CN101254185B - Application of iridoid compound in preparing medicine for promoting nerve cell proliferation and differentiation - Google Patents

Abstract

The invention discloses application of iridoid compounds in preparing medicaments for promoting proliferation and/or differentiation of nerve cells and a method for treating diseases related to proliferation and/or differentiation of nerve cells by using the iridoid compounds.

Description

Use of iridoids in the preparation of drugs for promoting nerve cell proliferation and differentiation

technical field

The invention relates to the use of iridoid compounds in the preparation of drugs for promoting the proliferation and/or differentiation of nerve cells and the prepared pharmaceutical composition. The present invention also relates to methods of treating or preventing diseases or conditions associated with promoting neural cell proliferation and/or differentiation with said compounds or pharmaceutical compositions.

technical background

Neural stem cells (NSCs) refer to multipotential cells in the central nervous system that have the ability to self-renew and can differentiate into mature brain cells (including neurons, astrocytes, and oligodendrocytes). Neural stem cells have self-renewal ability, high proliferation potential and multi-differentiation potential, and can be differentiated into neurons, oligodendrocytes and astrocytes, etc. The progeny cells produced by neural stem cells play an important role in the development, maintenance and Implementation of cell replacement therapy plays an important role. The discovery of NSC and the successful in vitro culture have broadened the way for the cell replacement therapy of central nervous system diseases. For example, treatment of neurodegenerative diseases such as Parkinson's disease (PD), Alzheimer's disease (AD) and Huntington's disease (HD) can significantly improve symptoms. At the same time, a large number of in vitro and in vivo experiments have confirmed that NSCs do exist in the adult brain, but they lose the stimulating effect of active factors after maturation. Under certain conditions, such as brain tissue injury, ischemia, growth factor stimulation, etc., these cells can proliferate, migrate and differentiate, and new neurons can replace lost neurons to perform certain functions. Therefore, it is of great significance to study effective methods for promoting the proliferation and/or differentiation of neural stem cells.

Iridoids are a special class of monoterpene compounds, the core of which is cyclic, with olefinic and etheric bonds. In 1925, iridone was first isolated from the defense secretions of ants. In 1958, O.Halpern and H.Schmid studied the structure of plumieride and determined the basic skeleton of iridoids. The iridoids in the plant kingdom are derived from geranyl pyrophosphate (GPP) through the biosynthetic pathway to iridodial, and then acetalized. Its basic skeleton is shown in the following structural formula.

1. Basic skeleton of iridoids

Among the iridoid molecules, C ₁ -OH is very active and is easy to combine with sugar. The naturally occurring iridoids are mostly glycosides, and most of them are D-glucosides. Including but not only:

(1) Common iridoid glycosides: ①C-8 iridoid glycosides. ②C-9 iridoid glycosides (the ninth C is connected to C-4). ③C-9 iridoid glycosides (the ninth C is connected to C-8). ④ C-10 iridoid glycosides. Such as 6'-acetyl carniroside, morroniside, loganin, geniposide, catalpol, cicadine, cicadine, caroside, aucubin, catalpaside, deoxy Tribenoside, Genipin, Frangipani, Verbenain, Jinjiside.

(2) Split-ring iridoid glycosides: such as gentiopicroside, gentiopicroside, kailian glucoside, swerberin, etc.

(3) Valeriana-type iridoids, such as 7, 10, 2'-3 acetyl pateroside, and 10-acetyl pateroside.

(4) Plumeria-type iridoids: such as citrusin A.

(5) The iridoid glycoside crescentoside B that forms an oxygen bridge between the 3 and 10 positions, such as the iridoid glycoside B that forms an oxygen bridge between the 3 and 10 positions.

(6) Nepetalactone-related types of iridoids, including laurin.

(7) Deformed iridoids, such as crescentoside A, B, and citrusin B with a three-membered oxygen spiro ring structure at the 8-position.

(8) It is composed of several iridoids directly connected by ester bonds or by terpenoids and phenols. Such as valerian ether esters, deacetylated carnelinolate methyl esters, etc.

Through the research on loganin and morroniside, the inventors of the present invention found that iridoids have a good effect of promoting proliferation and/or promoting differentiation of neural stem cells, thus completing the present invention.

Contents of the invention

One aspect of the present invention provides the use of iridoid compounds such as loganin and/or morroniside in the preparation of drugs for promoting the proliferation and/or differentiation of neural stem cells. Another aspect of the present invention also provides a pharmaceutical composition containing an iridoid compound and a method for treating diseases related to nerve cell proliferation and/or differentiation with the compound or composition.

With the rapid update and progress of modern molecular biology, it will be possible for human beings to develop the proliferation and differentiation technology for mature adult nerve cells. Based on the pro-proliferation and differentiation effects of iridoids on neural stem cells, iridoids may also have pro-proliferation and differentiation effects on adult nerve cells, and then treat neuronal loss characterized by various reasons. Nervous system disease.

The above-mentioned methods and uses of iridoids can be used alone to promote the proliferation and differentiation of stem cells remaining in the nervous system of patients with neurological diseases, or to transplant exogenous stem cells into the nervous system of patients with neurological diseases to proliferate and differentiate To achieve the purpose of treating or preventing neurological diseases.

The morroniside and loganin of the present invention both have the basic skeleton of iridoids, and can be extracted from Cornus officinalis according to known methods, or obtained from the market.

Loganin Morroniside

The compounds of the present invention can also be synthesized according to methods known to those skilled in the art.

The present invention is intended to cover homologues and analogs of such compounds, in addition to the compounds listed above. In this context, homologues are molecules that have substantial structural similarity to the aforementioned compounds, and analogs are molecules that have substantial biological similarity independent of structural similarity.

The present invention also encompasses pharmaceutical compositions comprising pharmaceutically acceptable salts, such as acid addition salts, of iridoids with organic and inorganic acids such as hydrochloric acid, sulfuric acid, methanesulfonic acid, Fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, oxalic acid, citric acid, tartaric acid, carbonic acid, phosphoric acid, etc. Pharmaceutically acceptable salts can also be prepared by treatment with inorganic bases such as sodium, potassium, ammonium, calcium or iron hydroxides, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol , histidine, procaine, etc.

The present invention also encompasses pharmaceutical compositions comprising hydrates of iridoids. The term "hydrate" includes, but is not limited to, hemihydrate, monohydrate, dihydrate, trihydrate, and the like.

The present invention also encompasses pharmaceutical compositions comprising any solid or liquid physical form of an iridoid compound. For example, iridoids may be in crystalline form, amorphous, and have any particle size. The iridoid particles may be micronized, or may be in the form of agglomerated particulate particles, powder, oil, oil suspension, or any other solid or liquid physical form.

pharmaceutical composition

The compounds of the present invention and their derivatives, fragments, analogs, homologues, pharmaceutically acceptable salts or hydrates can be prepared together with pharmaceutically acceptable carriers or excipients into pharmaceutical compositions suitable for oral administration. Such compositions generally comprise a therapeutically effective amount of any of the compounds described above and a pharmaceutically acceptable carrier. Preferably, the effective amount is an amount effective to promote neural cell proliferation and/or differentiation and less than an amount causing toxicity to a patient.

Any inert excipient commonly used as a carrier or diluent, such as gums, starches, sugars, cellulosic materials, acrylates or mixtures thereof, may be used in the formulations of the present invention. A preferred diluent is microcrystalline cellulose. The composition may further comprise a disintegrant (such as croscarmellose sodium) and a lubricant (such as magnesium stearate), and may additionally contain one or more additives selected from binders, buffers, proteases Inhibitors, surfactants, solubilizers, plasticizers, emulsifiers, stabilizers, viscosity increasers, sweeteners, film formers, or any combination thereof. Furthermore, the compositions of the present invention may be in the form of controlled release formulations or immediate release formulations.

One embodiment is a pharmaceutical composition for oral administration, comprising an iridoid compound or a pharmaceutically acceptable salt or hydrate thereof, microcrystalline cellulose, croscarmellose sodium and magnesium stearate . Another embodiment comprises 50-70% by weight of iridoids or pharmaceutically acceptable salts or hydrates thereof, 20-40% by weight of microcrystalline cellulose, 5-15% by weight of cross-linked carboxyl Sodium methylcellulose and 0.1-5% by weight of magnesium stearate. Another embodiment comprises about 50-200 mg of an iridoid.

In one embodiment, the pharmaceutical composition is administered orally and thus is formulated in a form suitable for oral administration, ie a solid or liquid preparation. Suitable solid oral formulations include tablets, capsules, pills, granules, pellets and the like. Suitable liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils and the like. In one embodiment of the invention, the composition is formulated as a capsule. According to this embodiment, the compositions of the invention comprise, in addition to the iridoid active compound and an inert carrier or diluent, a hard gelatine capsule.

As used herein, "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., which are compatible with pharmaceutical administration , such as sterile pyrogen-free water. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference work in the field, incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles, such as fixed oils, may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active compounds, their use in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

Solid carriers/diluents include, but are not limited to, gums, starches (e.g., cornstarch, pregelatinized starch), sugars (e.g., lactose, mannitol, sucrose, glucose), cellulosic materials (e.g., microcrystalline cellulose), acrylates (such as polymethacrylate), calcium carbonate, magnesium oxide, talc or mixtures thereof.

For liquid formulations, the pharmaceutically acceptable carrier can be an aqueous or non-aqueous solution, suspension, emulsion or oil. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Examples of oils are those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil and cod liver oil. Solutions or suspensions may also include the following components: sterile diluents, such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or p-hydroxybenzoic acid Methyl esters; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, or phosphates; and tonicity-adjusting agents such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.

Additionally, the composition may further comprise a binder (e.g., gum arabic, cornstarch, gelatin, carbomer, ethylcellulose, guar gum, hydroxypropylcellulose, hydroxypropylmethylcellulose, povidone ), disintegrants (such as corn starch, potato starch, alginic acid, silicon dioxide, croscarmellose sodium, crospovidone, guar gum, sodium starch glycolate, Primogel), different pH and Buffers of ionic strength (e.g. Tris-HCl, acetate, phosphate), additives to prevent surface absorption (e.g. albumin or gelatin), detergents (e.g. Tween 20, Tween 80, Pluronic F68, bile salts ), protease inhibitors, surfactants (such as sodium lauryl sulfate), penetration enhancers, solubilizers (such as glycerol, polyvinylglycerol), glidants (such as colloidal silicon dioxide), antioxidants (such as ascorbic acid, partial sodium bisulfite, butylated hydroxyanisole), stabilizers (e.g. hydroxypropyl cellulose, hydroxypropyl methylcellulose), viscosity builders (e.g. carbomer, colloidal silicon dioxide, ethyl cellulose , guar gum), sweeteners (such as sucrose, aspartame, citric acid), flavorings (such as peppermint, methyl salicylate, or orange flavor), preservatives (such as thimerosal, benzyl alcohol , parabens), lubricants (e.g. stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow aids (e.g. colloidal silicon dioxide), plasticizers (e.g. diethyl phthalate, triethyl citrate), emulsifiers (e.g. carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymer coatings (e.g. poloxamer or poloxamine), coating With film formers (eg ethyl cellulose, acrylates, polymethacrylates) and/or auxiliaries.

In one embodiment, the active compounds are formulated with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. Materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes directed to infected cells and monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example as described in US Patent No. 4,522,811.

It is especially advantageous to formulate oral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, as well as the limitations inherent in the art surrounding such an active compound with respect to individual therapy.

The pharmaceutical composition can be included in a container, pack or dispenser together with instructions for administration.

A compound or composition of the invention may be administered continuously on repeated daily basis for several days to several years. Oral therapy can be continued for one week up to the life of the patient. Preferably, dosing is for five consecutive days, after which time the patient can be evaluated to determine if further dosing is required. Administration can be continuous or intermittent, that is, treatment on consecutive days followed by a rest period.

The preparation of pharmaceutical compositions containing active ingredients is well known in the art, such as mixing, granulating or tabletting processes. The active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. For oral administration, the active agent is mixed with additives customary for the purpose, such as carriers, stabilizers or inert diluents, and converted by customary methods into dosage forms suitable for administration, such as tablets, coated tablets, Hard or soft gelatin capsules, aqueous, alcoholic or oily solutions, etc., as described above.

The total daily dose of the compounds of the invention administered orally is between 25 and 4000 mg/ ^m2 , eg about 25 to 1000 mg, 50-1000 mg, 100 mg, 200 mg, 300 mg, 400 mg, 600 mg, 800 mg, 1000 mg, etc. Typically, when up to 400 mg is administered to a patient, the compound is administered as a single dose. For higher total doses (ie, greater than 400 mg), the total amount is divided into doses, eg twice daily, three times daily, etc., preferably at equal intervals throughout the day. For example, two doses, for example 500 mg each, may be administered 12 hours apart, for a total dose of 1000 mg per day.

In a presently preferred embodiment, the total daily dose of the iridoid compound administered to the patient is 200 mg. In another presently preferred embodiment, the iridoid is administered to the patient in a total daily dose of 400 mg. In another presently preferred embodiment, the total daily dose of the iridoid compound administered to the patient is 600 mg.

The amount of compound administered to a patient is less than that which would cause toxicity to the patient. In certain embodiments, the amount of the compound administered to the patient is less than the amount that would result in the concentration of the compound in the patient's plasma equal to or exceeding the toxic level of the compound. Preferably, the concentration of the compound in the patient's plasma is maintained at about 10 nM. In another embodiment, the concentration of the compound in the plasma of the patient is maintained at about 25 nM. In another embodiment, the concentration of the compound in the plasma of the patient is maintained at about 50 nM. In another embodiment, the concentration of the compound in the plasma of the patient is maintained at about 100 nM. In another embodiment, the concentration of the compound in the plasma of the patient is maintained at about 500 nM. In another embodiment, the concentration of the compound in the plasma of the patient is maintained at about 1000 nM. In another embodiment, the concentration of the compound in the patient's plasma is maintained at about 2500 nM. In another embodiment, the concentration of the compound in the plasma of the patient is maintained at about 5000 nM. The optimal amount of a compound that should be administered to a patient in the practice of this invention will depend on the particular compound employed and the type of condition being treated.

In a currently preferred embodiment of the present invention, the pharmaceutical composition comprises common iridoid glycosides; microcrystalline cellulose as a carrier or diluent; croscarmellose sodium as a disintegrant; and magnesium stearate as a disintegrant. lubricant. In a particularly preferred embodiment, the iridoid is morroniside or loganin.

The percentages of active ingredient and various excipients in the formulation can vary. For example, the composition may comprise 20 to 90% by weight, preferably 50-70% by weight, of common iridoid glycosides. Furthermore, the composition may comprise from 10 to 70% by weight, preferably from 20 to 40% by weight, of microcrystalline cellulose as a carrier or diluent. Furthermore, the composition may contain 1 to 30% by weight, preferably 5-15% by weight, of croscarmellose sodium as a disintegrant. In addition, the composition may contain 0.1-5% by weight of magnesium stearate as a lubricant. In another preferred embodiment, the composition comprises about 50-200 mg of the iridoid (eg, 50 mg, 100 mg and 200 mg of the iridoid). In a particularly preferred embodiment, the composition is in the form of a tablet or gelatin capsule.

A currently preferred embodiment of the invention is a solid of iridoids with microcrystalline cellulose NF (Avicel Ph 101), croscarmellose sodium NF (AC-Di-Sol) and magnesium stearate NF Preparation, contained in a gelatin capsule. A further preferred embodiment is 200 mg solid SAHA with 89.5 mg microcrystalline cellulose, 9 mg croscarmellose sodium and 1.5 mg magnesium stearate contained in a gelatin capsule.

The pharmaceutical composition of the present invention can not only be used to promote the proliferation and differentiation of nerve cells, but it should be obvious to those skilled in the art that these compositions can be used to treat various diseases for which iridoid compounds have been found useful.

For example, it has been found that iridoids can be used to treat diseases that cause neuron loss due to various reasons, including central nervous system damage, including but not limited to ischemic, traumatic, and alcoholic brain or spinal cord injuries; Systemic wasting diseases affecting the nervous system, degenerative diseases of the basal ganglia, dystonias, primary extrapyramidal diseases; and degenerative diseases of the nervous system. Ischemic injury, including cerebral ischemia (eg, brain injury as a result of trauma, epilepsy, hemorrhage, or stroke, all of which can cause neurodegeneration); amyotrophic lateral sclerosis (ALS); multiple sclerosis; myopathies, such as Muscle protein metabolism disease.

For example, iridoids have been found useful in the treatment of a variety of neurodegenerative diseases, a non-exhaustive list of which includes:

I. Disorders characterized by progressive dementia in the absence of other prominent neurological signs, such as Alzheimer's disease; Alzheimer's disease; and Pick's disease (lobar atrophy).

II. Syndromes with progressive dementia and other prominent neurologic abnormalities, such as A) Syndromes seen primarily in adults (eg, Huntington's disease, multiple system atrophy with dementia and ataxia, and/or Parkinson's disease manifestations, progressive supranuclear palsy (Steel-Richardson-Olszewski), diffuse Lewy body disease, and corticodentatonigral degeneration); and B) syndromes seen primarily in children or young adults (such as Hay-Schöd disease and progressive familial myoclonic epilepsy).

III. Syndromes of progressive abnormalities of position and movement, such as parkinsonism, striatonigral degeneration, progressive supranuclear palsy, torsional dystonia (torsion spasm; deformed dystonia), spastic torticollis With other movement disorders, familial tremor, and Tourette's syndrome.

IV. Syndromes of progressive ataxia, such as cerebellar degeneration (eg cerebellar cortical degeneration and olivopontocerebellar atrophy (OPCA)) and spinocerebellar degeneration (Friedreich's ataxia and related disorders).

V. Syndromes of Central Autonomic Nervous System Failure (Shy-Drager Syndrome).

VI. Syndromes of muscle weakness and wasting without sensory alterations (motor neuron diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy (eg, infantile spinal muscular atrophy (Werdnig-Hoffman), juvenile spinal muscular atrophy (Wohlfart-Kugelberg) -Welander) and other forms of familial spinal muscular atrophy), primary lateral sclerosis, and hereditary spastic paraplegia.

VII. Syndromes with muscle weakness and wasting with altered sensation (progressive neuromuscular atrophy; chronic familial polyneuropathy), eg A. Charcot-Marie-Tooth, B. Hypertrophic interstitial polyneuropathy Neuropathy (Dejerine-Sottas), and C. Various forms of chronic progressive neuropathy.

VIII. Syndromes of progressive visual loss, such as pigmentary retinal degeneration (retinitis pigmentosa) and hereditary optic atrophy (Leeber's disease).

The following examples in the Experimental Details section further illustrate the invention. This section is intended to aid in understanding the invention, but is not intended, nor should it be construed, to limit in any way the invention as defined by the following claims.

Description of drawings:

Figure 1. Effects of iridoids loganin and morroniside on the proliferation of neural stem cells.

A. Control group: cultured with DMEM/F12 medium without growth factors for 6 days, most of the cells died (×200); B. Added loganin 50 μg/ml to DMEM/F12 medium without growth factors , cultured for 6 days, the volume of neural stem cell spheres increased (×200); C. adding loganin 25 μg/ml to the DMEM/F12 culture medium without growth factors, and cultured for 14 days, the volume of neural stem cells further increased (×200 ); D. Morroniside 25 μg/ml was added to the DMEM/F12 culture medium without growth factors, cultured for 6 days, and the volume of neural stem cell spheres increased (×200); E. DMEM/F12 culture without growth factors Add 25 μg/ml of morroniside to the solution, culture for 14 days, the volume of neural stem cells further increased (×200).

Figure 2. The effect of iridoids loganin and morroniside on promoting the differentiation of neural stem cells.

A control group: DMEM/F12 medium containing 1% fetal bovine serum (FBS) was used to induce differentiation of neural stem cells for 7 days, and single cells migrated from the periphery of the cell ball, and the morphology of the migrated cells can be roughly divided into two types: ① small The shape of some cell bodies is close to oval, with 2 to 3 slender protrusions, which are similar to the shape of neurons; ② most of the cells are irregular in shape and have many protrusions. According to the analysis of the results of immunocytochemical experiments, they are neurons , glial cells and astrocytes (×200). B Induced differentiation with DMEM/F12 culture medium containing 50ug/ml loganin (without FBS) for 7 days, the cell spheres were almost completely adhered to the wall, and the differentiation was good. They are connected to each other into a network (×200). C was induced to differentiate with DMEM/F12 culture solution containing 50ug/ml morroniside (without FBS) for 7 days, the cell spheres were almost completely attached to the wall, and the differentiation was good, and the processes of the cells that migrated were connected end to end to form a "string of beads" (× 200).

Figure 3. The effect of the iridoid pharmaceutical composition on promoting the proliferation of neural stem cells after brain injury.

A Normal rat hippocampal dentate gyrus has scattered Brdu-positive neural stem cells, and the nuclei of the positive cells are stained, most of which are oval; B Normal rats have scattered Brdu-positive neural stem cells in the subventricular layer, and the nuclei of the positive cells are stained, Most of them are oval; C. The Brdu-positive neural stem cells in the hippocampal dentate gyrus of the rats in the cerebral ischemia model group are significantly increased compared with the normal group; D. The Brdu-positive neural stem cells in the subventricular layer of the rats in the cerebral ischemia model group are significantly higher than those in the normal group Increase; E gives the iridoid pharmaceutical composition (loganin: morroniside composition=1:1) the cerebral ischemia model group rat hippocampus dentate gyrus Brdu positive neural stem cell number than normal group and model Group significantly increased; F gave the iridoid pharmaceutical composition (loganin: morroniside composition = 1:1) after the cerebral ischemia model group rat ependymal Brdu positive neural stem cell number than normal group and The model group increased significantly.

Example 1

Loganin and Morroniside Promote the Survival and Proliferation of Neural Stem Cells

1. Purpose of the experiment

To observe the effects of loganin and morroniside on the survival and proliferation of neural stem cells.

2. Experimental method

Take a newborn SD rat one day old, take out the brain under sterile conditions, separate the hippocampus, and place it in a petri dish filled with a small amount of high-glucose DMEM/F12; peel off the meninges and vascular tissues, cut the tissues as much as possible, and add cells for culture Gently blow with a flame-polished Pasteur pipette to make a cell suspension, filter through a ⁴⁰⁰ -mesh sieve, and count the proportion of live cells by placepan blue staining. 25ml culture flask. Using basal culture medium DMEM/F12 (1:1) and B27 (2%), and adding loganin and morroniside at final concentrations of 10, 50, 100, 200, 400 μg/ml respectively, the culture plate was transferred into Culture in an incubator at 37°C, 5% CO2, and 95% air-balanced humidity, observe the growth of cells with an inverted microscope, and measure the diameter of cell spheres with a micrometer.

For the identification of neural stem cells, take the above-mentioned cell balls, remove the culture medium containing growth factors EGF and bFGF, add DMEM/F12 containing 10% FBS, and plant them in a 24-well culture plate covered with poly-lysine. After 16 hours, Immunocytochemical staining of nestin and doublecortin (DCX) was performed, and almost all cells were positive for nestin and doublecortin, indicating that the cultured cell spheres were neural stem/progenitor cells.

3. Experimental results

It can be seen from Figure 1 that most of the cells died when cultured in DMEM/F12 medium without growth factors for 6 days, and the cells were in a diffuse state. Add loganin 25-200ug/ml to the DMEM/F12 culture medium without growth factors, culture for 6 days, the volume of neural stem cell spheres increases, which is the effective range for promoting the survival and proliferation of stem cells, and 50-100ug/ml is the best; Morroniside 25-100ug/ml increases the volume of neural stem cell spheres, which is the effective range for promoting cell survival and proliferation, and 25-50ug/ml is the best. It shows that loganin and morroniside can effectively promote the survival and proliferation of neural stem cells.

Example 2

Effects of loganin and morroniside on promoting the differentiation of neural stem cells

1. Purpose of the experiment

Observing the effects of loganin and morroniside on the differentiation of neural stem cells

2. Experimental method

The second-generation neural stem cell spheres were taken, and the culture medium containing growth factors in the culture bottle was removed, and the cell spheres were randomly divided into two groups, ① fetal bovine serum (FBS) control group: the culture medium was DMEM/F12+10% FBS; ② Drug treatment group: Add loganin or morroniside at final concentrations of 10, 50, and 100 μg/ml to FBS-free culture medium DMEM/F12, respectively, and the above two groups of cells are cultured in 24 wells covered with polylysine In the plate, immunofluorescent staining was performed 7 days later, and observation and photography were taken.

3. Experimental results

In the fetal bovine serum control group, the cell balls were completely attached to the wall, and single cells migrated from the periphery of the cell balls. The morphology of the cells that migrated out could be roughly divided into two types: ① A small number of cell bodies were nearly oval in shape, and there were 2 to 3 elongated cells. Protrusion; ②The cell body of most of the cells is large, irregular in shape, and many protrusions. According to the analysis of the results of immunocytochemical experiments, they are neurons, glial cells and astrocytes.

In the cell culture plate in which loganin and morroniside were added to FBS-free culture medium DMEM/F12, the cell spheres of 10 and 50 μg/ml groups were almost completely adhered to the wall, well differentiated, and the cells that migrated out were arranged in a radial pattern , but the cells are connected to each other to form a network, or the protrusions of several cells are connected end to end to form a "string of beads"; the results of the 100μg/ml group are basically the same. The results showed that loganin and morroniside had good differentiation-promoting effects on neural stem cells (Fig. 2).

Example 3

The combination of loganin and morroniside promotes the proliferation of neural stem cells after brain injury

1. Purpose of the experiment

Observation of the effect of loganin and morroniside composition on neurogenesis in stroke rat model

2. Experimental method

SD rats were intragastrically given a composition of loganin and morroniside (1:1) (180 mg/kg in a large dose, 60 mg/kg in a medium dose, and 20 mg/kg in a small dose). For the rat model of cerebral ischemia, Brdu (10 mg/ml, 50 mg/kg/time, 2 times/d) was given intraperitoneal injection within 4 to 7 days after model establishment to label endogenously proliferating cells, perfused on the 8th day, fixed, The brains were taken out and serially frozen sections with a thickness of 30-40um were subjected to immunofluorescent staining.

3. Experimental results

There are scattered Brdu-positive cells in the dentate gyrus (DG) and subventricular zone (SVZ) of the hippocampus of normal rats, and the nuclei of the positive cells are stained, most of which are oval; Increase; the positive cells of the rats in the high-dose group of the composition of loganin and morroniside increased compared with the normal group and the model group. It shows that the combination of loganin and morroniside can promote the proliferation of neural stem cells after brain injury ( FIG. 3 ).

Claims (5)

1. morroniside and meliatin combine as the purposes of only active ingredient in the medicine of preparation promotion cell proliferation of nerve cord and/or differentiation.

2. according to the purposes of claim 1, wherein said promotion cell proliferation of nerve cord and/or differentiation are to be used to treat the nervous system degeneration disease.

3. according to the purposes of claim 2, wherein said disease is systemic wasting disease, Basal ganglia degenerative disease, myodystonia or the idiopathic extrapyramidal disease of parkinson disease, Alzheimer, Huntington Chorea, amyotrophic lateral sclerosis, movement disorder disease, the property amyotrophy of spinal cord source, the system that affects the nerves.

4. according to the purposes of claim 1, wherein said promotion cell proliferation of nerve cord and/or differentiation are to be used to treat central nervous system injury, and said central nervous system injury is traumatic, ethanol property brain or spinal cord injury.

5. according to the purposes of claim 4, wherein said central nervous system injury is a traumatic brain injury.

Images