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CN106176837B - Pharmaceutical composition, preparation method and application thereof - Google Patents

Pharmaceutical composition, preparation method and application thereof Download PDF

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Publication number
CN106176837B
CN106176837B CN201610676510.XA CN201610676510A CN106176837B CN 106176837 B CN106176837 B CN 106176837B CN 201610676510 A CN201610676510 A CN 201610676510A CN 106176837 B CN106176837 B CN 106176837B
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taxus
mairei
temperature
use according
leaf extract
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CN106176837A (en
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李娟�
汤伟彬
袁谱龙
王莉佳
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Wuxi Yew Pharmaceutical Co ltd
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JIANGSU YEW PHARMACEUTICAL CO Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/13—Coniferophyta (gymnosperms)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation or decoction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization

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  • Health & Medical Sciences (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Biotechnology (AREA)
  • Botany (AREA)
  • Medical Informatics (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Medicinal Preparation (AREA)

Abstract

The invention relates to a medicine, in particular to a medicine composition, which comprises a taxus mairei leaf extract and a pharmaceutically acceptable carrier, wherein the weight percentage of the taxus mairei leaf extract is 0.1-99.9%. The medicinal composition has natural source, simple preparation process and low toxicity to cells, has stronger effect of preventing and treating neurodegenerative diseases, and has wide application prospect.

Description

Pharmaceutical composition, preparation method and application thereof
Technical Field
The invention relates to the field of pharmacy, in particular to a pharmaceutical composition, a preparation method and application thereof.
Background
Neurodegenerative diseases (NDD) are chronic central nervous system diseases based on progressive degeneration and death of neurons, mainly affecting cognitive or motor functions of patients, and mainly include Alzheimer's Disease (AD), Parkinson's Disease (PD), Huntington's disease, spinocerebellar ataxia (spinocerebellar ataxia), amyotrophic lateral sclerosis (amyotrophic lateral sclerosis), and myeloatrophy. Although the clinical manifestations and pathological features of these diseases are different, they all share the common features: the progressive degeneration necrosis of the neuron and the obvious proliferation and activation of the glial cell have no ideal prevention and treatment medicine so far, and the patient has high disability and lethality rate.
At present, the pathogenesis of the diseases is not clear, multiple complex factors such as aging, inflammation, heredity, environmental toxins and the like are common pathogenic factors of the diseases, and the pathological mechanisms comprise neurons, impaired glial cell function, axonal transfer dysfunction, glutamate excitotoxicity, overhigh Reactive Oxygen Species (ROS) level, impaired metabolic pathways, reduced mitochondrial energy generation, increased formation of misfolded proteins or insufficient degradation of the misfolded proteins and the like. The neurodegenerative diseases seriously harm human health, bring great psychological and economic burden to patients, families and society, and develop medicaments for preventing and treating the neurodegenerative diseases are paid much attention.
Taxus chinensis var. mairei is also called as Taxus beauty, is a plant of Taxus genus of Taxus family of Taxaceae of Taxus order of Taxus class of Eugenia plant, is specially produced in China, is mainly distributed in Yangtze river basin, south mountain area, mountain areas of Henan, Shanxi (Qinling mountain), Gansu province and other mountainous regions or brook, and is a plant with fastest growth and widest distribution in Taxus genus.
Besides containing small-molecule active substances such as paclitaxel and the like, the leaves of the taxus mairei also find that active polysaccharides contained in the leaves of the taxus mairei have the activities of improving immunity, protecting liver, resisting tumor and the like, but no report of preventing and treating neurodegenerative diseases by the polysaccharides in the leaves exists at present.
Disclosure of Invention
The invention aims to provide a pharmaceutical composition, a preparation method and application thereof, wherein the pharmaceutical composition has the effect of immunosuppressive activity, has small toxic and side effects on cells, and has wide application prospects.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a pharmaceutical composition comprising the extract of taxus mairei leaves and a pharmaceutically acceptable carrier, wherein the weight percentage of the extract of taxus mairei leaves is 0.1-99.9%, such as 0.1%, 0.2%, 0.3%, 0.5%, 0.8%, 1%, 2%, 5%, 8%, 10%, 13%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 98%, 99% or 99.9%.
In the invention, the taxus mairei leaf extract has numerous chemical components and has the effect of compatibility use of traditional Chinese medicine compounds, and as a natural medicine, compared with a single medicine, the taxus mairei leaf extract has small toxic and side effects and the effect of enhancing immunity, and the function of preventing and treating neurodegenerative diseases is the specific effect of the taxus mairei leaf extract, and other parts of the taxus mairei do not have the effect.
Preferably, the weight percentage of the taxus mairei leaf extract is 1-90%, preferably 20-85%, and more preferably 40-80%.
Preferably, the taxus mairei leaf extract is a water extract and/or a traditional Chinese medicine extract.
Preferably, the pharmaceutically acceptable carrier is one or a mixture of at least two of mannitol, sorbitol, sorbic acid or potassium salt, sodium metabisulfite, sodium bisulfite, sodium thiosulfate, cysteine hydrochloride, thioglycolic acid, methionine, vitamin a, vitamin C, vitamin E, vitamin D, azone, disodium EDTA, calcium sodium EDTA, carbonates of monovalent alkali metals, acetates, phosphates and aqueous solutions thereof, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acids, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivatives, cellulose and derivatives, alginates, gelatin, polyvinylpyrrolidone, glycerol, propylene glycol, ethanol, tween 60-80, span-80, wax, lanolin, liquid paraffin, cetyl alcohol, gallic acid esters, agar, triethanolamine, basic amino acids, urea, allantoin, calcium carbonate, calcium bicarbonate, surfactants, polyethylene glycol, cyclodextrin, β -cyclodextrin, materials, kaolin, stearic acid, calcium stearate, magnesium stearate, vegetable oil, phospholipids, oleic acid, and ionic complexing agents thereof.
Preferably, the vegetable oil is any one or a mixture of at least two of soybean oil, medium-chain oil, olive oil, tea oil, palm oil, angelica oil, sea buckthorn oil, zedoary oil, ligusticum wallichii oil, coix seed oil, safflower oil, zanthoxylum oil and garlic oil.
Preferably, the phospholipid is one or a mixture of at least two of egg yolk lecithin, soybean phospholipid, hydrogenated egg yolk lecithin, hydrogenated soybean phospholipid and synthetic phospholipid.
Preferably, the polyethylene glycol phospholipid is any one or a mixture of at least two of polyethylene glycol-cephalin, polyethylene glycol-cholesterol, polyethylene glycol-di-fatty glyceride, polyethylene glycol-fatty acid ester, polyethylene glycol-fatty amine or polyethylene glycol-fatty alcohol.
Preferably, the antioxidant is vitamin E.
Preferably, the pharmaceutical composition is any one or a combination of at least two of tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, hard capsules, soft capsules, oral liquid, buccal agents, granules, medicinal granules, pills, powder, ointment, pellets, suspensions, solutions, injections, suppositories, ointments, plasters, creams, sprays, drops or patches.
In a second aspect, the present invention provides a method for preparing the pharmaceutical composition according to the first aspect, wherein the extraction method of the taxus chinensis var mairei leaf extract is any one or a combination of at least two of water extraction, chromatography, vacuum extraction, supercritical fluid extraction, ultrasonic extraction, enzymatic extraction, microwave extraction, charged extraction or semi-bionic extraction, preferably water extraction.
Preferably, the extraction method of the taxus mairei leaf extract comprises the following steps:
the extraction method of the taxus mairei leaf extract comprises the following steps:
decocting Taxus chinensis var mairei leaves in water, and cooling to room temperature; adding a precipitator into the decoction, standing and filtering to obtain a precipitate; mixing the precipitate with water, centrifuging, deproteinizing the supernatant with 15% trichloroacetic acid water solution, and centrifuging to obtain supernatant; adding a precipitator into the obtained supernatant, filtering to obtain a precipitate, washing with absolute ethyl alcohol, and drying in vacuum to obtain the taxus mairei leaf extract.
Preferably, the weight ratio of the taxus mairei leaves to water is 1: (5-15), for example, may be 1: 5. 1: 6. 1: 7. 1: 8. 1: 9. 1: 10. 1: 11. 1: 12. 1: 13. 1: 14 or 1: 15, preferably 1: (9-12).
Preferably, the number of times of decoction is 2-5, for example, 2, 3, 4 or 5, preferably 3.
Preferably, the time for decoction is 2-5h, for example, 2h, 3h, 4h or 5h, preferably 2 h.
Preferably, the precipitant is any one or a mixture of at least two of methanol, ethanol or acetone, preferably ethanol.
Preferably, the decoction is added with the precipitant to make the volume ratio of the precipitant 40-70%, for example, 40%, 41%, 42%, 43%, 45%, 48%, 50%, 52%, 55%, 58%, 60%, 62%, 65%, 68%, 69% or 70%, preferably 60%.
The deproteinization temperature is preferably 1 to 10 ℃ and may be, for example, 1 ℃, 2 ℃, 3 ℃, 4 ℃, 5 ℃, 6 ℃, 7 ℃, 8 ℃, 9 ℃ or 10 ℃, preferably 3 to 7 ℃, and more preferably 4 ℃.
Preferably, the vacuum drying temperature is 20-80 ℃, for example can be 20 ℃, 21 ℃, 22 ℃, 23 ℃, 25 ℃, 26 ℃, 28 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃ or 80 ℃, preferably 30-60 ℃, more preferably 50 ℃.
In a third aspect, the present invention provides an application of the pharmaceutical composition according to the first aspect in preparing a medicament for preventing and treating neurodegenerative diseases.
Preferably, the neurodegenerative disease includes any one of alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis, huntington's disease, multiple sclerosis, spinocerebellar atrophy or vascular dementia or a combination of at least two thereof.
Compared with the prior art, the invention has the following beneficial effects:
(1) compared with extracts of other parts of the taxus mairei, the extracted taxus mairei leaf extract has the specific effect of a medicament for preventing and treating neurodegenerative diseases, has the inhibition rate of activity on microglial cell activation of over 50 percent, has an inhibition effect on neuroinflammation caused by LPS, and has a protection effect on nerve injury caused by the LPS;
(2) the medicine provided by the invention has the advantages of simple preparation process and obvious drug effect, obviously improves the behavioral disturbance of a model animal with neurodegenerative diseases, inhibits the damage of dopaminergic neurons in the brain of the model animal, relieves the activation of glial cells, has the effect of treating the neurodegenerative diseases, provides more choices for clinical treatment of the neurodegenerative diseases, and has wide application prospects.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
EXAMPLE 1 preparation of a pharmaceutical composition of the invention
The extraction method of the taxus mairei extract comprises the following steps:
taking taxus mairei leaves, adding 10 times of water, decocting for three times, each time for 2 hours, combining decoction, and cooling to room temperature; adding a precipitator into the decoction until the volume ratio of the precipitator in the decoction reaches 60%, standing and filtering to obtain a precipitate; mixing the precipitate with water, centrifuging, deproteinizing the supernatant with 15% trichloroacetic acid water solution at 4 deg.C, and centrifuging to obtain supernatant; adding a precipitator into the obtained supernatant, filtering to obtain a precipitate, washing with absolute ethanol, and drying at 50 ℃ in vacuum to obtain the taxus mairei leaf extract.
Mixing the extracted Taxus chinensis var mairei leaf extract with polyethylene glycol, and making into capsule.
Experimental example 2 verification of inhibition of microglial activation
The BV2 cell is a mouse microglial cell line;
griess reagent: preparing 0.1% of naphthyl ethylenediamine by using distilled water, preparing 1% of sulfanilic acid by using 5% of phosphoric acid, and mixing the two in equal volume of 1:1 before use;
curcumin, prepared with DMSO;
lipopolysaccharide (LPS) is prepared by sterile PBS, and the final concentration is 300 ng/mL;
the experimental method comprises the following steps: (1) BV2 cells were cultured in DMEM-F12 medium containing 10% newborn calf serum at 37 ℃ with 5% CO295% air, 100% relative humidity;
(2) BV2 cells in logarithmic growth phase, after counting by digestion, at 2X 104Inoculating each well into a 96-well plate, adding a test object after 24 hours, adding LPS after 1 hour, continuously culturing for 24 hours, and collecting the culture medium supernatant, wherein the final concentration of the LPS is 300 ng/mL;
(3) collecting cell culture supernatant (100 μ L), adding equal volume Griess reagent, standing at room temperature for 20min, adjusting to zero with distilled water, measuring OD at 540nm on enzyme labeling instrument, measuring OD with sodium nitrate as standard, and calculating NO in sample to be measured2 -The concentration of (c) reflects the concentration of NO.
The results are shown in table 1 below.
TABLE 1
As can be seen from the results in the table, the drug of the invention shows obvious activity of inhibiting the activation of microglia at the administration concentration of 100 mug/mL, and the inhibition rate is more than 50%.
Experimental example 3 inhibition of neuroinflammation and neuronal injury induced by LPS
Lipopolysaccharide prepared with sterile PBS to a final concentration of 100 ng/mL;
preparing curcumin with DMSO;
the experimental method comprises the following steps:
(1) establishing a primary hippocampal neuron/glial cell mixed culture system: separating hippocampus of 18d pregnant SD rat embryos under a dissecting mirror, blowing by using a pipette until tissue blocks cannot be seen, filtering, and inoculating into a 24-well plate;
(2) cell administration treatment, namely culturing the primary cells for 7 days, respectively incubating the primary cells with a test object, adding 100ng/mL of LPS (LPS) serving as a stimulant after 3 hours, and taking a culture medium after 5 hours to measure IL-1 β and TNF- α by using an ELISA kit;
(3) after the cells were treated with the drug for 7 days, the culture medium was collected and LDH was detected with an LDH-detecting reagent.
The results are shown in table 2 below.
TABLE 2
The results in the table show that the medicament can obviously inhibit inflammatory reaction under the stimulation of LPS, reduce the levels of IL-1 β and TNF- α in a culture medium, and inhibit the inflammation to the same extent as that of a positive control medicament of curcumin (10 mu g/mL) at the concentration of 100 mu g/mL-5M) are equivalent; the release amount of LDH is obviously increased under the stimulation of LPS by the mixed culture system of hippocampal neurons/glial cells, and the neurons are obviously damaged.
In conclusion, the taxus chinensis leaf extract is used as a natural medicine, can obviously improve the behavioral disturbance of a neurodegenerative disease model animal, inhibit the damage of brain dopaminergic neurons in the model animal, and relieve the activation of glial cells, has the effect of treating neurodegenerative diseases, provides more choices for clinical treatment of the neurodegenerative diseases, and has wide application prospects.
The applicant states that the present invention is illustrated by the above examples of the process of the present invention, but the present invention is not limited to the above process steps, i.e. it is not meant that the present invention must rely on the above process steps to be carried out. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific modes and the like, which are within the scope and disclosure of the present invention, are contemplated by the present invention.

Claims (13)

1. An application of Taxus chinensis var mairei leaf extract in preparing microglia cell activation inhibitor or LDH release inhibitor is provided;
the taxus mairei leaf extract is extracted by adopting the following method, and the method comprises the following steps:
decocting Taxus chinensis var mairei leaves in water, and cooling to room temperature; adding ethanol as a precipitator into the decoction until the volume ratio of the ethanol to the precipitator reaches 60%, standing and filtering to obtain a precipitate; mixing the precipitate with water, centrifuging, deproteinizing the supernatant with 15% trichloroacetic acid water solution, and centrifuging to obtain supernatant; adding ethanol as precipitant into the supernatant, filtering to obtain precipitate, washing with anhydrous ethanol, and vacuum drying to obtain the Taxus chinensis var mairei leaf extract.
2. The use of claim 1, wherein the weight ratio of the taxus mairei leaves to water is 1: (5-15).
3. The use of claim 1, wherein the weight ratio of the taxus mairei leaves to water is 1: (9-12).
4. The use of claim 1, wherein the number of said decocts is 2-5.
5. The use of claim 1, wherein the number of said decocts is 3.
6. The use of claim 1, wherein the time of said decocting is 2-5 hours.
7. The use according to claim 1, wherein the time of decoction is 2 h.
8. Use according to claim 1, wherein the deproteinisation temperature is 1-10 ℃.
9. Use according to claim 1, wherein the deproteinisation temperature is 3-7 ℃.
10. Use according to claim 1, wherein the deproteinisation temperature is 4 ℃.
11. Use according to claim 1, wherein the temperature of the vacuum drying is 20-80 ℃.
12. Use according to claim 1, wherein the temperature of the vacuum drying is 30-60 ℃.
13. Use according to claim 1, wherein the temperature of the vacuum drying is 50 ℃.
CN201610676510.XA 2016-08-15 2016-08-15 Pharmaceutical composition, preparation method and application thereof Active CN106176837B (en)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20170002846A (en) * 2015-06-30 2017-01-09 (주)아모레퍼시픽 Composition containing extract of soybean leaves for suppressing and preventing myopathy
CN107213174A (en) * 2017-06-22 2017-09-29 江苏红豆杉药业有限公司 A kind of pharmaceutical composition and preparation method thereof and the application in the medicine for preparing preventing and treating nerve degenerative diseases
CN107137434B (en) * 2017-06-22 2021-04-30 无锡紫杉药业有限公司 Pharmaceutical composition, preparation method thereof and application thereof in preparation of coxsackie virus resistant medicine
CN108187026A (en) * 2018-03-16 2018-06-22 南京财经大学 A kind of product that can promote wound healing

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101244991A (en) * 2008-03-21 2008-08-20 昆明自主择业集源生物科技有限公司 Method for extracting and purifying sequoyitol by using solvent deposition
CN101735330A (en) * 2009-10-12 2010-06-16 于荣敏 Preparation method of yew polysaccharide with function of resisting tumors and application thereof
CN104248649A (en) * 2014-09-24 2014-12-31 江苏红豆杉药业有限公司 Preparation method for taxus chinensis extractive and application of taxus chinensis extractive

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101244991A (en) * 2008-03-21 2008-08-20 昆明自主择业集源生物科技有限公司 Method for extracting and purifying sequoyitol by using solvent deposition
CN101735330A (en) * 2009-10-12 2010-06-16 于荣敏 Preparation method of yew polysaccharide with function of resisting tumors and application thereof
CN104248649A (en) * 2014-09-24 2014-12-31 江苏红豆杉药业有限公司 Preparation method for taxus chinensis extractive and application of taxus chinensis extractive

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