis " is a kind of progressive and fatal neurodegenerative diseases, involve upper motor neuron, have influence on again a kind of chronic progressive external degenerative disease of the trunk of lower motor neuron and domination thereof, extremity and Head And Face muscle.
As used herein, term " aids related dementia " refers to a kind of degenerative neural disease caused by HIV (human immunodeficiency virus), and clinical main manifestations is dull-witted.
As used herein, term " bovine spongiform encephalopathy " is also called mad cow disease, and being the neural infectious disease of a kind of Progressive symmetric erythrokeratodermia of the cattle caused by infectious agent, is a kind of Transmissible spongiform encephalopathy.The principal character of this disease is that spongy pathological changes occurs Medulla Bovis seu Bubali, and degenerates with brain function, and clinical manifestation is mentally deranged, movement disorder, dementia and dead.
As used herein, term " treatment " has its general sense, and refer to especially herein and adopt medicine of the present invention to process to the animal individual of the disease the suffering from microglia mediation particularly disease of microglia of the present invention mediation, effects such as treating to producing described disease, curing, alleviate, alleviate.Similarly, as used herein, term " prevention " has its general sense, and refer to especially herein the disease of microglia of the present invention mediation may be suffered from or adopt medicine of the present invention to process to the animal individual that the disease that microglia of the present invention mediates has a risk, preventing to producing described disease, preventing, stop, the effect such as partition.
The routine techniques in formulation art can be adopted, the effective ingredient of the raw material of pharmaceutical composition of the present invention is obtained by extraction separation and purification means conventional in pharmaceutical production, mix with one or more of pharmaceutically acceptable carrier, then form required dosage form, prepare pharmaceutical composition of the present invention.
As used herein, " pharmacy is acceptable " represents when not having tangible toxic action with during usual dosage use, thus by government or the international organization suitable with it approval or can be approved for animal, more specifically to people, or be registered in pharmacopeia.
" pharmaceutically acceptable carrier " available in pharmaceutical composition of the present invention can be the carrier of any routine in field of pharmaceutical preparations, and the selection of specific support will depend on the administering mode or disease type and state that are used for the treatment of particular patient.For the preparation method of the said synthetic processes of specific administration pattern completely in the ken of drug world technical staff.Such as, the solvent of pharmaceutical field routine, diluent, dispersant, suspending agent, surfactant, isotonic agent, thickening agent, emulsifying agent, binding agent, lubricant, stabilizing agent, hydrating agents, emulsifying accelerator, buffer agent, absorbent, coloring agent, ion-exchanger, releasing agent, smears, correctives and antioxidant etc. can be comprised as pharmaceutically acceptable carrier.If desired, flavouring agent, preservative and sweetener etc. can also be added in pharmaceutical composition.
As used herein, term " effective dose " refers to the amount of active component of the present invention, and the disease that this amount is enough to microglia of the present invention mediates effectively treats and/or prevents.
As used herein, term " pharmaceutical composition " has its general sense.In addition, " pharmaceutical composition " of the present invention can also exist with forms such as health product, functional food, food, food additive or provide.The routine techniques of pharmaceutical field particularly in formulation art can be adopted, the effective ingredient of the raw material of pharmaceutical composition of the present invention is obtained by extraction separation and purification means conventional in pharmaceutical production, optionally mix with one or more of pharmaceutically acceptable carrier, then form required dosage form, prepare pharmaceutical composition of the present invention.According to pharmaceutical composition of the present invention, it is the pharmaceutical preparation going for oral administration, parenteral or topical, topical administration.Pharmaceutical composition of the present invention can make the various ways such as tablet, powder, granule, capsule, oral liquid.The medicine of above-mentioned various dosage form all can be prepared according to the conventional method of pharmaceutical field.Specifically, according to pharmaceutical composition of the present invention, described pharmaceutical dosage form includes but not limited to: tablet, capsule, granule, powder, injection, injectable powder, transdermal patch, ointment, gel, suppository, oral administration solution, oral administration mixed suspension, injectable emulsion, Orally taken emulsion etc., slow releasing tablet, controlled release tablet.The medicine of above-mentioned various dosage form all can be prepared according to the conventional method of pharmaceutical field.
Such as tablet, pill, hard or soft capsule, solution, suspensoid, Emulsion, syrup, powder, powder, granula subtilis, granule, pilule, elixir etc. can be comprised for Orally administered dosage form, be not limited to this.Except active component, these preparations also can comprise diluent (such as lactose, dextrose, sucrose, mannitol, Sorbitol, cellulose and glycine), lubricant (such as silicon dioxide, Talcum, stearic acid or its magnesium salt, calcium salt and Polyethylene Glycol).Tablet also can comprise binding agent, such as aluminium-magnesium silicate, gelatinized corn starch, gelatin, tragacanth, methylcellulose, sodium carboxymethyl cellulose and polyvinylpyrrolidine.If desired, it also can comprise medical additive, such as disintegrating agent (as starch, agar, alginic acid or its sodium salt), absorbent, coloring agent, flavouring agent, sweeting agent etc.Tablet can according to the method preparation of conventional mixing, pelletize or coating.
Dosage form for using outside intestinal can comprise such as injection, medicinal drops, ointment, lotion, gel, emulsifiable paste, spray, suspensoid, Emulsion, suppository, patch etc., is not limited to this.
According to pharmaceutical composition of the present disclosure can oral or non-bowel such as per rectum, through local, through skin, through intravenous, through intramuscular, through intraperitoneal or through subcutaneous administration.
As used herein, term " individuality " or " animal individual " have its general sense, and individuality or the animal individual of the disease maybe may suffering from microglia of the present invention mediation be can refer to suffer from this article, the individuality in order to certain object such as uses in order to scientific research object or animal individual can also refer to.Specifically, described individuality is such as animal individual, particularly mammalian subject, such as people, pig, Canis familiaris L., cat, cattle, sheep, horse, rat, mice, rabbit, Cavia porcellus, monkey etc.More particularly, individuality of the present invention is people.
The acceptable dosage of pharmacy of active component, i.e. application dosage, can change according to the judgement of the order of severity of the age of object to be treated, sex and body weight, disease specific to be treated or pathological state, disease or pathological state, route of administration and diagnosis person.Consider that these factor determination application dosages are in the horizontal extent of those skilled in the art.General dosage can be 0.01-1000mg/kg/ day, particularly 1-100mg/kg/ day.But the scope of the present disclosure is limited to described application dosage never in any form.
Accompanying drawing explanation
In order to be illustrated more clearly in the present invention, will be briefly described accompanying drawing below, wherein:
Fig. 1: cryptogenin is on the impact of microglia cell viability.BV-2 cell adds after cryptogenin (1,5,10 μm of ol/L) hatches 24h, abandons supernatant, adds CCK-8, continue to hatch 0.5h, measure absorbance in 450nm wavelength place.
Fig. 2: cryptogenin produces the impact of inflammatory mediator (NO) level on activating mice microglia system BV-2.Cryptogenin (1,5,10 μm of ol/L) the preincubate BV-2 cell 0.5h of variable concentrations, then add bacteria cell wall lipopolysaccharide (Lipopolysaccharide, hereinafter referred to as LPS) (0.1 μ g/mL) to stimulate.Collecting cell culture supernatant after cultivation 24h, Greiss method detects the concentration of NO.#p < 0.05, compared with matched group; * p < 0.05, compares with model group.
Fig. 3 a-3d: the impact that cryptogenin is expressed microglia IL-1 β, IL-6, iNOS, COX-2mRNA is shown respectively.
Mice microglia system BV-2 adds cryptogenin (10 μm of ol/L) preincubate 0.5h, then adds LPS (0.1 μ g/mL) and carries out stimulation 8h.Wash 1 time with PBS, extract cell total rna with Trizol reagent.TaqMan Reverse Transcription Reagents test kit is adopted to be cDNA (20 μ l system) by total serum IgE (1.5 μ g) reverse transcription.The cDNA product getting 1 μ l is used as the template of quantitative pcr amplification.House-keeping gene GAPDH is as internal reference.Value represents by mean+standard deviation (mean+SD), n=3.#p < 0.05, compared with matched group; * p < 0.05, compares with model group.
Fig. 4: cryptogenin produces the impact of inflammatory mediator TNF-alpha levels on activating microglia system BV-2.
Cryptogenin (10 μm of ol/L) preincubate BV-2 cell 0.5h, then adding LPS (0.1 μ g/mL) stimulates.Collecting cell culture supernatant after cultivation 24h, Elisa method detects cytokine TNF-alpha content.Value represents by mean+standard deviation (mean+SD), n=3.#p < 0.05, compared with matched group; * p < 0.05, compares with model group.
Detailed description of the invention
Microglia in central nervous system derives from brephic myelomonocyte, is immunoreactive antigen presenting cell and main immune effector cell in brain, plays very crucial effect in central nervous system's immunoreaction process.Under normal circumstances, the microglia in brain remains static.In the neurodegenerative diseases pathogenic processes such as apoplexy, Alzheimer, parkinson disease, cerebral trauma, multiple sclerosis, amyotrophic lateral sclerosis, aids related dementia, bovine spongiform encephalopathy, microglia abnormal activation, its effect is except engulfing the neuron of removing degeneration death, also secrete proinflammatory factor and neurotrophic factor, inflammatory reaction and neuronic reparation and regeneration may be participated in.The form of microglia has plasticity, be branch-like in resting state, accepting damage signal stimulates rear cell volume to increase, projection is extended, is increased, become the microglia of hyperbranchedization, this is that they develop into microglia and the brain phagocyte of activation very soon, so can assist judge whether microglia is in state of activation according to the change of form between static and between activating a kind of intermediateness.
Under LPS effect, NO is formed and burst size obviously increases.NO can suppress the activity of Mitochondria complex I, and make mitochondrion synthesize ATP and reduce, cell is dead due to energy exhaustion.NO can also react with superoxide ion and form the larger nitrite free radical of toxicity, and the biomacromolecule such as nucleic acid, protein, lipid of direct aggression cell, causes cell death.
Except NO, activate microglia also can secrete cytokine profiles such as TNF-α, IL-1 α, IL-1 β, IFN-γ, IL-6, epidermal growth factor etc., and the generation of cytokine and inflammatory reaction and neuronic injury repairing, regenerate closely related.Wherein TNF-α has very important effect in inflammatory reaction process.LPS can stimulate microglia TNF secretion-α, and TNF-α can promote astrocyte and microglia synthesis colony stimulating factor (colony stimulating factor, CSF).CSF can attract the granulocyte in blood circulation and macrophage to arrive the inflammation part of central nervous system as LCF, participates in inflammatory reaction, thus is exaggerated inflammation.TNF-α also can Induction of neuronal MHC I up-regulated, makes them be subject to MHC I restrictive cell cytotoxic T cell and attacks.In addition, TNF-α can disturb the electrophysiological characteristics of neuron and star horn cell, and in vitro study finds that TNF-α can affect Ca in cell membrane potential, cell
2+balance and long term potentia￣tion, therefore abnormal with the cell function in inflammatory process about (see the foundation of the parkinson disease inflammation/dysimmunity cell model of the people such as Zhou Huifang, Chinese Journal of Neuromedicine, 2003 years, 2nd volume, the 1st phase: 48-51 page).
Below in conjunction with accompanying drawing, in the mode of specific embodiment, technical scheme of the present invention is clearly and completely described.Obviously, described embodiment is only a part of embodiment of the present invention, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
Experiment material used in following embodiment and method, if no special instructions, be conventional material and method.
Preparation embodiment 1: prepare cryptogenin tablet
Cryptogenin 5mg
Corn starch 10mg
Lactose 10mg
Magnesium stearate 0.2mg
Vitamin C 5mg
Mix above composition according to common method and make tablet.
Preparation embodiment 2: prepare cryptogenin capsule
Cryptogenin 5mg
Corn starch 10mg
Lactose 10mg
Magnesium stearate 0.2mg
Vitamin C 5mg
Serine 5mg
Mix above composition according to common method and load in gelatine capsule.Preparation embodiment 3: the tablet preparing pharmaceutical composition of the present invention
Cryptogenin 3mg
Levodopa 2mg
Corn starch 10mg
Lactose 10mg
Magnesium stearate 0.2mg
Vitamin C 5mg
Mix above composition according to common method and make tablet.
Preparation embodiment 4: the capsule preparing pharmaceutical composition of the present invention
Cryptogenin 2mg
Benzatropine 3mg
Corn starch 10mg
Lactose 10mg
Magnesium stearate 0.2mg
Vitamin C 5mg
Serine 5mg
Mix above composition according to common method and load in gelatine capsule.
Embodiment 1: cryptogenin is on the impact of microglia cell viability
BV-2 cell (purchased from consonance medical university cell bank) adds after cryptogenin (1,5,10 μm of ol/L) cultivates 24h, abandon supernatant, add CCK-8 (please source be supplement, such as, purchased from colleague's chemistry), continue to hatch 0.5h, measure absorbance in 450nm wavelength place.Result is as shown in table 1.
Table 1: cryptogenin is on the impact of microglia cell viability
Corresponding result is also shown in Figure 1.
As illustrated clearly in Fig. 1, cryptogenin can not T suppression cell vigor, does not cause cell death.
Embodiment 2: cryptogenin is on the impact of microglia secretion inflammatory mediator
The ultimate principle of Greiss method: change NO into very soon after the NO release in biosystem
2 -and NO
3 -, both stable in properties rear, therefore can by measuring NO
2 -or NO
3 -indirectly reflect NO growing amount.NO
2 -with its additive reaction of p-anilinesulfonic acid. under sour environment, its product and N-(1-naphthyl)-ethylenediamine react, and product has absworption peak at 548nm place.
Complete DMEM culture medium (purchased from invitrogen company) cultivates the rat microglia cells of mice microglia system BV-2 and original cuiture.Adjustment cell concentration is 4 × 10
5/ mL, is inoculated in 48 well culture plates and spends the night, and adds cryptogenin (final concentration is 1,5,10 μm of ol/L) the preincubate 0.5h of variable concentrations, and then adding endotoxin (LPS, final concentration 0.1 μ g/mL) stimulates.Experiment grouping: Normal group, LPS group, positive controls minocycline (Mino) and cryptogenin add LPS group.The concentration of NO is detected by Greiss method.
Table 2: cryptogenin is on the impact of microglia secretion inflammatory mediator
Result: the release of the NO that positive control minocycline (Mino) can suppress LPS to cause.The release of the NO that cryptogenin intervention also can obviously suppress LPS to cause, and in dose dependent in 1-10 μm of ol/L concentration range, see Fig. 2.
Embodiment 3: the impact that cryptogenin is expressed microglia IL-1 β, IL-6, iNOS, COX-2mRNA.
Mice microglia system BV-2, plants and spends the night in 12 well culture plates, adds cryptogenin (10 μm of ol/L) preincubate 0.5h, then adds LPS (0.1 μ g/mL) and carries out stimulation 8h.Wash 1 time with PBS, extract cell total rna with Trizol reagent (purchased from invitrogen company).Sample thief carries out the ultraviolet detection that wavelength is 260nm and 280nm, analyzes the degree of total serum IgE and carries out quantitatively.TaqMan Reverse Transcription Reagents test kit (by Applied Biosystems sold) and oligod (T) 16 primer (purchased from Applied Biosystems company) is adopted to be cDNA (20 μ l system) by total serum IgE (1.5 μ g) reverse transcription.The cDNA product of 1 μ l is used as the template of quantitative pcr amplification.Pcr amplification reaction adopts SYBR Green PCR Master Mix reagent kits test kit (Applied Biosystems company) and corresponding special primer.。
Table 3: the impact that cryptogenin is expressed microglia IL-1 β, IL-6, iNOS, COX-2mRNA
| Group | IL-1βmRNA | IL-6mRNA | iNOS?mRNA | COX-2mRNA |
| Normal group | 1.009 | 1.016 | 1.027 | 1.075 |
| LPS group | 133.041 | 100.530 | 18.155 | 14.791 |
| LPS+ cryptogenin group | 5.570 | 7.923 | 6.894 | 2.745 |
Result: LPS can increase the expression of BV-2 cell IL-1 β, IL-6, iNOS, COX-2mRNA significantly; And IL-1 β, IL-6, iNOS, COX-2mRNA that cryptogenin significantly reduces LPS induction express, see Fig. 3.
Embodiment 4: cryptogenin produces the impact of inflammatory mediator TNF-alpha levels on activating microglia system BV-2
Elisa method ultimate principle: with the ELISA Plate absorption determined antigen of specific monoclonal antibody bag quilt, again in turn by biotin labeled monoclonal antibody, be connected to determined antigen in conjunction with the chain enzyme avidin of horseradish peroxidase, add substrate TMB to develop the color, wavelength 450nm place surveys absorbance.
Complete DMEM culture medium (containing 10% hyclone, 100U/mL penicillin and 100 μ g/mL streptomycins) cultivates the rat microglia cells of mice microglia system BV-2 and original cuiture.Adjustment cell concentration is 4 × 10
5/ mL, is inoculated in 48 well culture plates and spends the night, and adds cryptogenin (final concentration 10 μm of ol/L) preincubate 0.5h, and then adding endotoxin (LPS, final concentration 0.1 μ g/mL) stimulates.Experiment grouping: Normal group, LPS group and cryptogenin add LPS group.Cultivate 24h and collect each processed group cell culture supernatant, detect cytokine TNF-alpha content by Elisa method.Operating procedure completes by test kit description (R & D Systems company).
Table 4: cryptogenin produces the impact of inflammatory mediator TNF-alpha levels on activating microglia system BV-2
| Group | TNF-α(pg/ml) |
| Normal group | -46.458 |
| LPS group | 2104.578 |
| LPS+ cryptogenin group | 342.138 |
[0108]result: in microglia BV-2 culture supernatant, TNF-alpha content is extremely low under normal circumstances, after stimulating with 0.1 μ g/mL LPS, the content of TNF-α significantly increases.And the release of the TNF-α that cryptogenin intervention can obviously suppress LPS to cause, see Fig. 4.
In sum, cryptogenin can not only reduce the generation of the cytotoxicity inflammatory mediators such as activated microglia NO, TNF-α, and significantly can reduce the expression of IL-1 β, IL-6, iNOS, COX-2mRNA that LPS induces.This shows that cryptogenin may be used for preventing and treating the disease of Activated Microglia mediation, especially the nerve immunity inflammatory diseases caused by neurotoxic effect of microglia mediation, such as apoplexy, Alzheimer, parkinson disease, cerebral trauma, multiple sclerosis etc.
Claims (10)
1. following formula cryptogenin for the preparation of prevent and/or treat Activated Microglia mediation disease medicine in purposes, described disease especially microglia mediation the nerve immunity inflammatory diseases caused by neurotoxic effect, preferably described disease is selected from: apoplexy, Alzheimer, parkinson disease, cerebral trauma, multiple sclerosis, amyotrophic lateral sclerosis, aids related dementia, bovine spongiform encephalopathy
2. purposes according to claim 1, every daily dose that wherein said medicine gives object in need counts 0.01-1000mg/kg body weight with cryptogenin, is preferably 0.1-100mg/kg body weight, is more preferably 1-100mg/kg body weight.
3., according to the purposes of claim 1 or 2, wherein said cryptogenin provides with the form of cryptogenin monomer, or provide with the form of the plant extract of the preferred Fructus Tribuli of the plant extract comprising cryptogenin and Trillium tschonoskii Maxim.
4. purposes according to claim 3, wherein said cryptogenin is that the form of the Fructus Tribuli crude saponin comprising cryptogenin provides.
5. according to the purposes of claim 3 or 4, the cryptogenin that wherein said plant extract, preferably Fructus Tribuli crude saponin comprise more than 30% by weight, are preferably more than 60%, are more preferably more than 90%.
6. one kind for preventing and/or treating the pharmaceutical composition of disease of microglia mediation, it contains the cryptogenin preventing and/or treating effective dose or the plant extract comprising cryptogenin, preferably comprises Fructus Tribuli crude saponin, optional other drug and the optional pharmaceutically acceptable carrier of cryptogenin.
7. pharmaceutical composition according to claim 6, wherein, based on the gross weight of active component in described pharmaceutical composition,
The content of described cryptogenin is 1-99%; Be preferably 20-80%; Be more preferably 40-60%;
The content of described other drug is 99-1%; Be preferably 80-20%; Be more preferably 60-40%;
Described other drug is selected from especially: levodopa, carbidopa, Selegiline, benzhexol, benzatropine, amantadine, bromocriptine, pergolide, tacrine, huperzine A, galantamine, xanomeline, donepezil hydrochloride, rivastigmine, memantine, piracetam, Dihydroergotoxine Mesylate, pentoxifylline, nicergoline, aspirin, melatonin, curcumin, desferrioxamine, idebenone, Tirilazad Mesylate and vitamin C.
8., according to the pharmaceutical composition of claim 6 or 7, wherein said pharmaceutically acceptable carrier is selected from solvent, diluent, dispersant, suspending agent, surfactant, isotonic agent, thickening agent, emulsifying agent, antiseptic, binding agent, lubricant, stabilizing agent, hydrating agents, emulsifying accelerator, buffer agent, absorbent, coloring agent, flavouring agent, sweeting agent, ion-exchanger, releasing agent, smears, correctives and antioxidant.
9. the pharmaceutical composition any one of claim 6-8, it is the dosage form of unit dosage, the cryptogenin comprise 0.001mg-5000mg in the dosage form of this unit dose, being preferably 0.1-1000mg, being more preferably 1-100mg, its dosage form is tablet, capsule, powder, granule, lozenge, pill, solution, suspensoid, Emulsion, syrup, powder, granula subtilis, pilule, elixir, injection, medicinal drops, ointment, lotion, gel, emulsifiable paste, spray, suppository or patch especially.
10. the pharmaceutical composition any one of claim 6-9, the disease of wherein said microglia mediation is the nerve immunity inflammatory diseases caused by neurotoxic effect of microglia mediation, and the disease of described microglia mediation is especially selected from: apoplexy, Alzheimer, parkinson disease, cerebral trauma, multiple sclerosis, amyotrophic lateral sclerosis, aids related dementia and bovine spongiform encephalopathy.
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| CN107375310A (en) * | 2017-03-06 | 2017-11-24 | 许迎春 | Cryptogenin is preparing the purposes in treating heart failure drugs |
| CN110604736A (en) * | 2019-08-22 | 2019-12-24 | 西南医科大学 | Application of trillium saponin in preparing nerve protection medicine |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105168785A (en) * | 2015-10-08 | 2015-12-23 | 湖北民族学院 | Traditional Chinese medicine preparation for treating AD (Alzheimer's disease) and preparation method |
| CN105168235A (en) * | 2015-10-22 | 2015-12-23 | 江西中医药大学 | Preparation methods of trillium saponin substance and preparation thereof as well as application of trillium saponin substance and preparation thereof in preparation of medicine for treating senile dementia and Alzheimer's disease |
| CN107648430A (en) * | 2015-10-22 | 2018-02-02 | 江西中医药大学 | A kind of preparation method of the effective constituents of steroid saponin containing trilliaceae capsule preparations and its application in various kinds of drug is prepared |
| CN107890470A (en) * | 2015-10-22 | 2018-04-10 | 江西中医药大学 | A kind of preparation method of the effective constituents of steroid saponin containing trilliaceae tablet and its application in various kinds of drug is prepared |
| CN107375310A (en) * | 2017-03-06 | 2017-11-24 | 许迎春 | Cryptogenin is preparing the purposes in treating heart failure drugs |
| CN110604736A (en) * | 2019-08-22 | 2019-12-24 | 西南医科大学 | Application of trillium saponin in preparing nerve protection medicine |
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