CN109498596B - Composition for treating senile dementia and preparation method and application thereof - Google Patents
Composition for treating senile dementia and preparation method and application thereof Download PDFInfo
- Publication number
- CN109498596B CN109498596B CN201811622989.4A CN201811622989A CN109498596B CN 109498596 B CN109498596 B CN 109498596B CN 201811622989 A CN201811622989 A CN 201811622989A CN 109498596 B CN109498596 B CN 109498596B
- Authority
- CN
- China
- Prior art keywords
- parts
- coenzyme
- composition
- preparation
- brain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/12—Ketones
- A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/5123—Organic compounds, e.g. fats, sugars
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5169—Proteins, e.g. albumin, gelatin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
Abstract
The invention provides a composition for treating senile dementia and a preparation method and application thereof, belonging to the technical field of biological medicines. The composition provided by the invention comprises the following components in parts by mass: 0.5-1 part of coenzyme Q10, 0.05-0.2 part of polysorbate, 0.3-0.8 part of medium-chain fatty acid triglyceride, 3-8 parts of gelatin, 3-8 parts of glycerol and 3-8 parts of sorbitol. The composition provided by the invention can effectively penetrate through a blood brain barrier to enter deep part of brain and reach target neuron under the packaging specification of less than or equal to 50 nm; the nerve cell mitochondria are efficiently activated, the activity of the mitochondrial cytochrome C is improved, the ATP synthesis is enhanced, and energy is provided for neurons; reducing formaldehyde in brain, reducing promotion effect of formaldehyde on senile plaque, removing intercellular space blockage, and recovering flow of intercellular fluid; has no toxicity to human body, has the capability of enhancing immunity, and can improve the memory of dementia model mice.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a composition for treating senile dementia and a preparation method and application thereof.
Background
Senile dementia (AD) is a progressive degenerative disease of the nervous system with occult onset. Clinically, the overall dementia such as dysmnesia, aphasia, disuse, agnosia, impairment of visual spatial skills, dysfunction in execution, and personality and behavior changes are characterized, and the etiology is unknown. Development of AD drugs is done worldwide at billions of dollars per year, and none of them enters phase four. Dementia faces the embarrassment of no medication and failure to reverse memory decline. At present, clinical dementia drugs such as memantine, donepezil, curcumin and the like have obvious curative effect in the early stage of dementia, but gradually lose the clinical curative effect after 5 years. AD imposes a heavy economic and social burden on every country, family.
Clinical studies suggest that extracellular deposition of a β to form senile plaques and intracellular oligomerization to produce toxicity are positive and key causes of the induction of genetic family AD. However, the most authoritative british journal "lancet" 2016 announced: at present, the existing development of AD drugs fails. Antibodies, small molecule compounds, and polypeptides that bind to a β, which are directed against the production, aggregation, and elimination of a β, have not achieved clinically expected therapeutic effects. These disastrous failures, high economic costs, compel us to consider-what causes failed AD drug development. The current view is that all 95% of antibodies, synthetic polypeptides and macromolecules are difficult to permeate blood brain barrier, which is the main reason for the failure of most developed AD drugs. However, there are also studies showing that: some of the developed AD drugs can penetrate the blood-brain barrier, and it is difficult to understand that the drugs still cannot rescue neuronal death deep in the brain. However, our recent studies have now revealed the reasons for drug failure: the medicine has too large diameter to penetrate the intercellular space less than 50nm, and reaches the neurons in the deep part of brain via the flow of intercellular fluid, so as to protect the neurons.
Disclosure of Invention
In view of the problems in the background art, the present invention aims to provide a composition for effectively treating senile dementia, a preparation method and an application thereof.
The invention provides a composition for treating senile dementia, which comprises the following components in parts by mass: 0.5-1 part of coenzyme Q10, 0.05-0.2 part of polysorbate, 0.3-0.8 part of medium-chain fatty acid triglyceride, 3-8 parts of gelatin, 3-8 parts of glycerol and 3-8 parts of sorbitol.
Preferably, the composition comprises the following components in parts by mass: 0.7-0.85 part of coenzyme Q10, 0.08-0.12 part of polysorbate, 0.4-0.6 part of medium-chain fatty acid triglyceride, 4-6 parts of gelatin, 4-6 parts of glycerol and 4-6 parts of sorbitol.
Preferably, the composition is powder, and the particle diameter of the powder is less than or equal to 50 nm.
The invention provides a preparation method of the composition, which comprises the following steps:
(1) mixing coenzyme Q10, polysorbate, medium-chain fatty acid triglyceride, gelatin, glycerol, sorbitol and anhydrous ethanol to obtain an organic phase;
(2) mixing the organic phase with water which is 3-8 times of the volume of the organic phase to obtain a mixed solution;
(3) heating the mixed solution to 60-80 ℃, stirring, evaporating and concentrating to 0.3-0.8 time of the volume of the organic phase to obtain a nano emulsion;
(4) placing the nano emulsion in an environment of 0-4 ℃ and continuously stirring to obtain a nano coenzyme Q10 suspension;
(5) and homogenizing the nano-coenzyme Q10 suspension, and performing vacuum freeze drying to obtain the composition for treating senile dementia.
Preferably, the ratio of the mass of the coenzyme Q10 to the volume of the absolute ethyl alcohol in the step (1) is 0.5-1 g: 100 mL.
Preferably, the stirring speed in the step (3) or (4) is 1000-2000 rpm.
Preferably, the stirring time in the step (4) is 1-2 h.
Preferably, the temperature for homogenizing in the step (5) is 45-55 ℃, and the pressure for homogenizing is 200-1500 bar.
Preferably, the number of homogenization is 2-4, and the pressure of homogenization is gradually increased.
The invention also provides application of the composition for treating senile dementia in preparation of a medicine for treating senile dementia.
Has the advantages that: the invention provides a composition for treating senile dementia, which comprises the following components in parts by mass: 0.5-1 part of coenzyme Q10, 0.05-0.2 part of polysorbate, 0.3-0.8 part of medium-chain fatty acid triglyceride, 3-8 parts of gelatin, 3-8 parts of glycerol and 3-8 parts of sorbitol. The research of the invention finds that: senile plaques formed by a β aggregation block the intercellular space (ECS) of adjacent neurons, blocking the flow of intercellular fluid (ISF) from the superficial layer of the brain to the deep part of the brain. This means that blockage of neuronal intercellular spaces is a key cause of the inability of drugs to enter deep in the brain with interstitial fluid. Therefore, the invention achieves the aim of treating the dementia by developing the medicine with the diameter less than 50nm to reach the deep part of the brain along with the flow of intercellular fluid, and provides a new idea for treating the dementia. The composition provided by the invention can effectively penetrate through a blood brain barrier to enter deep part of brain and reach target neuron under the packaging specification of less than or equal to 50 nm; the nerve cell mitochondria are efficiently activated, the activity of the mitochondrial cytochrome C is improved, the ATP synthesis is enhanced, and energy is provided for neurons; reducing formaldehyde in brain, reducing promotion effect of formaldehyde on senile plaque, removing intercellular space blockage, and recovering flow of intercellular fluid; has no toxicity to human body, has the capability of enhancing immunity, and can improve the memory of dementia model mice.
Drawings
FIG. 1 is a schematic representation of a composition provided by the present invention upon crossing the blood-brain barrier;
FIG. 2 is a schematic diagram of the treatment of Alzheimer's disease with the composition of the present invention;
FIG. 3 shows the results of the detection in example 3 of the present invention;
FIG. 4 shows the results of the detection in example 4 of the present invention;
FIG. 5 shows the results of the detection in example 5 of the present invention;
FIG. 6 shows the results of the detection in example 6 of the present invention.
Detailed Description
The invention provides a composition for treating senile dementia, which comprises the following components in parts by mass: 0.5-1 part of coenzyme Q10, 0.05-0.2 part of polysorbate, 0.3-0.8 part of medium-chain fatty acid triglyceride, 3-8 parts of gelatin, 3-8 parts of glycerol and 3-8 parts of sorbitol.
The composition provided by the invention comprises coenzyme Q10, polysorbate, medium-chain fatty acid triglyceride, gelatin, glycerol and sorbitol. In the invention, the amount of the coenzyme Q10 is 0.5-1 part by mass, preferably 0.7-0.85 part by mass, and more preferably 0.78 part by mass; the dosage of the polysorbate is 0.05-0.2 part, preferably 0.08-0.12 part, and more preferably 0.1 part; the dosage of the medium-chain fatty acid triglyceride is 0.3-0.8 part, preferably 0.4-0.6 part, and more preferably 0.5 part; the using amount of the gelatin is 3-8 parts, preferably 4-6 parts, and more preferably 5 parts; the using amount of the glycerol is 3-8 parts, preferably 4-6 parts, and more preferably 5 parts; the sorbitol is used in an amount of 3-8 parts, preferably 4-6 parts, and more preferably 5 parts. In the invention, the components and the dosage ratio can effectively improve the wrapping rate and the drug loading rate of the coenzyme Q10 (especially, the effect is better under the condition that the drug-fat ratio is 1: 20). The sources of the above components are not particularly limited in the present invention, and any products conventionally commercially available in the art may be used. In the embodiment of the invention, the purchasing channels of the components correspond to the following: coenzyme Q10(CAS:303-98-0, Sigan Siji Biotechnology Co., Ltd.), polysorbate-80 (CAS:9005-65-6, Hunan Guanao Biotechnology Co., Ltd.), medium-chain fatty acid triglyceride (CAS:73398-61-5, Shanghai eosin oil Co., Ltd.), gelatin (CAS:9000-70-8, Qihuaton chemical products Co., Ltd. Zhengzhou), glycerin (CAS:56-81-5, Shanghai Yien chemical technology Co., Ltd.), sorbitol (CAS:142-77-8, Meilan chemical brand is Meilan practical (Shanghai) Co., Ltd.), and absolute ethyl alcohol (CAS:64-17-5, Shanghai Square wild chemical Co., Ltd. Prita division Co., Ltd.).
In the present invention, the composition is preferably in the form of a powder. The particle diameter of the powder is preferably less than or equal to 50nm, more preferably less than or equal to 40nm, and more preferably 25-35 nm. The research of the invention finds that the coenzyme Q10 packaged with the diameter less than or equal to 50nm can effectively penetrate through the blood brain barrier to enter the deep part of the brain, efficiently activate the nerve mitochondria, generate ATP and improve the memory of dementia mice.
The invention provides a preparation method of the composition, which comprises the following steps:
(1) mixing coenzyme Q10, polysorbate, medium-chain fatty acid triglyceride, gelatin, glycerol, sorbitol and anhydrous ethanol to obtain an organic phase;
(2) mixing the organic phase with water which is 3-8 times of the volume of the organic phase to obtain a mixed solution;
(3) heating the mixed solution to 60-80 ℃, stirring, evaporating and concentrating to 0.3-0.8 time of the volume of the organic phase to obtain a nano emulsion;
(4) placing the nano emulsion in an environment of 0-4 ℃ and continuously stirring to obtain a nano coenzyme Q10 suspension;
(5) and homogenizing the nano-coenzyme Q10 suspension, and performing vacuum freeze drying to obtain the composition for treating senile dementia.
The method comprises the steps of mixing coenzyme Q10, polysorbate, medium-chain fatty acid triglyceride, gelatin, glycerol, sorbitol and absolute ethyl alcohol to obtain an organic phase. In the present invention, the ratio of the mass of coenzyme Q10 to the volume of absolute ethanol is preferably 0.5 to 1 g: 100mL, more preferably 0.7-0.85 g: 100mL, more preferably 0.78 g: 100 mL. The mixing order of the above components is not particularly limited in the present invention. In the invention, ultrasonic treatment is preferably used after the mixing, and the intensity of the ultrasonic treatment is preferably 150-250W, and more preferably 200W. The time of ultrasonic treatment is preferably 15-25 min, and more preferably 20 min. The ultrasonic treatment is more beneficial to the full dispersion of each component in the absolute ethyl alcohol.
After the organic phase is obtained, the organic phase is mixed with water to obtain a mixed solution. In the invention, the water is preferably distilled water, and the amount of the water is preferably 3-8 times, and more preferably 5 times of the volume of the organic phase. The present invention preferably uses a syringe to slowly inject the organic phase into the water. During the injection, the present invention preferably performs stirring. The stirring speed is preferably 1000-2000 rpm, and more preferably 1500 rpm.
After the mixed solution is obtained, the mixed solution is heated, stirred, evaporated and concentrated to obtain the nano emulsion. In the invention, the heating temperature is preferably 60-80 ℃, more preferably 65-75 ℃, and more preferably 70 ℃. The stirring speed is preferably 1000-2000 rpm, and more preferably 1500 rpm. The heating and stirring can promote the evaporation of the solvent in the mixed solution, and the concentration of the mixed solution is realized. In the present invention, the degree of concentration is preferably 0.3 to 0.8 times the volume of the organic phase, and more preferably 0.5 times the volume of the organic phase.
Concentrating to obtain nanometer emulsion. The nano-emulsion is placed in a low-temperature environment and continuously stirred to obtain nano-coenzyme Q10 suspension. In the invention, the low-temperature stirring can enable the coenzyme Q10 to be quickly wrapped, and the drug loading capacity is improved. In the invention, the low temperature is preferably 0-4 ℃, and more preferably 0-2 ℃. The low temperature environment is preferably an ice water bath. The speed of the continuous stirring is preferably 1000-2000 rpm, and more preferably 1500 rpm. The time for continuing stirring is preferably 1-2 h, and more preferably 1.5 h.
Stirring to obtain suspension of nanometer coenzyme Q10. The invention homogenizes the suspension of the nano-coenzyme Q10. In the invention, the homogenizing temperature is preferably 45-55 ℃, and more preferably 50 ℃. The homogenizing pressure is preferably 200-1500 bar. In the present invention, the number of homogenization is preferably 2 to 4, and more preferably 3. The homogenizing pressure is increased gradually, and when the homogenizing time is 3 times, the homogenizing pressure is preferably 250-350 bar, 700-900 bar, 1100-1300 bar, more preferably 300bar, 800bar and 1200bar in sequence.
The invention homogenizes the suspension of the nano-coenzyme Q10, and then the suspension is frozen and dried in vacuum to obtain white powder. The diameter of the white powder detected by a cryoelectron microscope is 30-40 nm. After obtaining the white powder, the present invention preferably stores the white powder in a closed environment at 4 ℃. The white powder is the composition for treating senile dementia. As shown in fig. 1, the white powder has a smaller diameter than the intercellular space and can penetrate the blood-brain barrier into the deep brain.
The invention also provides the application of the composition in preparing a medicament for treating senile dementia. In the present invention, the medicament is preferably a capsule; the capsule adopts oral administration, preferably one capsule per day. In the present invention, each capsule preferably contains 10 micrograms of nano-coenzyme. In the invention, the mass content of the nano-coenzyme Q10 in the capsule finished product is preferably 5-20%, and more preferably 10%.
In the present invention, the applied treatment principle is shown in fig. 2. The molecular mechanism of AD generation is as follows: familial dementia produces excessive a β, which deposits in the intercellular space (ECS) between two neurons to form Senile Plaques (SP), which in turn block the flow of intercellular fluid (ISF). The A beta can be oligomerized into cell mitochondria to be combined with the FDH of formaldehyde dehydrogenase to inhibit the activity of the FDH, so that the formaldehyde can not be degraded and accumulated. Excessive formaldehyde in conjunction with a β induces an increase in intracellular calcium and excessive reactive oxygen species are produced to induce neuronal death. The composition provided by the invention has the effect of treating AD: on one hand, the coenzyme Q10 packaged with less than or equal to 50nm (especially 30nm) can reduce formaldehyde in brain, reduce the promotion and formation effect of formaldehyde on senile plaques, achieve the purpose of clearing intercellular space blockage and recover the flow of intercellular fluid; on the other hand, coenzyme Q10 packaged at 50nm or less (especially 30nm) can flow to deep part of brain along with intercellular fluid in intercellular space, provide ATP energy for neuron in apoptosis or death, provide mitochondrial function, and save neuron activity, thereby gradually recovering brain memory.
Experiments show that the composition provided by the invention can effectively penetrate through a blood brain barrier to enter deep part of brain and reach target neurons; the nerve cell mitochondria are efficiently activated, the activity of the mitochondrial cytochrome C is improved, the ATP synthesis is enhanced, and energy is provided for neurons; reducing formaldehyde in brain, reducing promotion effect of formaldehyde on senile plaque, removing intercellular space blockage, and recovering flow of intercellular fluid; has no toxicity to human body, has the capability of enhancing immunity, and can improve the memory of dementia model mice.
The composition for treating senile dementia and the preparation method and application thereof provided by the present invention will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Preparation of Nanoprockaged coenzyme Q10
The main raw materials are as follows: coenzyme Q10(CAS:303-98-0, Sigan Siji Biotechnology Co., Ltd.), polysorbate-80 (CAS:9005-65-6, Hunan Guanao Biotechnology Co., Ltd.), medium-chain fatty acid triglyceride (CAS:73398-61-5, Shanghai eosin oil Co., Ltd.), gelatin (CAS:9000-70-8, Zhengzhou Qihuaton chemical products Co., Ltd.), glycerol (CAS:56-81-5, Shanghai Yien chemical technology Co., Ltd.), sorbitol (CAS:142-77-8, Meilan chemical brand is Meilan industry Co., Ltd.), absolute ethyl alcohol (CAS:64-17-5, Shanghai Square chemical industry Co., Ltd.), purified water (laboratory deionized water).
The preparation method comprises the following steps: preparation of nano-packaged coenzyme Q10 by original emulsification-low temperature-high pressure method
Precisely weighing 0.78g of coenzyme Q10, 0.1g of polysorbate-80, 0.5g of medium-chain fatty acid triglyceride, 5g of gelatin, 5g of glycerol and 5g of sorbitol;
dissolving the weighed components in 100mL of absolute ethyl alcohol, and performing 200W ultrasonic treatment twice for 10 minutes to form an organic phase;
thirdly, slowly injecting the organic phase into 200mL of distilled water (1500r/min) by using an injector, stirring at a constant temperature of 70 ℃, evaporating the solvent and concentrating to 50mL to obtain a semitransparent nano emulsion;
quickly adding the nano emulsion into a 50mL ice-water bath container at the temperature of 0-2 ℃, and continuously stirring for 1.5h to obtain nano coenzyme Q10 suspension;
and fifthly, homogenizing the obtained nano-coenzyme Q10 suspension (crude milk) for 3 times under high pressure, wherein the homogenizing pressure is 300bar, 800bar and 1200bar respectively. Homogenizing at 50 deg.C, and vacuum freeze drying. The white powder after drying is the nano-packaged coenzyme Q10. The diameter is 30-40 nm directly by the detection of a cryoelectron microscope. Sealing at 40 deg.C for storage.
Example 2
Research on penetration of nano-packaged coenzyme Q10 into blood brain barrier
The detection method comprises the following steps: high Performance Liquid Chromatography (HPLC)
And (3) detection process:
instruments and reagents: HP1100 high performance liquid chromatograph (HP1100, Agilent Technologies co. ltd, USA); nitrogen blowing instrument (KL512J, Beijing kang science and technology, Inc.). Coenzyme Q10 standard (BBI company, purity greater than 99.9%). Preparing a coenzyme Q10 standard solution: accurately weighing coenzyme Q100.0250g in 25mL volumetric flasks, dissolving coenzyme Q10 in ethanol, metering to a certain volume, shaking up for later use, and preparing into 1g/L standard stock solution. Before the determination, the solution is diluted by a mobile phase according to a standard curve to be used concentration.
Chromatographic conditions are as follows: the chromatographic column adopts a PhenomenexC18 column (5 μm,250mm × 4.6mm i.d.); methanol-ethanol (volume ratio is 20: 80) is used as a mobile phase; detection wavelength switching procedure: 292nm in 0-8 min and 275nm in 8-16 min; the flow rate is 1L/min; the sample volume is 20 mu L; the column temperature was 25 ℃.
Drawing a standard curve: absorbing a proper amount of vitamin E and coenzyme Q10 standard stock solution, diluting with a mobile phase to be a standard series of 0, 2.0, 5.0, 10.0, 20.0 and 50.0mg/L, taking 20 mu L of sample injection for determination, and drawing a standard curve by taking the mass concentration of the standard solution as a horizontal coordinate and the peak area as a vertical coordinate.
Sample treatment: because the coenzyme Q10 contains isopentenyl, the coenzyme Q10 is easy to degrade when exposed to light, and therefore sample pretreatment is carried out under the condition of keeping out of the sun. Taking 0.2mL of blood sample in a centrifuge tube, adding 0.2mL of ethanol to precipitate protein, and mixing uniformly by vortex oscillation for 1 min. Adding 1mL of n-hexane, and uniformly mixing for 2min by vortex oscillation. And taking 800 mu L of supernatant liquid to be placed in a centrifuge tube, and drying by nitrogen. The residue is dissolved by the mobile phase (methanol-ethanol volume ratio is 20: 80), centrifuged at 12000r/min for 2min, and the supernatant is taken for 50 μ L injection determination.
Test site: institute of biophysics, china academy of sciences;
the test result is shown in fig. 3, the content of coenzyme Q10 in the brain of the mouse is increased by about 3 times after the gastric lavage for 15 days.
Example 3
Effect of nano-packaged coenzyme Q10 on ATP
The detection method comprises the following steps: mouse ATP kit (96T, mouse Adenosine Triphosphate (ATP) ELISA kit, Shanghai Heart word Biotech limited)
And (3) detection procedures: mouse brain tissue specimen, cut specimen, and weigh. An amount of PBS, pH7.4 was added. And (5) rapidly freezing and storing the extract by using liquid nitrogen for later use. The temperature of the specimen is still kept between 2 and 8 ℃ after the specimen is melted. An amount of PBS (pH7.4), or a tissue protein extraction reagent, is added and the specimen is homogenized by hand or by a homogenizer. Centrifugation was carried out for about 20 minutes (2000-. The supernatant was carefully collected. Subpackaging the obtained product for detection, freezing the rest product for later use, and detecting according to the kit indication program.
The results are shown in FIG. 4, which shows that the ATP content in brain is increased rapidly after acute injection by more than 3 times compared with the ATP content in brain without package.
Example 4
Action of nano-packaged coenzyme Q10 on formaldehyde in brain
The detection method comprises the following steps: high Performance Liquid Chromatography (HPLC)
And (3) detection procedures:
materials: mouse whole brain, acetonitrile (liquid chromatography grade, Fisher Scientific, usa), 2, 4-dinitrophenylhydrazine (analytical reagent, beijing chemical research institute), formaldehyde (analytical reagent, Sigma-Aldrich, usa), trichloroacetic acid (analytical reagent, west longa chemical corporation), dichloromethane (analytical reagent, beijing chemical plant), sodium chloride (analytical reagent, west longa chemical corporation), acetic acid (analytical reagent, west longa chemical corporation).
Reagent: 2, 4-dinitrophenylhydrazine acetonitrile solution (1.0 g/L): weighing 100mg of 2, 4-dinitrophenylhydrazine, dissolving in acetonitrile to reach the constant volume of 100 ml. 10% trichloroacetic acid solution: 10g of trichloroacetic acid was weighed out and made to a volume of 100ml with ultrapure water.
Sample preparation: preparation of brain samples: taking 1g of brain tissue of different parts, adding 10% trichloroacetic acid solution (10ml), homogenizing, and centrifuging (13000r/min, 4 deg.C, 30 min). 0.4ml of the supernatant, 0.1ml of 2, 4-dinitrophenylhydrazine (1g/L) and 0.5ml of acetonitrile are taken. After mixing, the mixture was incubated at 60 ℃ for 30min, centrifuged (13000r/min, 4 ℃ C., 10min) and the supernatant was taken for HPLC analysis. Retention extraction step (control): 2 volumes of dichloromethane were added, vortexed at vortex for 1min, centrifuged (3000r/min, room temperature, 10min), and the lower layer of extract was collected and placed in a stoppered glass tube. And (3) carrying out water bath at 80 ℃ for 1-2 h, cooling to room temperature after all the extract liquid is evaporated to dryness, adding 1ml of acetonitrile, carrying out vortex oscillation for 1min by vortex, centrifuging (3000r/min, room temperature, 10min), and taking the supernatant for HPLC analysis. High performance liquid chromatography conditions: LC-20A high Performance liquid chromatograph UV-HPLC, SPD-M20A diode array Detector (Shimadzu, Japan). A chromatographic column: LiChrospher 100RP-18(250 mm. times.4.6mm. times. 5 μm) (Merck, Germany); mobile phase: acetonitrile: ultrapure water: 65: 35(v/v), and ultrasonic degassing; flow rate: 0.8 ml/min; sample introduction amount: 20 mu l of the mixture; detection wavelength: 355 nm; column temperature: 35 ℃ is carried out.
Test site: institute of biophysics, china academy of sciences;
the detection results are shown in fig. 5: the nano-packaged coenzyme Q10 can rapidly reduce the content of formaldehyde in the brain of a mouse in an acute gavage mouse.
Example 5
Enhancing effect of nano-packaged coenzyme Q10 on APP mouse memory
The detection method comprises the following steps: morris water maze
The Morris water maze experiment system consists of a circular water pool and an automatic image acquisition and processing system. The automatic image acquisition and processing system mainly comprises a camera, a computer and an image monitor, wherein the monitoring device is started after the animal enters water, the movement track of the animal is recorded, and the relevant parameters of the report are automatically analyzed after the experiment is finished. The Morris water maze round pool is a round ABS engineering plastic bucket, the diameter is 100cm, the height is 50-60cm, the water depth of the pool is 30-40cm, and a round platform, the diameter is 9cm, and the round platform is hidden under the water surface of 2 cm. The inside of the water pool can not be marked, and a proper amount of fresh milk or milk powder is added into the water, so that the water pool becomes opaque milky white. The upper edge of the barrel is equidistantly provided with 4 marking points of east, south, west and north which are used as water inlet points of the animal water inlet pool, and the projection points of the 4 water inlet points on the water surface and the bottom of the bucket divide the water surface and the bucket part into 4 quadrants. The platform can be arbitrarily arranged in the middle of a certain quadrant according to experimental requirements. The water temperature is kept at 23-25 ℃ by proper thermostatic equipment. The water maze pool should be equipped with good water filling and draining equipment, and the location of the pool, once determined, should not be easily changed, especially in the same round of water maze test. The water maze image automatic acquisition and processing system can automatically acquire parameters such as the water entry position of an animal, the swimming speed, the time required by target searching, the running track, the searching strategy and the like, and can carry out statistics and analysis on various acquired data. The experimental training phase was continued for 3 days with 4 trains per day. During training, the rat is placed into the water pool from four water inlet points facing the pool wall, the time from the water inlet of the rat to the finding of an underwater hidden platform and the standing of the rat on the hidden platform is recorded and used as the latency period which is expressed by seconds(s), and the rat is allowed to stand on the platform for 10s after finding the platform. If the rat fails to find the platform 60s after entering the water, it is gently pulled up the platform from the water and stays for 10s before the next training. Each mouse is respectively put into the water pool from four water inlet points for one training, and the interval between the two training is 30 s.
Test site: the research center of brain major diseases of the university of capital medical science;
the detection results are shown in fig. 6: the learning ability of the dementia transgenic mouse is sharply reduced in the training period of 1-6 days, which is shown in that the time for finding the platform hidden under the water surface is increased, and the time for finding the platform is obviously shortened after the nano-package Q10 is treated. After the platform was removed on day 7, the time to find the target quadrant at the original platform position decreased, but the time to stay in the target quadrant was significantly increased after nanocapsule Q10, indicating that memory was significantly increased.
Examples 1 to 5 provided by the present invention show that:
1) the diameter of the nano-packaged coenzyme Q10 is smaller than the diameter of intercellular space, and the coenzyme Q10 can flow with intercellular fluid, and further reach target neurons and enter deep parts of brain.
2) The coenzyme Q10 packaged in nanometer can enter neuron to improve activity of mitochondrial cytochrome C, enhance ATP synthesis, and provide energy for neuron.
3) The coenzyme Q10 packaged in nanometer can reduce formaldehyde in brain, reduce promotion effect of formaldehyde on senile plaque, remove intercellular space blockage, and recover flow of intercellular fluid.
4) The nano-packaged coenzyme Q10 provided by the application does not adopt synthesized external drugs, uses the coenzyme Q10 which is possessed by effective active human neurons, has no toxicity to human bodies and has the function of enhancing the immunity.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (9)
1. The composition for treating senile dementia is characterized by comprising the following components in parts by mass: 0.5-1 part of coenzyme Q10, 0.05-0.2 part of polysorbate, 0.3-0.8 part of medium-chain fatty acid triglyceride, 3-8 parts of gelatin, 3-8 parts of glycerol and 3-8 parts of sorbitol; the composition is powder, and the particle diameter of the powder is less than or equal to 50 nm.
2. The composition according to claim 1, characterized by comprising the following components in parts by mass: 0.7-0.85 part of coenzyme Q10, 0.08-0.12 part of polysorbate, 0.4-0.6 part of medium-chain fatty acid triglyceride, 4-6 parts of gelatin, 4-6 parts of glycerol and 4-6 parts of sorbitol; the composition is powder, and the particle diameter of the powder is less than or equal to 50 nm.
3. A process for the preparation of a composition according to any one of claims 1 or 2, characterized in that it comprises the following steps:
(1) mixing coenzyme Q10, polysorbate, medium-chain fatty acid triglyceride, gelatin, glycerol, sorbitol and anhydrous ethanol to obtain an organic phase;
(2) mixing the organic phase with water which is 3-8 times of the volume of the organic phase to obtain a mixed solution;
(3) heating the mixed solution to 60-80 ℃, stirring, evaporating and concentrating to 0.3-0.8 time of the volume of the organic phase to obtain a nano emulsion;
(4) placing the nano emulsion in an environment of 0-4 ℃ and continuously stirring to obtain a nano coenzyme Q10 suspension;
(5) and homogenizing the nano-coenzyme Q10 suspension, and performing vacuum freeze drying to obtain the composition for treating senile dementia.
4. The method according to claim 3, wherein the ratio of the mass of coenzyme Q10 to the volume of absolute ethanol in step (1) is 0.5 to 1 g: 100 mL.
5. The method according to claim 3, wherein the stirring speed in the step (3) or (4) is 1000 to 2000 rpm.
6. The preparation method according to claim 5, wherein the stirring time in the step (4) is 1-2 h.
7. The method according to claim 3, wherein the temperature for homogenization in step (5) is 45 to 55 ℃ and the pressure for homogenization is 200 to 1500 bar.
8. The method according to claim 7, wherein the number of the homogenization is 2 to 4, and the pressure of the homogenization is gradually increased.
9. Use of the composition of any one of claims 1 or 2 or the composition prepared by the preparation method of any one of claims 3 to 8 in the preparation of a medicament for treating senile dementia.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811622989.4A CN109498596B (en) | 2018-12-28 | 2018-12-28 | Composition for treating senile dementia and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811622989.4A CN109498596B (en) | 2018-12-28 | 2018-12-28 | Composition for treating senile dementia and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109498596A CN109498596A (en) | 2019-03-22 |
| CN109498596B true CN109498596B (en) | 2021-03-02 |
Family
ID=65756652
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811622989.4A Active CN109498596B (en) | 2018-12-28 | 2018-12-28 | Composition for treating senile dementia and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109498596B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101066260A (en) * | 2006-11-17 | 2007-11-07 | 姚瑶 | Coenzyme Q10 emulsion and its freeze dried prepn and their prepn process |
| CN106727441A (en) * | 2016-12-29 | 2017-05-31 | 厦门金达威生物科技有限公司 | Water-soluble nano slow-release function Co-Q10 microcapsules and preparation method and application |
-
2018
- 2018-12-28 CN CN201811622989.4A patent/CN109498596B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101066260A (en) * | 2006-11-17 | 2007-11-07 | 姚瑶 | Coenzyme Q10 emulsion and its freeze dried prepn and their prepn process |
| CN106727441A (en) * | 2016-12-29 | 2017-05-31 | 厦门金达威生物科技有限公司 | Water-soluble nano slow-release function Co-Q10 microcapsules and preparation method and application |
Non-Patent Citations (1)
| Title |
|---|
| Coenzyme Q10 Reduces β-Amyloid Plaque in an APP/PS1 Transgenic Mouse Model of Alzheimer’s Disease;Xifei Yang等;《Journal of Molecular Neuroscience》;20091016;第1-5页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109498596A (en) | 2019-03-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| James et al. | Phencyclidine: tissue distribution in the rat | |
| CN105168235B (en) | Application of pennogenin compound in preparing medicament for treating senile dementia and Alzheimer disease | |
| Mikov et al. | 3a, 7a-Dihydroxy-12-oxo-5b-cholanate as blood-brain barrier permeator | |
| Lee et al. | Standardization of the manufacturing process of bee venom pharmacopuncture containing melittin as the active ingredient | |
| Hu et al. | Effect of kai xin san on learning and memory in a rat model of paradoxical sleep deprivation | |
| CN109498596B (en) | Composition for treating senile dementia and preparation method and application thereof | |
| WO2004014409A1 (en) | Developing method for separating d-amygdalin and neoamygdalin by reversed-phase hplc and optimum conditions for inhibition of racemization of amygdalin | |
| CN106491680B (en) | A Chinese medicinal composition for preventing or treating senile dementia, and its preparation method | |
| CN101085295B (en) | Freeze dried injection containing muskone and preparation method thereof | |
| CN1977878A (en) | Chinese medicine composition and its use | |
| CN107213157A (en) | A kind of medicine with potential treatment Parkinson's | |
| CN101628086A (en) | Gastrodiaelata Blume plant extract for preventing and treating vascular dementia and preparation method thereof | |
| CN103330941B (en) | Pharmaceutical composition of paclitaxel injection | |
| Fernandez et al. | Stereoselective distribution and stereoconversion of zopiclone enantiomers in plasma and brain tissues in rats | |
| Xin et al. | Antipyretic and anti-inflammatory activities of Thais luteostoma extracts and underlying mechanisms | |
| CN102670670A (en) | Preparation method of ginkgo dipyridolum injection with high content of ginkgo terpene lactones | |
| CN103536589B (en) | The preparation method of the compound recipe nanometer liposome of catechin bioavailability can be improved | |
| Li et al. | HPLC study of tissue distribution of loganin in rats | |
| CN102125548A (en) | Use of butyl phthalide and derivatives thereof in preparation of medicines for treating Parkinson disease | |
| CN106866696B (en) | Sauchinone sulfonate and its geniposide composition are applied in medicine preparation | |
| CN115645464B (en) | Traditional Chinese medicine composition for treating atherosclerosis and fatty liver and application thereof | |
| Lin et al. | Osthole Improves Spatial Memory Deficits in Rats via Hippocampal α 1-Adrenergic and D1/D2 Receptors | |
| CN117503876A (en) | Pharmaceutical composition for resisting senile dementia and preparation method thereof | |
| CN102178751B (en) | Brain clearing and pain relieving preparation | |
| CN106841464A (en) | Ellagic acid and 3,3` dimethoxy ellagic acid detection method of content in a kind of China fir parasitism |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |