CN110123744A - A kind of method and its application preparing erythricine injection using rough gentian as raw material - Google Patents
A kind of method and its application preparing erythricine injection using rough gentian as raw material Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
- A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
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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
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/51—Gentianaceae (Gentian family)
- A61K36/515—Gentiana
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/12—Carboxylic acids; Salts or anhydrides thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
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- 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
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- 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
- A61K9/08—Solutions
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- 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
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- 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
- A61P25/08—Antiepileptics; Anticonvulsants
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
- C07D491/04—Ortho-condensed systems
- C07D491/044—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
- C07D491/052—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring the oxygen-containing ring being six-membered
Abstract
A kind of to can be used for anticonvulsion and erythricine injection with cerebral protection, the injection is made of following components: 5-10 parts of erythricine crystalline powder, 6-15 parts of cosolvent, 1-5 parts of antioxidant, 50-120 parts of osmotic pressure regulator, 1000 parts of water；Its preparation process is characterized in that using ascorbic acid or tartaric acid as cosolvent; it can be improved the solubility of erythricine; the stability that erythricine can be effectively improved simultaneously truly has specific cerebral injury protective effect from pathological section angle (Change of Ultrastructure, organelle variation) research discovery erythricine.
Description
Technical field
The present invention relates to pharmaceutical preparation fields, and in particular to a method of erythricine injection is prepared by raw material of rough gentian
And its application.
Background technique
Hyperpyretic convulsion is also known as febrile convulsion, is children's period one of the most common type convulsive disorder, frequently or high for a long time
Febrile convulsion breaking-out can cause infant's central lesion, and make to convulsions neurological susceptibility at a specified future date, learning and memory function
At influence.Currently, hyperpyretic convulsion still lacks ideal therapeutic agent: diazepam is anticonvulsion choice drug, but a drug
It is difficult to fully control convulsions, application easily leads to respiration inhibition repeatedly, and with dependence and withrawal symptom etc..Sodium phenobarbital is made
Long with the time, Small side effects, the two drug combination can learn from other's strong points to offset one's weaknesses, but prolonged application can still generate dependence.
For motherland's medical angle, hyperpyretic convulsion belongs to acute infantile convulsions scope, in therapy with heat-clearing, slit phlegm, it is relieving convulsion, relieve dizziness, high fever, infantile convulsions, epilepsy, etc.
For fundamentum.Chinese medicine rough gentian is northeast genunie medicinal materials, price it is low and, it is clear liver and gallbladder damp-heat that safety is good, rush down lower burnt stagnated fire it
Conventional Chinese medicine, the contained rough gentian of Shennong's Herbal " main frightened epilepsy ".Using rough gentian as example cool frightened ball of many compound preparations of key agents, work as hank
Luxuriant growth ball treats the effective means of infantile convulsion in this way.Erythricine is that gentiamarin is transformed under alkaline condition, has been reported that table
Bright, gentiamarin is quickly generated erythricine after entering degradation in vivo.Applicant studies have shown that erythricine have anticonvulsant action,
Compared to diazepam the drugs such as sodium phenobarbital, there is more reliable, safer advantage；On the other hand, erythricine still has solution
The effects of heat, cerebral injury protection, can occur to prevention disease before disease for faint from fear caused by high fever, is played, resist in disease it is frightened it is only frightened,
Alleviate various effects such as damage after being ill, there is certain advantage.
Rough gentian is the common relieving convulsion Chinese medicine of heat-clearing, and main pharmacodynamics ingredient is gentiamarin, modern research shows that its main medicine
Effect ingredient is gentiamarin, and deriving erythricine has the effect of anti-inflammatory, analgesia, be depressured, be hypoglycemic.Research Literature is main at present
Erythricine is prepared by raw material of gentianae macrophyllae, rough gentian can be converted under certain condition by the iridoid glycoside of representative of gentiamarin
Alkali.Costly, the research on the other hand currently for erythricine pharmacological effect is still insufficient for one side cost of material.
It is currently used in the raw medicinal material gentianae macrophyllae resource scarcity for preparing erythricine, causes market price of raw material high, seeks
A kind of raw material that can reduce cost is sought, there is important value for the industrialization of erythricine.
Erythricine poor solubility is an important factor for restricting clinical administration, how to improve its solubility asking in order to administration
It inscribes urgently to be resolved.
Summary of the invention
It is water-soluble very poor since erythricine has stronger lipophilicity, it is not easy to administration when child's hyperpyretic convulsion, the present invention
Provide it is a kind of using rough gentian as raw material prepare erythricine injection method and it is a kind of safely and effectively, for the note of intramuscular injection
Liquid is penetrated, to improve its bioavilability, improves its anticonvulsion, cerebral injury protective effect.
It is an object of the present invention to provide a kind of erythricine injection, injection is made of following components: erythricine knot
5-10 parts of crystalline flour end, 6-15 parts of cosolvent, 1-5 parts of antioxidant, 50-120 parts of osmotic pressure regulator, 1000 parts of water；It is described to help
Solvent is one of ascorbic acid, citric acid, tartaric acid, acetic acid, lactic acid, hydrochloric acid, phosphoric acid or any combination thereof；The antioxygen
Agent be one of sodium pyrosulfite, sodium hydrogensulfite, sodium sulfite, sodium thiosulfate or disodium ethylene diamine tetraacetate or its
Any combination；The osmotic pressure regulator is mannitol, sorbierite, dextran, glucose, lactose, xylitol or sodium chloride
One of or any combination thereof.
The injection preparation step is as follows:
(1) it takes 80% water for injection to filter deoxygenation, is heated to 40-50 DEG C, cosolvent and antioxidant is added, stirring is complete
Dissolution；
(2) erythricine crystalline powder is added in the solution obtained to step (1), stirring is completely dissolved；
(3) osmotic pressure regulator is added in the solution obtained to step (2), stirring and dissolving supplies water for injection, uses
0.5% active carbon stirring and adsorbing；
(4) de- charcoal is carried out by the way of three-stage filtration processing:
First order filtering, is filtered processing to medical fluid using plate and frame filter press；
Second level filtering is filtered processing to the medical fluid after by-pass filtration using thin number husky filter stick；
Third level filtering, is filtered processing to the medical fluid after secondary filtration using 0.22 μm of aperture filter membrane；
(5) 121 DEG C at a temperature of, sterilize 15-20min, and nitrogen charging is filling.
Erythricine crystalline powder preparation process are as follows:
1) Gentiana adds 5-10 times of alkali organic solvent measured to impregnate, 80 DEG C refluxing extraction 2~3 times, 0.5~2h every time
It filters, merging filtrate；
2) above-mentioned filtrate is recovered under reduced pressure, measures lipophylic organic solvents with 1-5 times and is extracted to water layer, divides and takes organic solvent layer,
Organic solvent is recovered under reduced pressure, and drying to constant weight, obtains medicinal extract, and above-mentioned organic solvent is acetone or ethyl acetate or ether or three chloromethanes
Alkane or methylene chloride or carbon tetrachloride or hexamethylene or petroleum ether；
3) sour water is measured with 2-5 times, hydrolyzes 2-12h, filtered off precipitating, obtain rufous liquid, aforesaid liquid is poured into preparatory dress
In good cation exchange resin column, successively with water, 50% ethanol elution to colourless, eluent is discarded, with alkaline ethanol solution
8-15 times of column volume is eluted, alkaline ethanol solution is collected, after ethyl alcohol is recovered under reduced pressure, measures lipophylic organic solvents extraction with 1-5 times
Water layer is recovered under reduced pressure organic solvent and obtains compound I crude product,
4) crude product progress silica gel column chromatography, dry column-packing, dry method loading, silica gel are mixed by 2: 1 with compound I crude product and are mixed
Sample, chloroform: methanol elution gradient is first eluted with chloroform, is then successively changed eluent polarity, is finally collected trichlorine
Methane: methanol=150: 1 flow point merges same composition after thin-layer qualitative, is recovered under reduced pressure, obtains white, needle-shaped crystals.
Further, ammonium hydroxide and methanol, ethyl alcohol, propylene glycol, isopropanol, just that the alkali organic solvent is 5%-25%
Butanol, dimethylformamide, dimethyl acetamide one of which reagent mixed solvent.
Further, the lipophylic organic solvents are ethyl acetate or ether or chloroform or methylene chloride or tetrachloro
Change carbon or hexamethylene or petroleum ether.
The invention has the advantages that:
1, the solubility of erythricine can be improved as cosolvent using ascorbic acid or tartaric acid in the present invention, while can
Effectively improve the stability of erythricine.
2, the resource pressure of gentianae macrophyllae can be alleviated by preparing erythricine to a certain extent using rough gentian as raw material, reduce injection
Preparation cost.
Detailed description of the invention
Attached drawing 1 is erythricine purifying, refined process flow chart；
Attached drawing 2 is the HPLC map of compound；
Attached drawing 3 is the UV map (HPLC full wavelength scanner) of compound；
Attached drawing 4 is the ESI-MS map of compound 1；
Attached drawing 5 is the 13C-NMR map of compound 1；
Attached drawing 6 is the 1H-NMR map of compound 1；
Attached drawing 7 is structural formula of compound；
Attached drawing 8 is hyperpyretic convulsion Ultrastructural observation on CA 1 region in hippocampus result；
A:NG group transmission electron microscope × 16500；B:FC group transmission electron microscope × 16500；C:GT group transmission electron microscope × 16500
Attached drawing 9 is each group neuron and organelle denaturation histogram.
Specific embodiment
Only invention is further described in detail for following examples, but does not constitute any limitation of the invention.
All ingredients are commercial goods in following embodiments.
Embodiment 1
Rough gentian 10kg, Gentiana originate from Liaoning Province, are purchased from Harbin City rural area medicinal material company.It is accredited as certified Radix Gentianae
The root and rhizome of (Gentiana scabra Bge.), quality meet one relevant regulations of the Pharmacopoeia of the People's Republic of China.
High performance liquid chromatograph (Waters2695-2998 type)；ODSC18 chromatographic column (250mm × 4.6mm)；Water bath with thermostatic control
Pot (Medical Equipment Plant, Beijing)；Adjust the temperature electronically electric jacket (Tianjin Stettlen Instrument Ltd.)；Rotary evaporator (RE-
5298A type, Shanghai Yarong Biochemical Instrument Plant)；Circulating water type vacuum pump (Zhengzhou Greatwall Scientific Industrial & Trading Co., Ltd.)；SHB-3 double frequency
Numerical control ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.)；Vacuum desiccator (DZF-6050 type, the permanent scientific and technological instrument in Shanghai one
Device Co., Ltd)；Four use uv analyzer (ZF-II type, Shanghai Gu Cun electric light instrument plant)；The exchange of 732 type highly acidic cations
Resin (fine chemistry industry research institute is recovered in Tianjin), column chromatography silica gel (SILVER REAGENT), thin layer silica gel (GF254), carboxymethyl cellulose
Plain sodium；Ethyl alcohol, methanol, chloroform, ammonium hydroxide, petroleum ether and acetone are that analysis is pure.
1. extraction process process
Gentiana 10kg is weighed, is crushed, 20~40 meshes is crossed, obtains Gentiana crude product.
Medicinal material crude product 1kg is taken, is impregnated for 24 hours with 95% ethyl alcohol and 10% ammonium hydroxide by 50: 7, solvent volume and medicinal material weight ratio
It is 10: 1.
Next day refluxing extraction 2 times at 80 DEG C, each 2h are filtered, merging filtrate.
It is recovered under reduced pressure to no alcohol taste, chloroform 2: 1 is extracted to water layer improvement bismuth potassium iodide reaction and does not develop the color, and is recovered under reduced pressure
Chloroform obtains medicinal extract, and drying to constant weight, obtains thick rufous medicinal extract.
10% sour water (HCl) dissolution, places 12h, adds 2~3 times of amount water dissolutions, filters off precipitating, obtain rufous liquid.
Aforesaid liquid is poured into pre-packed cation exchange resin column, successively with water, 50% ethanol elution to nothing
Color, flow control are discarded in 20ml/min, eluent.Be followed by 95% ethanol elution containing ammonia, thin layer track to eluent without
Target component and stop.After resin regeneration can secondary use, with chloroform extraction after being recovered under reduced pressure, chloroform layer be recovered under reduced pressure to obtain change
Close object I crude product.
Crude product progress silica gel column chromatography, dry column-packing, dry method loading, silica gel mix by 2: 1 with crude product medicinal extract and mix sample, trichlorine
Methane: methanol elution gradient is first eluted with chloroform, is then successively changed eluent polarity, is finally collected 150: 1 parts,
Merge same composition after thin-layer qualitative, be recovered under reduced pressure, obtains white, needle-shaped crystals, erythricine purifying, process for refining process such as Fig. 1 institute
Show.
Compound purity detection and Structural Identification
Purity detecting
Appropriate compound I crystallization is dissolved in chloroform by the detection of TLC method.Each 5 μ l of draw solution is put in GF254 thin layer
On plate (German Mo Ke company), respectively with chloroform: methanol=50: 1, ether: acetone=3: 1 is solvent, and expansion is secondary,
It takes out, dries, set that inspect under ultraviolet lamp (254nm) be single-point, it is believed that be monomer component.
HPLC method detects the acicular crystals for taking the compound I obtained from silica gel column purification, accurately weighed, with methanol ultrasound
10ml is dissolved and is settled to, as test solution.(ultraviolet all-wave length detector) is measured using high performance liquid chromatography,
Chromatographic condition: chromatographic column is Diamonsil C18 chromatographic column (250mm × 4.6mm, 5 μm), and mobile phase is that methanol-water 40: 60 arrives
60: 40 gradient elution 20min；Detection wavelength: 220nm；Flow velocity is 1ml/min；28 DEG C of column temperature；It is first carried out with full wavelength scanner miscellaneous
Then the observation of mass peak carries out preliminary assay with normalization method.Chromatographic peak is single symmetrical, can be considered single component, sees figure
2。
Compound I Structural Identification
Physicochemical property compound I is white, needle-shaped crystals, and bismuth potassium iodide reaction is positive, and prompting it may be alkaloids
Compound is soluble in chloroform, is dissolved in ether, ethyl acetate, and acetone, methanol, ethyl alcohol are slightly soluble in water, does not dissolve in petroleum ether；
UV (Me0H): λ=max220nm is shown in Fig. 3.
The positivity ESI-MS spectrum of compound I provides [M+H]+quasi-molecular ions at m/z176, shows that molecular weight is 175, sees figure
4；Estimating its molecular formula in conjunction with 1H-NMR and 13C-NMR spectrum etc. is C10H9NO2.Calculating its degree of unsaturation is 7.
In 13C-NMR, the carbon signal of 1 lactone carbonyl can be observed at δ 163.5, δ 151.0,150.4,121.1,
144.9,7 olefinic carbon signals can be observed at 129.6,131.0 and 120.6；Carbon signal at δ 24.4 and 66.3 is respectively belonging to
Methylene and oxo mesomethylene carbon signal (attached drawing 5).
In 1H-NMR, hydrogen signal at 63.10 (2H, t, J=5.6Hz) and 4.57 (2H, t, J=5.6Hz), according to it
Coupling splits merotype and belongs to 2 adjacent methylene.Equally, δ 6.79 (1H, dd, J=16.2,11.2Hz), 5.60 (1H, d,
J=11.2Hz) and the hydrogen signal at 5.82 (1H, d, J=16.2Hz), show the structure for containing-CH=CH2 in compound structure
Segment (attached drawing 6).
In conjunction with the integration analysis of wave spectrum, in the whole proton signals and 13C-NMR spectrum to the 1H-NMR spectrum of compound I
All carbon signals have carried out detailed parsing and accurate ownership, are shown in Table 1.
Table -11H-and13C-NMR data of 1(CDCl3)
In summary data and analysis as a result, and by the spectral data of compound I and known compound gentianine into
Row compares, and the two is consistent, and the structure of final authenticating compound I is erythricine gentianine, sees Fig. 7.
2. injection preparation step is as follows:
It takes 80% water for injection to filter deoxygenation, is heated to 40-50 DEG C, cosolvent and antioxidant is added, stirring is completely molten
Solution；
Erythricine crystalline powder is added in the solution obtained to step (1), stirring is completely dissolved；
Osmotic pressure regulator is added in the solution obtained to step (2), stirring and dissolving supplies water for injection, with 0.5%
Active carbon stirring and adsorbing；
De- charcoal is carried out by the way of three-stage filtration processing:
First order filtering, is filtered processing to medical fluid using plate and frame filter press；
Second level filtering is filtered processing to the medical fluid after by-pass filtration using thin number husky filter stick；
Third level filtering, is filtered processing to the medical fluid after secondary filtration using 0.22 μm of aperture filter membrane；
121 DEG C at a temperature of, sterilize 15-20min, and nitrogen charging is filling.
3. the Detection of Stability of rough gentian alkali preparation
Content assaying method: it is detected according to aforementioned HPLC condition.
Clarity: in clarity detecting apparatus black background, using under 4500LX illumination, taking 5 bottles of each prescription finished product to be examined
It surveys.
PH value: finished product pH value is detected referring to " Chinese Pharmacopoeia " annex.
24 months stability of injection is as follows:
2 erythricine injection stability study of table
The result shows that prescription of the present invention can effectively improve the solubility of erythricine in water, and Primary Study is shown,
Gained injection can keep stablizing in 24 months.
4. the research of erythricine cerebral protection
Using hyperpyretic convulsion rat caused by hot bath method as animal pattern, it is divided into model group (FC) and administration group (GT).
GT group: in being induced after febrile convulsion in 30min every time, erythricine physiology is injected intraperitoneally by 100mg/kg rat body weight
Saline solution, totally 10 times.
FC group: in being induced after febrile convulsion in 30min every time, thank to 0.9% chlorination by 0.1ml/kg rat body weight abdominal cavity note
Sodium solution, totally 10 times.
NG group: blank group, not induced convulsions thank to 0.9% sodium chloride solution by 0.1ml/kg rat body weight abdominal cavity note, and totally 10
It is secondary.
After experiment, above-mentioned experimental rat is taken, is put to death, is separated brain tissue, be immersed in 0 DEG C of 3% glutaraldehyde and fix
It in liquid, is cut hippocampus site tissue piece (thick 1mm) in the horizontal Coronal of corpus geniculatum lateral, rapid power transmission mirror cell makes conventional transmission
Electron microscope specimen.Cerebral protection is checked, experimental result is as follows:
(1) rat hippocampus CA1Area's neuromal ultrastructure changes NG group: how oval hippocampal neuron mitochondria is or round
Shape, normal in size, ridge rule is clear, and rough surfaced endoplasmic reticulum (RER) and Ribosome Structure understand, Golgi complex form is normal, nucleus
Smoothly, cell membrane is smooth, (attached drawing 8, A)；FC group: most of hippocampal neuron mitochondrial swelling, the denaturation of part marrow sample, part ridge
Fracture, Golgi complex vacuole sample become, and rough surfaced endoplasmic reticulum (RER) is slightly expanded, cell membrane fractions fusion, and perivascular canal is broadening.(attached drawing
8, B)；GT group: hippocampal neuron mitochondrial fraction vacuolar degeneration partially tends to be normal, and rough surfaced endoplasmic reticulum (RER) remains static, not
See expansion, Golgi complex, which has no, to be substantially change.The fusion of nucleus nuclear membrane truncating.(attached drawing 8, C).
(2) it randomly selects 100 neurons under 2500 times of Electronic Speculum visuals field of neuron lesion observation to be observed, record denaturation
(cell nuclear swelling, Chromatin condensation side collection, kernel shrinkage deformation) and necrosis (karyopyknosis, fragmentation, dissolution, kernel disappear)
The number of neuron.The ratio of NG, FC, GT group CA 1 of Hippocampus neuronal degeneration necrosis be followed successively by 10% [(8+2)/
100], 48% [(28+20)/100], 36% [(18+10)/100].Prompt FC group rat hippocampus CA1 and other two groups of differences aobvious
Work property (χ 2=6.83, P < 0.05)
(3) it randomly selects 100 mitochondrias under 16500 times of Electronic Speculum visuals field of mitochondria lesion observation to be observed, record is swollen
(volume height expansion is in ball for swollen (volume increases, shape is rounded, ridge swelling shortens, number reduces, is disorganized) and vacuole change
Shape, ridge disappear substantially, essentially structureless vacuole spline structure substitution in mitochondria) number of mitochondria.NG, FC, GT, rat
The ratio of Hippocampal CA 1 mitochondria denaturation vacuole is followed successively by 32% [(22+10)/100], 64% [(20+44)/100], and 48%
[(18+30)/100].With other two groups of significances of difference (χ 2=12.86, P < 0.05)
(4) 100 groups of golgiosomes are randomly selected under 16500 times of Electronic Speculum visuals field of golgiosome lesion observation to be observed, is remembered
The lesion degree and number of record expansion (sachet expansion, structure disturbance) golgiosome.Gently, the expansion of weight golgiosome respectively refers to sachet
Bulked volume increases < 1/3, > 2/3NG, FC, GT, group CA 1 of Hippocampus Golgi complex and is denaturalized the ratio of vacuole successively
For 23% [(12+11)/100], 51% [(34+18)/100], 41% [(27+14)/100].With other two groups of significances of difference
(χ 2=7.89, P < 0.05) is shown in Table 3 and Fig. 9.
* three comparison among groups, there were significant differences, p < 0.05
The experimental results showed that erythricine truly has specific cerebral injury protective effect.
Claims (6)
1. a kind of erythricine injection, which is characterized in that the injection is made of following components: erythricine crystalline powder 5-10
Part, 6-15 parts of cosolvent, 1-5 parts of antioxidant, 50-120 parts of osmotic pressure regulator, 1000 parts of water；The cosolvent is anti-bad
One of hematic acid, citric acid, tartaric acid, acetic acid, lactic acid, hydrochloric acid, phosphoric acid or any combination thereof；The antioxidant is Jiao Ya
One of sodium sulphate, sodium hydrogensulfite, sodium sulfite, sodium thiosulfate or disodium ethylene diamine tetraacetate or any combination thereof；
The osmotic pressure regulator be one of mannitol, sorbierite, dextran, glucose, lactose, xylitol or sodium chloride or
Any combination thereof.
2. erythricine injection according to claim 1, which is characterized in that the injection preparation step is as follows:
(1) it takes 80% water for injection to filter deoxygenation, is heated to 40-50 DEG C, cosolvent and antioxidant is added, stirring is completely molten
Solution；
(2) erythricine crystalline powder is added in the solution obtained to step (1), stirring is completely dissolved；
(3) osmotic pressure regulator is added in the solution obtained to step (2), stirring and dissolving supplies water for injection, with 0.5%
Active carbon stirring and adsorbing；
(4) de- charcoal is carried out by the way of three-stage filtration processing:
First order filtering, is filtered processing to medical fluid using plate and frame filter press；
Second level filtering is filtered processing to the medical fluid after by-pass filtration using thin number husky filter stick；
Third level filtering, is filtered processing to the medical fluid after secondary filtration using 0.22 μm of aperture filter membrane；
(5) 121 DEG C at a temperature of, sterilize 15-20min, and nitrogen charging is filling.
3. erythricine injection according to claim 1, which is characterized in that the erythricine crystalline powder preparation process
Are as follows:
1) Gentiana adds 5-10 times of alkali organic solvent measured to impregnate, 80 DEG C refluxing extraction 2~3 times, 0.5~2h takes out every time
Filter, merging filtrate；
2) above-mentioned filtrate is recovered under reduced pressure, measures lipophylic organic solvents with 1-5 times and is extracted to water layer, divides and takes organic solvent layer, depressurizes
Recycling organic solvent, drying to constant weight, obtains medicinal extract, above-mentioned organic solvent be acetone or ethyl acetate or ether or chloroform or
Methylene chloride or carbon tetrachloride or hexamethylene or petroleum ether；
3) sour water is measured with 2-5 times, hydrolyzes 2-12h, filtered off precipitating, obtain rufous liquid, aforesaid liquid is poured into pre-packed
In cation exchange resin column, successively with water, 50% ethanol elution to colourless, eluent is discarded, and is eluted with alkaline ethanol solution
8-15 times of column volume collects alkaline ethanol solution, after ethyl alcohol is recovered under reduced pressure, measures lipophylic organic solvents aqueous layer extracted with 1-5 times,
Organic solvent is recovered under reduced pressure and obtains compound I crude product,
4) crude product progress silica gel column chromatography, dry column-packing, dry method loading, silica gel mix by 2: 1 with compound I crude product and mix sample, and three
Chloromethanes: methanol elution gradient is first eluted with chloroform, is then successively changed eluent polarity, is finally collected chloroform:
Methanol=150: 1 flow point merges same composition after thin-layer qualitative, is recovered under reduced pressure, obtains white, needle-shaped crystals.
4. erythricine injection according to claim 3, which is characterized in that the alkali organic solvent is 5%-25%'s
Ammonium hydroxide and methanol, ethyl alcohol, propylene glycol, isopropanol, n-butanol, dimethylformamide, dimethyl acetamide one of which reagent
Mixed solvent.
5. erythricine injection according to claim 3, which is characterized in that step (2) lipophylic organic solvents are
Acetone or ethyl acetate or ether or chloroform or methylene chloride or carbon tetrachloride or hexamethylene or petroleum ether；The step
(3) lipophylic organic solvents are ethyl acetate or ether or chloroform or methylene chloride or carbon tetrachloride or hexamethylene or petroleum
Ether.
6. erythricine injection described in claim 1 is in application anticonvulsion, in terms of cerebral injury protection.
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