CN113260358A - Application of composition containing ferrous amino acid chelate particles in preparing medicines for treating or relieving diseases related to nerve damage - Google Patents
Application of composition containing ferrous amino acid chelate particles in preparing medicines for treating or relieving diseases related to nerve damage Download PDFInfo
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- CN113260358A CN113260358A CN201980081513.0A CN201980081513A CN113260358A CN 113260358 A CN113260358 A CN 113260358A CN 201980081513 A CN201980081513 A CN 201980081513A CN 113260358 A CN113260358 A CN 113260358A
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- amino acid
- composition
- ferrous
- amyloid
- acid chelate
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Abstract
The invention provides an application of a composition in preparing a medicine for treating or alleviating diseases related to nerve damage, wherein the composition contains ferrous amino acid chelate particles sintered from ferrous amino acid chelates, the average particle size of the ferrous amino acid chelate particles is 500-2600 nanometers, the average molecular weight is 1,500 daltons (Dalton) to 600,000 daltons, and the medicine contains an effective dose of the composition and a pharmaceutically acceptable carrier. The composition has the effect of treating or relieving diseases related to nerve damage, particularly Alzheimer's disease and Parkinson's disease.
Description
The invention relates to an application of a composition containing ferrous amino acid chelate particles sintered by ferrous amino acid chelates, in particular to a composition for treating or relieving diseases related to nerve damage.
Nerve damage includes peripheral nerve damage and central nerve damage. When the peripheral nervous system is injured, the motor and sensory functions dominated by peripheral nerves have different degrees of disorders according to the severity of the injury, and clinically common disorders include muscle paralysis, soreness, tingling, or decline and disappearance of sensation. Common diseases or injuries caused by central nervous system injury are: stroke, brain trauma, Alzheimer's Disease (AD), spinal cord injury and Parkinson's Disease (PD).
Among them, alzheimer's disease is a chronic neurodegenerative disease, in which free Radicals (ROS) in brain of a patient are abnormally increased and tissue inflammation occurs, intracerebral pathological features show neurofibrillary tangles (NFTs) and massive β -amyloid (β -amyloid, Α β) precipitates in cerebral cortex and hippocampus to form plaques (sensory plaques), which may result in insufficient nutrition of brain nerve cells and loss of synaptic function of nerve cells. Parkinson's disease is caused by damage to nerve cells in the basal ganglia of the brain. The substantia nigra cells of basal ganglia produce dopamine, a nerve conduction substance required for movement, and when the substantia nigra cells die, dopamine is not produced, so that motor nerve information cannot be transmitted. In addition, melanocytes are damaged by MPP + (N-Methyl-4-phenylpyridinium Iodide, 1-Methyl-4-phenylpyridinium). Dopamine also modulates another nerve-conducting substance, acetylcholine, which increases when the amount of dopamine is insufficient, whereas excess acetylcholine causes tremors and muscle stiffness commonly seen in parkinson's patients.
The above diseases are common cranial nerve diseases in China, which not only affect the individual health and life of patients, but also have serious influence on the quality of family life of patients. Therefore, the need to find a drug for effectively treating or alleviating the diseases related to the nerve damage is a problem to be solved.
Disclosure of Invention
In order to achieve the above objects, the present invention provides a use of a composition for preparing a medicine for treating or alleviating diseases related to nerve damage, wherein the composition contains ferrous amino acid chelate particles sintered from ferrous amino acid chelate, and the ferrous amino acid chelate particles have an average particle size of 500 nm to 2600 nm and an average molecular weight of 1,500 Dalton (Dalton) to 600,000 Dalton, and the medicine contains an effective amount of the composition and a pharmaceutically acceptable carrier.
In one embodiment, the ferrous amino acid chelate particles preferably have an average molecular weight of 1,500 to 15,000 daltons; in another embodiment, the ferrous amino acid chelate particles preferably have an average molecular weight of 400,000 to 550,000 daltons, more preferably 1,500 to 550,000 daltons, and even more preferably 525,538 daltons.
Preferably, the weight ratio of ferrous iron to amino acid of the ferrous amino acid chelate in the composition is between 1: 1 to 1: 4 in the middle.
Preferably, the weight ratio of ferrous iron to amino acid of the ferrous amino acid chelate in the composition is between 1: 1.5 to 1: 2.5.
Preferably, the ferrous amino acid chelate in the composition is a ferrous amino acid chelate prepared by mixing an iron compound and an amino acid and heating at 60 ℃ to 90 ℃ for 8 hours to 48 hours, wherein the iron compound is preferably an inorganic iron compound, more preferably an inorganic ferrous compound. Wherein the weight ratio of iron compound to amino acid is between 1: 1.2 to 1: 1.5.
More preferably, the iron compound is ferrous sulfate, ferrous chloride, ferrous pyrophosphate or a combination thereof; the amino acid is glycine.
The invention further provides an application of a composition in preparing a medicine for treating or alleviating diseases related to nerve damage, wherein the composition consists of ferrous amino acid chelate particles sintered from ferrous amino acid chelates, the average particle size of the ferrous amino acid chelate particles is 500-2600 nanometers, the average molecular weight is 1,500-600,000 daltons, and the medicine consists of an effective dose of the composition and a pharmaceutically acceptable carrier.
The invention also provides a composition for treating or alleviating diseases related to nerve damage, wherein the composition contains ferrous amino acid chelate particles sintered from ferrous amino acid chelate, and the average particle size of the ferrous amino acid chelate particles is 500-2600 nm, and the average molecular weight is 1,500-600,000 dalton.
The invention also provides a composition for treating or alleviating diseases related to nerve damage, wherein the composition consists of ferrous amino acid chelate particles sintered from ferrous amino acid chelates, and the ferrous amino acid chelate particles have an average particle size of 500-2600 nm and an average molecular weight of 1,500-600,000 daltons.
The invention further provides a method for treating or alleviating a disease associated with nerve damage, comprising administering to a subject an effective amount of a composition and a pharmaceutically acceptable carrier, wherein the composition comprises ferrous amino acid chelate particles sintered from ferrous amino acid chelate, and the ferrous amino acid chelate particles have an average particle size of 500-2600 nm and an average molecular weight of 1,500-600,000 daltons.
The present invention further provides a method for treating or alleviating a disease associated with nerve damage, comprising administering to a subject an effective amount of a composition and a pharmaceutically acceptable carrier, wherein the composition comprises ferrous amino acid chelate particles sintered from ferrous amino acid chelate, and the ferrous amino acid chelate particles have an average particle size of 500 nm to 2600 nm and an average molecular weight of 1,500 dalton to 600,000 dalton.
The "effective dose" of the present invention refers to the dose required to achieve the effect of treating or alleviating the disease associated with nerve damage; according to some embodiments of the present invention, the composition containing sintered ferriamino acid chelate particles can reduce the damage of β -amyloid (β) protein to human neuroblastoma cells and/or reduce the damage of MPP + (N-Methyl-4-phenylpyridinium Iodide, 1-Methyl-4-phenylpyridium) to human neuroblastoma cells, or further has a repairing effect, some embodiments of the present invention are directed to compositions that are capable of recovering from decreased locomotor activity, impaired memory retention, impaired spatial learning, and increased acetylcholinesterase activity caused by beta-amyloid (β -amyloid) protein by administering a specific range of amounts of a composition comprising sintered ferrous amino acid chelate particles. The recovery means that the composition of the present invention can reduce and increase the locomotor activity caused by beta-amyloid (beta-amyloid, Abeta), improve the memory retention impairment and spatial learning ability disorder caused by beta-amyloid (beta-amyloid, Abeta), and reduce the increase of acetylcholinesterase activity caused by beta-amyloid (beta-amyloid, Abeta).
According to the present invention, the effective dose is calculated by converting the dose of the composition of the present invention in the experimental examples of the present specification based on the concentration of 8mg/kg to 36mg/kg, based on the estimated method at the initial stage of the experiment announced by the U.S. food and drug administration, based on an adult of 60 kg, into a dose of 6.2 times the recommended daily intake per kg body weight (/ kg b.w./d) of a human body, and into a dose of 1 time that of rats.
Preferably, the subject to which the composition of the present invention is administered may be a human, and the effective dose of the composition may be 1.3mg/kg to 5.8 mg/kg.
The "pharmaceutically acceptable carrier" of the present invention includes, but is not limited to, reducing agent (reducing agent), solvent (solvent), emulsifier (emulisifier), suspending agent (suspending agent), disintegrating agent (decongestor), binding agent (binding agent), excipient (excipient), stabilizing agent (stabilizing agent), diluent (diluent), gelling agent (gelling agent), preservative (preserving agent), lubricant (lubricating agent), surfactant (surfactant), and other carriers similar to or suitable for the present invention.
Preferably, the reducing agent includes, but is not limited to, ascorbic acid (ascorbic acid), citric acid (citric acid), acetic acid (acetic acid), propionic acid (propionic acid), butyric acid (butric acid), lactic acid (lactic acid), hydroxysuccinic acid (malic acid), sulfonic acid (sulfonic acid), succinic acid (succinic acid), or a combination thereof.
The "pharmaceuticals" of the present invention may exist in a variety of forms including, but not limited to, liquid, semi-solid, and solid pharmaceutical forms such as solutions (solutions), emulsions (emulsions), suspensions (suspensions), powders (powders), tablets (tablets), pills (pils), lozenges (dragees), troches (troches), chewing gum (chewing gum), capsules (capsules), liposomes, suppositories, and other similar or suitable dosage forms for use in the present invention.
Preferably, the medicament is in an enteral or parenteral dosage form.
More preferably, the enteral dosage form is an oral dosage form which is a solution, emulsion, suspension, powder, lozenge, pill, lozenge, tablet, chewing gum or capsule.
Preferably, the disease associated with nerve damage includes, but is not limited to, alzheimer's disease.
Preferably, the neurological impairment related disorder includes, but is not limited to, parkinson's disease.
The composition can reduce nerve cell damage caused by beta-amyloid protein and MPP +, generate repairing effect, clear free radicals generated by the beta-amyloid protein, protect nerve cells from being attacked by the free radicals, and can recover the motor activity reduction, memory retention damage, space learning ability disorder and acetylcholinesterase activity increase caused by the beta-amyloid protein (beta-amyloid, Abeta) in animal experiments, so that the composition can effectively treat or slow down diseases related to nerve damage, particularly Alzheimer disease and Parkinson disease.
FIG. 1 shows the effect of β -amyloid on the cell growth of SH-SY5Y cells at 0, 24 and 48 hours.
FIG. 2 shows the relative ratio of the number of cells of SH-SY5Y cells co-treated with beta-amyloid in the composition of the invention.
FIG. 3 shows the relative ratio of nitric oxide concentration in SH-SY5Y cells co-treated with beta-amyloid in the composition of the present invention.
FIG. 4 shows the relative proportion of MPP + that affects the cell growth of SH-SY5Y cells at 0, 24, and 48 hours.
FIG. 5 shows the relative proportion of cell growth after co-treatment of SH-SY5Y cells with MPP + in the composition of the present invention. Expressed as mean ± standard deviation.
Fig. 6 is an animal experimental schedule.
FIG. 7A shows that the composition of the present invention improves the locomotor activity of rats injected with β -amyloid protein: the distance was moved in 15 minutes. Expressed as mean ± standard deviation of 5 or 6 independent experiments. P <0.01 compared to sham group; # is p <0.05 compared to rats injected with β -amyloid (Student's t-test).
FIG. 7B shows that the composition of the present invention improves the locomotor activity of rats injected with β -amyloid protein: moving average speed. Expressed as mean ± standard deviation of 5 or 6 independent experiments. P <0.01 compared to sham group; # and # # are p <0.05 and p <0.01, respectively, compared to rats injected with β -amyloid protein (Student's t-test).
FIG. 8 is a graph of the composition of the present invention improving the bright-room residence time of rats injected with beta-amyloid in the memory retention test for inhibitory avoidance. Expressed as mean ± standard deviation of 5 or 6 independent experiments. P <0.001 compared to sham group; # and # are p <0.01 and p <0.001, respectively, compared to rats injected with β -amyloid (Student's t-test).
FIG. 9A is a composition of the invention that ameliorates memory impairment caused by β -amyloid: the mean time to arrival at the platform was determined using the Morris water maze. Expressed as mean ± standard deviation of 5 or 6 independent experiments. And p <0.01, p <0.001 compared to sham group; # #, # # and # # # are p <0.05, p <0.01, p <0.001 (Student's t-test) compared to rats injected with beta-amyloid.
FIG. 9B is a composition of the invention that ameliorates memory impairment caused by β -amyloid: the first arrival time at the platform was determined using the Morris water maze. Expressed as mean ± standard deviation of 5 or 6 independent experiments. P <0.05 compared to sham group; # and # # # are p <0.05 and p <0.001 (Student's t-test) compared to rats infused with β -amyloid.
FIG. 10 shows that the composition of the present invention improves the activity of cerebral cortical acetylcholinesterase in rats injected with β -amyloid. Expressed as mean ± standard deviation of 5 or 6 independent experiments. P <0.05 compared to sham group; # is p <0.05 compared to rats injected with β -amyloid (Student's t-test).
The invention will be further illustrated by the following examples, which are not intended to limit the invention as disclosed hereinbefore. Those skilled in the art can make various modifications and alterations without departing from the scope of the invention.
Preparation example 1 preparation of a composition containing ferrous amino acid particles
The composition containing ferrous amino acid chelate particles of the present invention was manufactured by taiwan ligand corporation (batch number: F171001; manufacturing date: 2017, 10 months and 5 days), and the composition was a freeze-dried powder, which was prepared in the following manner. Firstly, mixing ferrous sulfate and glycine (purity more than 98%) in a weight ratio of 1: 1.3 mixing and heating at 60 ℃ to 90 ℃ for 8 hours to 48 hours to obtain ferrous amino acid chelate, wherein the ferrous to amino acid chelating ratio of ferrous amino acid chelate is between 1: 1 to 1: 4, then, the ferrous amino acid chelate is sintered at the temperature of 200-240 ℃ to obtain ferrous amino acid chelate particles. The mean Particle Size of the ferriamino acid chelate particles was 1465.90 ± 132.29 nm as measured by dynamic light scattering in water using a laser Particle Size Analyzer (Beckman Coulter, N5, Submicron Particle Size Analyzer). The number average molecular weight (Mn), weight average molecular weight (Mw), peak average molecular weight (MP), and Polydispersity (PDI) were measured by Gel Permeation Chromatography (GPC) using a Waters Alliance 2695System and were 68188 daltons (Dalton), 525538 daltons (Dalton), 286426 daltons (Dalton), and 7.707205, respectively.
Preparation example 2 laboratory animal Care and treatment
Six-week-old Sprague-Dawley male rats (Lesco Biotechnology, Taiwan, China) were housed in groups of three rats per cage, and the rats had free access to food and water. The use of experimental animals has been ethically proven. Over a 2-week familiarity period, the rats were randomly divided into five groups of 6 rats: (1) control group (sham operated group); (2) a separate treatment group of beta-amyloid protein; (3) treatment of beta-amyloid and low concentrations of the composition of the invention (8 mg/kg); (4) treatment of beta-amyloid and moderate concentrations of the composition of the invention (24 mg/kg); (5) treatment of beta-amyloid protein and high concentration of the composition of the invention (36 mg/kg). The concentrations of the composition of the present invention were provided by Taiwan ligand co, Ltd, China, and the composition of the present invention was dissolved in sterile distilled water and administered to rats by feeding tube feeding for 21 days once a day. Three weeks after pretreatment with the composition of the present invention, beta-Amyloid (Tocris, Amyloid beta-Peptide (1-42)) was injected into the ventricle.
Example 1 Effect of beta-Amylactin on the neuroblastoma cell line SH-SY5Y
Human neuroblastoma cell line SH-SY5Y (
CRL2266 TM) At a rate of 1X 10 per hole5Cell densities of the cells were plated in 24-well cell culture dishes with 10% FBS DMEM/F12 medium (Gibco11330032) at 37 ℃ in 5% CO2The culture was carried out in an incubator for 24 hours. Beta-amyloid 1-42peptide (Taiclone tcsc0032566) dissolved in PBS at 500. mu.M was added to the medium in different amounts, and SH-SY5Y cells were treated with 0, 1, 2.5 and 5. mu.M beta-amyloid, respectively, and the 0. mu.M beta-amyloid treated group was used as a control group, and each treatment condition was triplicated. Then at 0 hr or 37 deg.C, 5% CO2At the time points of 24 hours and 48 hours in the incubator, the collected cells were stained with Trypan blue (Trypan blue), the growth of the cells was observed through a microscope and the number of non-stained living cells was calculated, and as shown in fig. 1, the relative proportion of the cell growth decreased with the increase of the treatment concentration of the β -amyloid and the increase of the time, that is, the cells were significantly damaged, based on the control group. In the case of 24 and 48 hours of cell growth, 2.5. mu.M and 5. mu.M β -amyloid treatment groups caused damage to SH-SY5Y cells similarly and both caused more severe cell damage than the 1. mu.M treatment group, so that a 2.5. mu.M β -amyloid concentration was used as a condition for subsequent SH-SY5Y cell damage experiments.
EXAMPLE 2 treatment of the cell growth of human neuroblastoma cell line SH-SY5Y with the composition of the invention and beta-amyloid protein
Will be the spirit of human(ii) cell line SH-SY5Y (
CRL2266 TM) At a rate of 1X 10 per hole5Cell densities of the cells were plated in 24-well cell culture dishes with 10% FBS DMEM/F12 medium (Gibco11330032) at 37 ℃ in 5% CO2After 24 hours of incubation in an incubator, the compositions of the invention of preparation 1 dissolved in 0.1M citric acid (pH3.5) were added in different amounts to the plates to subject the cells to 0, 2.5, 5 and 10. mu.g/mL of the compositions of the invention for 1 hour, followed by replacement of the medium with a medium containing 2.5. mu.M of β -amyloid 1-42peptide (Taiclone tcsc0032566) and the aforementioned different concentrations of the compositions of the invention, with three replicates of treatment per concentration, with the group not treated with the composition of the invention and β -amyloid being the control group. Then at 37 ℃ and 5% CO2After culturing in an incubator for 24 hours, the collected cells were stained with Trypan blue (Trypan blue), the growth of the cells was observed through a microscope and the number of unstained living cells was calculated, and plotted on the basis of a control group, and as a result, as shown in fig. 2, it can be seen that the relative ratio of the number of cells of the group treated with the composition of the present invention was higher than that of the group damaged with β -amyloid and was comparable to that of the undamaged control group, indicating that the treatment of the composition of the present invention has the effect of repairing damaged nerve cells. The compositions of the invention are therefore useful for protecting nerve cells.
EXAMPLE 3 Co-treatment of the nitric oxide concentration of human neuroblastoma cell line SH-SY5Y with the composition of the invention and beta-amyloid
The damage of nerve cells caused by nitric oxide radicals is closely related to neuropathy diseases such as Alzheimer's disease, and the present example tests the degree of attack of cells by nitric oxide radicals by measuring the concentration of nitric oxide concentration in a culture medium.
Human neuroblastoma cell line SH-SY5Y (
CRL2266 TM) At a rate of 1X 10 per hole5Cell densities of the cells were plated in 24-well cell culture dishes with 10% FBS DMEM/F12 medium (Gibco11330032) at 37 ℃ in 5% CO2After 24 hours of incubation in an incubator, the cells were treated with 0, 2.5, 5 and 10 μ g/mL of the composition of the present invention in preparation example 1 dissolved in 0.1M citric acid (pH3.5) in different amounts to the plates for 1 hour, and then the medium was replaced with a medium containing 2.5 μ M of β -amyloid (β -amyloid 1-42peptide, Taiclone tcsc0032566) and the composition of the present invention at the different concentrations described above, and the group not treated with the composition of the present invention and β -amyloid were used as control groups, and treatment was repeated three times at each concentration. Then at 37 ℃ and 5% CO2After culturing in the incubator for 20 hours, the cell culture fluid was collected, and the concentration of the nitric oxide was measured using a Biovision K262 reagent set and a manual, and the concentration of the radical was measured for each set. And plotted against the nitric oxide concentration of the control group, the results are shown in fig. 3, and the relative ratio of the nitric oxide concentrations of the groups treated with the composition of the present invention is significantly lower than that of the group damaged by β -amyloid protein and lower than that of the control group. The composition of the present invention can be used for scavenging free radicals and protecting nerve cells from free radical attack.
Example 4 Effect of SH-SY5Y cell treatment on MPP + on cell survival
This example uses the enzyme of viable cell mitochondria to reduce MTT reagent (Thiazoly Blue Tetrazolium Bromide) to formazan crystals, and measures the absorbance after dissolution to reflect the viability of the cells.
Human neuroblastoma cell line SH-SY5Y (
CRL2266 TM) At a rate of 1X 10 per hole5Cell densities of the cells were seeded into 24-well cell culture dishes with 10% FBS DMEM/F12 medium (Gibco11330032),and 5% CO at 37 deg.C2After 24 hours of incubation in an incubator, cells were treated with MPP + at 0, 1, 1.25, 1.5 and 2mM by adding different amounts of 100mM MPP + (Sigma D048) in sterile secondary water to the plates, and the 0mM MPP + treated group was used as a control group, with triplicate treatments at each concentration, and 5% CO at 0 hours or 37 ℃ respectively2MTT was treated at 24-48-hour culture time points in an incubator to observe cell growth, specifically, the culture medium of the culture dish was replaced with 500. mu.L of 1mg/mL MTT solution, and the resulting mixture was incubated at 37 ℃ with 5% CO2The culture was carried out in an incubator for 4 hours. Further, formazan crystals were collected into a 1.5mL centrifuge tube and centrifuged at 14,000rpm for five minutes, and then the supernatant was removed and stored in a-80 ℃ refrigerator. After dissolving the formazan crystals in 100. mu.L of 100% ethanol and DMSO (volume ratio 1: 1), shaking the solution on a shaker for 20 minutes, sucking 50. mu.L of the completely dissolved formazan crystals into a 96-well cell culture dish, reading the absorbance at 565nm with an enzyme immunoassay reader, and plotting the absorbance of the control group as a reference, the result is shown in FIG. 4, the MPP + treatment damages SH-SY5Y cells, and the survival rate of SH-SY5Y cells is lower as the concentration and time of the MPP + treatment are increased.
Example 5 SH-SY5Y Effect of cell treatment on the composition of the invention and MPP + on cell survival
In this example, MTT (Thiazoly Blue Tetrazolium Bromide) reagent can be reduced to formazan crystal by using enzyme in viable cell mitochondria, and the absorbance value after dissolution is measured to reflect the survival rate of cells.
Human neuroblastoma cell line SH-SY5Y (
CRL2266 TM) At a rate of 1X 10 per hole5Cell densities of the cells were plated in 24-well cell culture dishes with 10% FBS DMEM/F12 medium (Gibco11330032) at 37 ℃ in 5% CO2After 24 hours in an incubator, the cells were subjected to a temperature of 0, 0M citric acid (pH3.5) by adding various amounts of the composition of the present invention dissolved in 0.1M citric acid to the culture dish,5. 10 and 25. mu.g/mL of the composition of the present invention in preparation example 1 were treated for 1 hour, and then the medium was replaced with a medium containing 1.5M MPP + (Sigma D048) prepared from 100mM MPP + (Sigma D048) in sterile secondary water, and then the treatment was continued at 37 ℃ with 5% CO2After 24 hours in the incubator, MTT was treated and cell growth was observed, in which each treatment was repeated twice, and the group not treated with the composition of the present invention and β -amyloid was used as a control group. Specifically, the culture medium in the culture dish was replaced with 500. mu.L of 1mg/mL MTT solution, and the mixture was incubated at 37 ℃ with 5% CO2The culture was carried out in an incubator for 4 hours. Further, formazan crystals were collected into a 1.5mL centrifuge tube, centrifuged at 14,000rpm for five minutes, the supernatant was removed, and the mixture was stored in a-80 ℃ refrigerator. After dissolving the formazan crystals in 100. mu.L of 100% ethanol and DMSO (volume ratio 1: 1), shaking for 20 minutes on a shaker, sucking 50. mu.L of the completely dissolved formazan crystals into a 96-well cell culture dish, reading the absorbance at 565nm with an enzyme immunoassay reader, and obtaining a graph of the relative ratio of absorbance based on a control group as shown in FIG. 5.
Example 6 neurosurgery and Experimental Schedule
The neurosurgical method is adjusted according to Wu, C.R., Lin, H.C., & Su, M.H (2014), reverse by aqueous extracts of national tuff from biological defects in Alzheimer's disease-like model: recurrence for analog deposition and central neurostimulator function, BMC compensated and alternative media, 14(1), 202: after 21 days of pretreatment with the composition of the present invention, all rats of preparation example 2 were anesthetized with suetat (Zoletil) and fixed in a stereotaxic apparatus (David Kopf). Wherein an infusion cannula was implanted into the left ventricle of the rat (Kawayota AP-1.5mm, ML +0.8mm, V-3.6mm), the infusion of beta-amyloid (280 pmol/day) was maintained for 29 days by connecting the infusion cannula to a mini osmotic pump (Alzet 2002; Alza, Palo Alto, CA, USA, mini-osmotic pump), the injection of beta-amyloid was started after the infusion cannula was implanted and connected to the mini osmotic pump, and this day was designated as day 0 (day 0), and the control group (sham) was not injected with beta-amyloid. The composition of the invention was administered to the rats one hour prior to the behavioral test comprising the motor test (lococotor test) (on day 21 after β -amyloid injection), the inhibitory avoidance test (inhibitoray avoidance) (on days 22-23 after β -amyloid injection), and the morris water maze (morris water maze) (on days 24-28 after β -amyloid injection). On day 29, all rats were sacrificed one hour after the last administration of the composition of the present invention to measure the activity of acetylcholinesterase (AChE) in the cerebral cortex. The schedule of the operation, the treatment with the composition of the present invention, and the behavior test is shown in fig. 6.
Example 7 locomotor Activity
The locomotor activity of rats was tested as described in Wu, C.R., Tsai, C.W., Chang, S.W., Lin, C.Y., & Huang, L.C. (2015.) Carnosic acid technologies acquisition 6-hydroxydopamin-induced neurooxidation in vivo and in vitro model of Parkinson's disease: innovative of oxidative enzymes induction, chemical-Biological Interactions,225,40-46, and determined by open space Instruments (coulomb Instruments L.L.L.C., PA, USA). Each rat was placed independently in an open space apparatus for 15 minutes and allowed to move freely therein, the distance and speed of movement being recorded by Truscan software (Coulbourn Instruments l.l.c., PA, USA, Truscan software v 2.07).
The locomotor activity results are shown in fig. 7A and 7B, the moving distance and average speed of the rats injected with β -amyloid protein were significantly decreased compared to those of the sham-operated group, and the rats pretreated with the composition of the present invention at low, medium and high concentrations were largely restored to the decrease in moving distance and speed caused by β -amyloid protein.
Example 8 inhibitory avoidance test
The protocol for the inhibition avoidance test in rats is described in Wu, C.R., Lin, H.C., & Su, M.H, (2014.) reverse by aqueous extracts of Cistan tuff from biological assays in Alzheimer's disease-like model: release for amplified placement and central nervous system function, BMC complex and alternative media, 14, 202. Briefly, the operating room was maintained in a dark environment during the experiment, and at the training day, each rat was independently placed in the light room of the apparatus for 5 seconds, the gate was opened, the residence time of the rat in the light room (step-through latency) was measured, and unavoidable electrical stimulation was given when the rat entered the dark room (0.8mA for 2 seconds). After the shock, the rats were removed from the dark room and returned to the home cage. If the rat did not enter the dark room after 90 seconds, it was forced into the dark room and given an electrical stimulus. After 24 hours, the residence time in the bright room was recorded using a similar procedure to assess the retention of memory. If the time of staying in the bright room exceeds 300 seconds, the learning and memory are considered to be normal.
Evaluation of memory retention as shown in fig. 8, the retention time of beta-amyloid-infused rats in the open compartment was significantly decreased (p <0.001) compared to the sham-operated group, while the pre-treated low, medium, and high concentrations of the composition of the present invention increased the retention time of the open compartment decreased by beta-amyloid 254%, 201%, and 198%, respectively, compared to the beta-amyloid-only rats.
Example 9 Morris Water maze
All rats were tested in the Morris water maze to determine spatial learning ability, as described in Wu, C.R., Lin, H.C., & Su, M.H. (2014.) reverse by aqueous extracts of national tuff from biological definitions in Alzheimer's disease-like model, BMC comparative and adaptive media, 14(1),202. Briefly, a black round stainless steel water bath (165 cm diameter and 60 cm high) was filled to 35 cm and the water temperature was controlled at 23. + -. 1 ℃. The pool was divided into equal 4 quadrants, each containing a release point. Each rat was trained twice a day for four consecutive days to find a plastic glass platform (10 cm in diameter) centered in the northeast quadrant, hidden 1 cm below the water surface. In each training, rats are placed in sequence at the release point of one of the quadrants of the pool and each rat can swim up to 120 seconds to find the platform, if the rat does not find the platform within the termination time, the rat is guided to the platform by the operator. Rats may rest on the platform for 15 seconds, either failure or success. In addition, in the spatial probe test (probe test), the plastic glass platform was removed to check the reference memory of the last training. Each rat was released from the opposite quadrant of the removal platform quadrant and the rat was left to swim for 60 seconds, the time to first cross to the original platform-placed quadrant was measured, and data was recorded with a video camera and an automated video tracking system equipped with EthoVision XT (Noldus Information Technology, Leesburg, VA, USA) software.
Results of spatial learning capacity for eight of the four training days referring to both fig. 9A and 9B, the rats injected with β -amyloid spend more time finding the platform. In particular, rats injected with β -amyloid were significantly increased in mean time to find the platform compared to sham group (sham group) on the first and third days of training (p <0.01 and p < 0.001). In contrast, the composition of the present invention reduced the average time to find the plateau (p <0.05, p <0.01 and p <0.001) at all concentrations compared to the treatment of the β -amyloid group alone (fig. 9A). This shows that the composition of the present invention improves the steric learning disability caused by the injection of β -amyloid. After the last training, a spatial probation assay was performed to assess reference memory, as shown in fig. 9B, rats injected with β -amyloid took significantly more time to reach the quadrant of the original platform for the first time (p <0.05) than sham operated groups. The time for the low-concentration group, the medium-concentration group and the high-concentration group of the composition of the invention to reach the quadrant of the original platform is respectively reduced by 79%, 49% and 67% compared with the time for treating only the beta-amyloid group for the first time (p <0.05 and p < 0.01). In conclusion, the composition of the present invention improves memory impairment caused by β -amyloid.
Example 10 Acetylcholinesterase Activity measurement
Acetylcholine is an important nerve transfer substance in the brain, and the excessively high activity of acetylcholinesterase can decompose and reduce the concentration of acetylcholine, which seriously affects memory and cognitive functions, and is considered to be closely related to alzheimer's disease.
Rat cerebral cortex from the previous experiment was homogenized in ice Phosphate Buffered Saline (PBS), the homogenate was centrifuged at 12000rpm for 15 minutes at 4 ℃ and the supernatant collected and stored at-80 ℃ for determination of acetylcholinesterase activity. The determination of acetylcholinesterase activity was carried out according to the method described by Ellman et al, 1961. Briefly, 20 μ L of each cortical supernatant was added to a 96-well plate containing 90 μ L of 50mM ice PBS buffer, followed by 20 μ L of 10mM 5,5 '-dithio (2-nitrobenzoic acid) [5, 5' -dithiobis (2-nitrobenzoic acid) ] and 20 μ L of 75mM thioacetyl choline iodide (acetylthiocholine) matrix, yielding a yellow 5-thio-2-nitrobenzoic acid product after three minutes and reading the absorbance at 412 nm. The acetylcholinesterase activity of sham group (sham group) was ordered to be 100%
The results are shown in fig. 10, where β -amyloid significantly increased the acetylcholinesterase activity of cerebral cortex by about 41.1% (p <0.05) compared to the sham group. Whereas the pretreatment of the low and medium concentration compositions of the present invention reduced the increase in enzyme activity by β -amyloid by about 26% and 32%, respectively, compared to the β -amyloid alone (p < 0.05).
In summary, the composition containing ferrous amino acid particles (the composition of the present invention) can treat or alleviate diseases related to nerve damage. In particular, the compositions of the present invention can repair nerve cell damage caused by β -amyloid, which is closely associated with alzheimer's disease; and the composition can repair nerve cell damage caused by MPP +, which is known to cause Parkinson's disease in the literature, so the composition can be used for treating Alzheimer's disease and Parkinson's disease. In addition, animal experiments prove that the composition can improve the acetylcholinesterase activity increase, memory impairment and behavior performance caused by beta-amyloid, and the composition can be used for protecting the neurodegeneration caused by the Alzheimer disease.
Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the scope and spirit of this invention. Although the invention has been described in connection with specific preferred embodiments, it is to be understood that the invention is not to be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the following claims.
Claims (10)
- Use of a composition for preparing a medicament for treating or alleviating a disease associated with nerve damage, wherein the composition comprises ferrous amino acid chelate particles sintered from a ferrous amino acid chelate, the ferrous amino acid chelate particles have an average particle size of 500 nm to 2600 nm and an average molecular weight of 1,500 daltons (Dalton) to 600,000 daltons, and the medicament comprises an effective amount of the composition and a pharmaceutically acceptable carrier.
- The use of claim 1, said ferrous amino acid chelate particles having an average molecular weight of from 1,500 daltons to 550,000 daltons.
- The use of claim 1, wherein the weight ratio of ferrous iron to amino acid of the ferrous amino acid chelate is between 1: 1 to 1: 4 in the middle.
- The use of claim 1, wherein the weight ratio of ferrous iron to amino acid of the ferrous amino acid chelate is between 1: 1.5 to 1: 2.5.
- The use according to claim 4, wherein the ferrous amino acid chelate is prepared by mixing an iron compound and an amino acid and heating at 60 ℃ to 90 ℃ for 8 hours to 48 hours, wherein the weight ratio of the iron compound to the amino acid is 1: 1.2 to 1: 1.5.
- The use of claim 5, wherein the iron compound is ferrous sulfate, ferrous chloride, ferrous pyrophosphate, or a combination thereof.
- The use according to claim 1, wherein said ferrous amino acid chelate is ferrous glycine chelate.
- The use according to claim 1, wherein the disease associated with nerve damage is alzheimer's disease.
- The use according to claim 1, wherein the nerve damage related disease is parkinson's disease.
- The use according to any one of claims 1, 8, 9, wherein the effective dose is from 1.3mg/kg to 5.8 mg/kg.
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| PCT/CN2019/123869 WO2020125462A1 (en) | 2018-12-20 | 2019-12-09 | Use of composition comprising ferrous amino acid chelate particles in the preparation of medicament for treating or relieving diseases associated with nerve damage |
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| CN101102762A (en) * | 2004-12-22 | 2008-01-09 | 药物技术公司 | Compositions including iron |
| CN104769426A (en) * | 2012-07-27 | 2015-07-08 | 卢特波尔德药品公司 | Method of treating iron deficiency anemia |
| CN104955452A (en) * | 2013-09-05 | 2015-09-30 | 普惠德生技股份有限公司 | Use of composition containing ferrous amino acid chelate in preparation of an anti-cancer medicament |
| TWI587856B (en) * | 2016-05-26 | 2017-06-21 | Profeat Biotechnology Co Ltd | Compositions containing ferrous amino acid chelates are used in the manufacture of reduced cancer production The use of lactic acid medicine |
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| US20090035385A1 (en) * | 2004-12-22 | 2009-02-05 | Drugtech Corporation | Compositions including iron |
| JP5098051B2 (en) * | 2007-04-05 | 2012-12-12 | Ｓｂｉファーマ株式会社 | Mitochondrial disorder brain disease therapeutic agent and diagnostic agent |
| CN103476419A (en) * | 2011-01-07 | 2013-12-25 | 梅里翁第三研究有限公司 | Pharmaceutical compositions of iron for oral administration |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101102762A (en) * | 2004-12-22 | 2008-01-09 | 药物技术公司 | Compositions including iron |
| CN104769426A (en) * | 2012-07-27 | 2015-07-08 | 卢特波尔德药品公司 | Method of treating iron deficiency anemia |
| CN104955452A (en) * | 2013-09-05 | 2015-09-30 | 普惠德生技股份有限公司 | Use of composition containing ferrous amino acid chelate in preparation of an anti-cancer medicament |
| TWI587856B (en) * | 2016-05-26 | 2017-06-21 | Profeat Biotechnology Co Ltd | Compositions containing ferrous amino acid chelates are used in the manufacture of reduced cancer production The use of lactic acid medicine |
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| WO2020125464A1 (en) | 2020-06-25 |
| TWI717139B (en) | 2021-01-21 |
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| WO2020125462A1 (en) | 2020-06-25 |
| TWI710370B (en) | 2020-11-21 |
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