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CN104510902A - Compound traditional Chinese medicine preparation for treating cardiovascular and cerebrovascular diseases - Google Patents

Compound traditional Chinese medicine preparation for treating cardiovascular and cerebrovascular diseases Download PDF

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Publication number
CN104510902A
CN104510902A CN201310464242.1A CN201310464242A CN104510902A CN 104510902 A CN104510902 A CN 104510902A CN 201310464242 A CN201310464242 A CN 201310464242A CN 104510902 A CN104510902 A CN 104510902A
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rhizoma polygonati
folium crataegi
extract
ethanol
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肖延斌
肖云天
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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)
    • A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
    • A61K36/734—Crataegus (hawthorn)
    • 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)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/896—Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
    • A61K36/8969—Polygonatum (Solomon's seal)
    • 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
    • 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
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1617—Organic compounds, e.g. phospholipids, fats
    • A61K9/1623—Sugars or sugar alcohols, e.g. lactose; Derivatives thereof; Homeopathic globules
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • 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
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2022—Organic macromolecular compounds
    • A61K9/205—Polysaccharides, e.g. alginate, gums; Cyclodextrin
    • A61K9/2059—Starch, including chemically or physically modified derivatives; Amylose; Amylopectin; Dextrin
    • 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
    • A61K9/4841—Filling excipients; Inactive ingredients
    • A61K9/4858—Organic compounds
    • 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
    • A61K9/4841—Filling excipients; Inactive ingredients
    • A61K9/4866—Organic macromolecular compounds
    • 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
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    • 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
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    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
    • A—HUMAN NECESSITIES
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    • 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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Abstract

To satisfy clinical requirement, to treat cardiovascular and cerebrovascular diseases better and to improve health level of people, the invention provides a new traditional Chinese medicine composition for treating the cardiovascular and cerebrovascular diseases and a preparation method thereof, wherein the Chinese medicine composition is mainly prepared from hawthorn leaves and polygonatum kingianum. The Chinese medicine composition, when being used for preparing a drug for treating the cardiovascular and cerebrovascular diseases, has an unexpected effect.

Description

A kind of Chinese traditional compound medicine for the treatment of cardiovascular and cerebrovascular disease
Technical field
The invention belongs to medical art, relate to and be a kind ofly used for the treatment of preparation of the pharmaceutical composition made primarily of Folium Crataegi, Rhizoma Polygonati of cardiovascular and cerebrovascular disease and preparation method thereof.
Background technology
Cardiovascular disease, be also called blood circulation diseases, a series of diseases relating to blood circulation, blood circulation refers to hemophoric Organ and tissue in human body, mainly comprise heart, blood vessel (tremulous pulse, vein, blood capillary), can be subdivided into acute and chronic, be all generally relevant with arteriosclerosis.
Cardiovascular disease is a kind of serious threat mankind, the particularly commonly encountered diseases of more than 50 years old middle-aged and elderly people health, along with the change of growth in the living standard and rhythm of life, " three-hypers disease " (i.e. hypertension, hyperglycemia and the hyperlipidemia) being called as " affluenza " is increasing.With advancing age, Prevalence of Hypertension increases gradually.In more than 60 years old old peoples, 40% ~ 45% suffers from hypertensive also suffer from hyperglycemia or hyperlipidemia simultaneously, and according to the display of external data, the diabetes patient of about 50% is associated with the multiple infirmities of age such as hypertension, hyperlipidemia.The common pathologic basis such as angina pectoris, myocardial infarction, ischemic heart desease are all myocardial ischemia, blood supply of cardiac muscle causes myocardial metabolism disorderly for hypoxgia, energy is under-supply, myocardium shrinkage function declines, blood output reduces, and then affect the function of whole body, and even cause cardiomyocyte cell death.Cerebral ischemia re-pouring is the key of cerebrovascular disease therapy clinically.Affect energy metabolism after cardiac-cerebral ischemia, secondary lactic acid piled up, calcium exceeds standard, the multiple change such as radical damage; Mutiple Targets reverses or improves these changes, improves the important goal that comprehensive therapeutic effect is Drug therapy.
Folium Crataegi is the dried leaves of rosaceous plant Fructus Pyri Pashiae Crataegus pinnatifida Bge.var.major N.E.Br. or Fructus Crataegi Crataegus pinnatifida Bge..Folium Crataegi main component is vetexin-glucoside, vitexin rhamnoside, rutin, vitexin, hyperin, ursolic acid and the number of chemical such as Quercetin, vitexin composition, pharmacological action is mainly reflected in anti-inflammatory and antalgic, blood fat reducing, prevents and treats atherosclerosis, the protection to myocardial ischemia, the protection to cerebral ischemia, to liver protecting, to the aspect such as protection renal, inhibition tumor cell.
Rhizoma Polygonati is the dry rhizome of liliaceous plant P. kingianum Polygonatum kingianum Coll.et Hemsl., Rhizoma Polygonati Polygonatumsibiricum Red. or Polygonatum cyrtonema Hua Polygonatum cyrtonema Hua.Rhizoma Polygonati main component is polysaccharide, steroidal saponin, flavone, anthraquinone analog compound, aminoacid isoreactivity composition.The pharmacological action of Rhizoma Polygonati mainly contains the aspects such as hypoglycemic, the protection to myocardial damage, the protection to cerebral ischemia, antioxidation, slow down aging, Improving memory, antitumor, resisting fatigue, antibacterial and antioxidation.
Utilize the interaction of Folium Crataegi, Rhizoma Polygonati at present, composition of prescription is used for the treatment of cardiovascular and cerebrovascular disease, have not been reported.
Summary of the invention
In order to meet clinical needs, better treatment cardiovascular and cerebrovascular disease, improve the health level of the people, the invention provides a kind of pharmaceutical composition being used for the treatment of cardiovascular and cerebrovascular disease newly and preparation method thereof, this pharmaceutical composition is prepared from primarily of Folium Crataegi, Rhizoma Polygonati, for the preparation of in treatment cardiovascular and cerebrovascular disease, create beyond thought effect.
According to one aspect of the present invention, the invention provides a kind of herbal mixture for the treatment of cardiovascular and cerebrovascular disease, this Chinese medicine is made up of the raw material of Chinese medicine comprising following composition and parts by weight:
Folium Crataegi 1 ~ 100 part of Rhizoma Polygonati 1 ~ 100 part.
Preferably the active raw materials of this Chinese medicine is primarily of Folium Crataegi and Rhizoma Polygonati composition.
More preferably the active raw materials of this Chinese medicine is made up of Folium Crataegi and Rhizoma Polygonati, and the parts by weight of this raw material of Chinese medicine are:
Folium Crataegi 1 ~ 10 part of Rhizoma Polygonati 1 ~ 5 part
More preferably the parts by weight of described raw material of Chinese medicine are further:
Folium Crataegi 5 parts of Rhizoma Polygonatis 1 part
According to another aspect of the present invention, the invention provides a kind of preparation method for the treatment of the herbal mixture of cardiovascular and cerebrovascular disease, comprise the following steps:
With any solvent being suitable for carrying out traditional Chinese medicine extyaction, each of Folium Crataegi and Rhizoma Polygonati is carried out separately to the extracts active ingredients of one or many;
Or the extracts active ingredients of one or many is carried out with the mixture of any solvent being suitable for carrying out traditional Chinese medicine extyaction to Folium Crataegi and Rhizoma Polygonati.
Folium Crataegi in pharmaceutical composition mentioned above, Rhizoma Polygonati crude drug can with suitable solvent and method list be carried or mixed carrying prepares extract, and total extract is mixed and made into any preparation with pharmaceutically acceptable adjuvant again.Wherein said Extraction solvent preferred water or ethanol, extracting method can be infusion process, percolation, decocting method, reflux extraction or continuous extraction.As preferred technical scheme, the preparation method of the herbal mixture for the treatment of cardiovascular and cerebrovascular disease of the present invention comprises the following steps:
(1) Folium Crataegi is taken by proportional quantity, after 0.1 ~ 10 hour solvent soaking time by Folium Crataegi weight 2 times ~ 40 times, extract 0.1 ~ 10 hour under the condition of 30 DEG C ~ 100 DEG C, described solvent soaking and extraction operation can be carried out 1 time or repeat nearly 6 times, merge extractive liquid, distillating recovering solvent, distillate is centrifugal, with the solvent wash of Folium Crataegi medical material amount 0.1 times ~ 100 times of volumes precipitation once or repeated washing reach five times, merge centrifugal after supernatant and the supernatant of washing precipitation, distillation and concentration near dry after, dry, pulverize, to obtain final product; Described solvent is that water or any other are suitable for carrying out the solvent of traditional Chinese medicine extyaction.
(2) Rhizoma Polygonati is taken by proportional quantity, under 50 DEG C ~ 100 DEG C conditions, first time adds Rhizoma Polygonati weight 2 times ~ 40 times solvent extractions 0.1 ~ 10 hour, leaching process can carry out once or nearly 5 times, united extraction gained decocting liquid, and distillation and concentration becomes thick paste, dry, pulverize, obtain Rhizoma Polygonati extract; Described solvent is that water or any other are suitable for carrying out the solvent of traditional Chinese medicine extyaction;
(3) after the Rhizoma Polygonati extract mix homogeneously that the Folium Crataegi extract obtained according to above-mentioned steps (1) and above-mentioned steps (2) obtain, add suitable additives, make injection, oral solid formulation or oral liquid, described oral solid formulation is tablet, capsule, granule, soft capsule, described oral liquid is oral liquid or syrup, and described additives are one or more in filler, excipient, correctives or antiseptic.
According to preferred technical scheme, above-mentioned steps (2) is undertaken by following:
Take Rhizoma Polygonati by proportional quantity, under 50 DEG C ~ 100 DEG C conditions, first time adds the water extraction 0.1 ~ 10 hour of Rhizoma Polygonati weight 2 times ~ 40 times, and leaching process can carry out once or nearly 5 times, united extraction gained decocting liquid, filters and filtrate is concentrated 0.1 times ~ 20 times of Rhizoma Polygonati weight, adding ethanol and make alcohol content be 20% ~ 90%, static or centrifugal, get supernatant, filter, Distillation recovery ethanol, continue distillation and concentration and become thick paste, dry, pulverize, obtain Rhizoma Polygonati extract.
According to a kind of preparation method for the treatment of cardiovascular and cerebrovascular disease herbal mixture of optimal technical scheme of the present invention, comprising:
(1) Folium Crataegi 5 parts is taken, after the 45% soak with ethanol time 2 h of 8 times for the first time by Folium Crataegi weight, extract 2 hours under the condition of 80 DEG C, 45% ethanol of 5 times of second time Folium Crataegi weight extracts 1 hour the condition of 80 DEG C, third time, Folium Crataegi weight 4 times amount 45% ethanol extracted 0.5 hour the condition of 80 DEG C, 0.5 hour is extracted the condition of 80 DEG C 4th time with Folium Crataegi weight 3 times amount 45% ethanol, merge extractive liquid, Distillation recovery ethanol, distillate centrifuge is centrifugal 30min under the rotating speed of 3000R per minute, with the purified water washing precipitation three times of medical material amount 2 times of volumes, merge centrifugal after supernatant and the supernatant of washing precipitation, distillation and concentration is near dry rear dry, pulverize, obtain.
(2) take Rhizoma Polygonati 1 part, under 100 DEG C of conditions, first time adds 10 times of soak by water 2 hours of Bulbus Allii Macrostemonis and Rhizoma Polygonati gross weight, and second time adds Bulbus Allii Macrostemonis and Rhizoma Polygonati gross weight 8 times of soak by water 1 hour, collecting decoction, filtrate is concentrated into medical material gross weight 1 times, adds ethanol and makes alcohol content be 55%, static or centrifugal, get supernatant, filter, Distillation recovery ethanol, continue distillation and concentration and become thick paste, dry, pulverize, to obtain final product.
(3) after the Rhizoma Polygonati extract mix homogeneously that Folium Crataegi extract step (1) obtained and step (2) obtain, add suitable additives, make 10 parts of oral solid formulations, soft capsule or oral liquids, described additives are: starch, lactose, sodium carboxymethyl cellulose, magnesium stearate, one or more in sucrose, stevioside, sodium benzoate, soybean oil or soybean phospholipid.
According to optimal technical scheme of the present invention a kind of preparation method for the treatment of cardiovascular and cerebrovascular disease herbal mixture, characterized by further comprising:
A Folium Crataegi extract that abovementioned steps (1) obtains by (), after defat with petroleum ether by 1/2 amount, discard petroleum ether liquid, be extracted with ethyl acetate again, extract reclaim under reduced pressure ethyl acetate is also concentrated into dry, add suitable quantity of water and make dissolving, be added on polyamide column, first use the water elution of 2 times of column volumes, discard water lotion, then use 80% ethanol elution of 3 times of column volumes, collect eluent, reclaim ethanol and be concentrated into the concentrated solution of 60 DEG C of relative densities about 1.02 ~ 1.08, dry, obtain Folium Crataegi total flavones extract.
B Rhizoma Polygonati extract that abovementioned steps (2) obtains by (), the alcohol settling of amass with diploid 95%, repeatedly carry out 4 times, small-molecule substance is fallen with dialyzer dialysis, precipitate with ethanol again, finally use the absolute ether of equal volume and appropriate washing with acetone precipitate, dry coarse solomon's seal polysaccharide extract.
After the coarse solomon's seal polysaccharide extract mix homogeneously that c Folium Crataegi total flavones extract that above-mentioned steps (a) obtains by () and above-mentioned steps (b) obtain, add suitable additives, make 10 parts of injectable powder, small-volume injection or bulk capacity injections.
The invention provides Folium Crataegi Optimized extraction techniques, Folium Crataegi extract can obtain according to by following method, but is not limited only to following method:
Take Folium Crataegi 2 parts, after the solvent soaking time 0.1 ~ 10 hour of 2 times ~ 40 times of first time by medical material gross weight, extract 0.1 ~ 10 hour under the condition of 30 DEG C ~ 100 DEG C, second time 45% ethanol of 2 times ~ 20 times of the medical material gross weight condition at 30 DEG C ~ 100 DEG C is extracted 0.1 ~ 10 hour, third time extracts 0.1 ~ 10 hour by 2 times ~ 20 times amount 45% ethanol of the medical material gross weight condition at 30 DEG C ~ 100 DEG C, extract 0.1 ~ 10 hour by 2 times ~ 20 times amount 45% ethanol of the medical material gross weight condition at 30 DEG C ~ 100 DEG C for 4th time, merge extractive liquid, distillating recovering solvent, distillate is centrifugal, three times are precipitated with the solvent wash of medical material amount 0.1 times ~ 100 times of volumes, merge centrifugal after supernatant and the supernatant of washing precipitation, distillation and concentration is near dry rear dry, pulverize, obtain.
The extract yield prepared by above-mentioned technique is 10% ~ 35%, and the content of total flavones is with anhydrous rutin (C 21h 20o 12) meter, be not less than 20%; Vitexin rhamnoside (C 27h 34o 14) content be not less than 2%.
The invention provides the Optimized extraction techniques of Rhizoma Polygonati, Rhizoma Polygonati extract can obtain according to by following method, but is not limited only to following method:
Take Bulbus Allii Macrostemonis 1 part, under 50 DEG C ~ 100 DEG C conditions, first time adds 2 times ~ 40 times water extraction 0.1 ~ 10 hour of medical material gross weight, and second time adds medical material gross weight 2 times ~ 20 times water extraction 0.1 ~ 10 hour, collecting decoction, filtrate is concentrated into medical material gross weight 0.1 times ~ 20 times, adds ethanol and makes alcohol content be 20% ~ 90%, static or centrifugal, get supernatant, filter, Distillation recovery ethanol, continue distillation and concentration and become thick paste, dry, pulverize, to obtain final product.
The Bulbus Allii Macrostemonis extract yield prepared by above-mentioned technique is 25% ~ 55%, containing polysaccharide with anhydrous glucose (C 6h 12o 6) meter be not less than 4%.
More than composition is by weight as proportioning, can increase according to corresponding proportion when producing or reduce, as large-scale production can with kilogram for raw material, or in units of ton, small-scale production also can be in grams, weight can increase or reduce, but the constant rate of weight proportion between each component.
The ratio of above weight proportion is through that science screening obtains, for especial patient, can the ratio of corresponding adjustment composition, and increase or reduce being no more than 100%.
The consumption of medicine components of the present invention is through inventor to carry out lot of experiments and gropes to sum up and draw, each component consumption all has good therapeutic effect within the scope of above-mentioned weight portion.
The invention provides a kind of pharmaceutical composition for the preparation for the treatment of cardiovascular and cerebrovascular disease aspect, be mainly used in the aspect diseases such as treatment cerebral thrombosis, coronary heart diseases and angina pectoris, vasculitis, myocardial infarction and hyperlipemia.
Pharmaceutical composition of the present invention can add one or more pharmaceutically acceptable carriers, is applicable in mode that is oral or parenteral the patient needing this treatment.For time oral, conventional solid preparation can be made into, as tablet, capsule, dispersible tablet, oral liquid, granule, chewable tablet, oral cavity disintegration tablet, drop pill, slow releasing tablet, slow releasing capsule, controlled release tablet, controlled release capsule, make liquid preparation if water or oil-suspending agent or other liquid preparations are as syrup etc.; During for parenteral, the solution of injection, water or oil-suspending agent etc. can be made into, as liquid drugs injection, freeze-dried powder, aseptic powder injection, transfusion etc.Preferred form is injection, Tablet and Capsula.
Pharmaceutical composition of the present invention can adopt the conventional method in existing pharmaceutical field to produce, and can add various pharmaceutically acceptable carrier when needs.Described carrier comprises the diluent, excipient, filler, binding agent, wetting agent, disintegrating agent, absorption enhancer, surfactant, absorption carrier, lubricant etc. of pharmaceutical field routine.
Pharmaceutical composition of the present invention, when making injection, in order to increase its dissolubility, can add the solubilizing agents such as tween 80.The isoosmotic adjusting agent for regulating osmotic pressure can be added in transfusion, such as, sodium chloride, potassium chloride, magnesium chloride, calcium chloride, sodium lactate, glucose, xylitol, sorbitol and dextran etc., preferred sodium chloride or glucose.Can excipient be added in powder pin, such as, mannitol, glucose etc.
Pharmaceutical composition of the present invention has the following advantages:
(1) provide a kind of pharmaceutical composition being used for the treatment of cardiovascular and cerebrovascular disease newly and preparation method thereof, meet urgent clinical needs.
(2) carried out pharmacodynamic study to the interaction of pharmaceutical composition of the present invention and composition of prescription first, result is as follows:
1. this pharmaceutical composition can improve S-T section and raises; Obviously can reduce the activity of creatine kinase in Serum fibrosis markers (CK), lactic acid dehydrogenase (LDH), aspartate amino transferase (AST); Atpase activity is reduced, and lactic acid (LD) and free fatty (NEFA) content reduce; Malonaldehyde (MDA) content is reduced, and the activity of total number born (SOD), glutathion peroxidase (GSH-Px) raises; Each administration group whole blood viscosity reduces, plasma viscosity reduces, packed cell volume reduces, erythrocyte sedimentation rate reduces, this pharmaceutical composition energy inhibiting erythrocyte aggregation is described, reduce erythrocyte fragility, strengthen its morphotropism, make that the heart rate of rising is tending towards normally, left ventricular systolic pressure raise, left room diastolic pressure reduces, the maximum climbing speed in left room raises, the maximum fall off rate in left room raises, eventually last diastolic pressure reduce.Treatment group pathological changes and model group obviously alleviate myocardial cell disorder, breaking degree is lighter, karyopycnosis and eosinophilic cytoplasmic apparition reduce, myocardial infarction area obviously reduces, and the improvement that the change of this pharmaceutical composition to myocardial infarction and ischemia model rat indices has and therapeutical effect are described.
2. this pharmaceutical composition obviously can reduce the content of T-CHOL in serum (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and apolipoprotein B (ApoB), increases HDL-C (HDL-C) and ApoA 1(ApoA 1) content; Obviously can reduce malonaldehyde (MDA) content, increase the content of nitric oxide (NO), raise total number born (SOD), glutathion peroxidase (GSH-Px) is active; Treatment group fatty degeneration of liver Leukopenia, endochylema lactone drips and reduces or disappear, and cell volume is close to normal group hepatocyte.Illustrate that the change of this pharmaceutical composition to Hyperlipemia model rat indices has improvement and therapeutical effect.
3. result of the test shows that medicament composition capsule agent curative effect of the present invention is obviously better than alone Folium Crataegi, Rhizoma Polygonati.Prompting Folium Crataegi, the application of Rhizoma Polygonati compatibility have the effect of Synergistic, and consequently those skilled in the art institute is beyond thought.
(3) pharmacodynamic study has been carried out to each proportioning of the present composition, drawn the optimal proportion of the present composition.
(4) the present invention can feed intake with raw material, and preparation technology is simple, and between different batches medicine, mass discrepancy is little, and drug quality is evenly stable.
(5) acute toxicity testing carried out shows that the maximum tolerated dose of medicament composition capsule agent of the present invention is equivalent to 356 times of 70kg body weight day for human beings research on maximum utilized quantity, and indicate pharmaceutical composition low toxicity of the present invention, safety is high.
(6) stability experiment carried out shows that medicament composition capsule agent indices of the present invention is all more stable, ensure that the safety of clinical application.
(7) present composition combination drug determined curative effect, and decrease relative dosage, be with a wide range of applications.
The beneficial effect of pharmaceutical composition of the present invention is set forth further below by way of experimental example.
Embodiment
The detailed description of the invention of form by the following examples, is described in further detail foregoing of the present invention.But this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following embodiment.All technology realized based on foregoing of the present invention all belong to scope of invention.In above example, the adjuvant of each dosage form can be replaced with pharmaceutically acceptable adjuvant, or reduce, increase.
Embodiment 1: the preparation of Folium Crataegi extract
Take Folium Crataegi 2 parts, after the 45% soak with ethanol time 2 h of 8 times for the first time by medical material gross weight, extract 2 hours under the condition of 80 DEG C, 45% ethanol of 5 times of second time medical material gross weight extracts 1 hour the condition of 80 DEG C, third time, 4 times amount 45% ethanol of medical material gross weight extracted 0.5 hour the condition of 80 DEG C, 0.5 hour is extracted the condition of 80 DEG C 4th time with 3 times amount 45% ethanol of medical material gross weight, merge extractive liquid, Distillation recovery ethanol, distillate centrifuge is centrifugal 30min under the rotating speed of 3000R per minute, with the purified water washing precipitation three times of 2 times of volumes of medical material gross weight, merge centrifugal after supernatant and the supernatant of washing precipitation, distillation and concentration is near dry rear dry, pulverize, obtain.
The discriminating of Folium Crataegi extract
Get this product 50mg, add ethanol 5ml, shake up, supersound process 5 minutes, filter, get filtrate as need testing solution.Separately get control substance of Rutin, hyperin reference substance, add ethanol respectively and make the solution of every 1ml containing 0.2mg, product solution in contrast.Test according to thin layer chromatography (" Chinese Pharmacopoeia " annex VIB in 2010), draw each 1 μ l of above-mentioned three kinds of solution, put respectively on same polyamide film, with ethanol-acetone-water (7:5:6) for developing solvent, launch, take out, dry, spray with aluminum chloride test solution, dry up, place after 1 hour, inspect under putting ultra-violet lamp (365nm).In test sample chromatograph, with on reference substance chromatograph relevant position, show the fluorescence speckle of same color.
The assay of Folium Crataegi extract
The preparation precision of reference substance solution takes at 120 DEG C of drying under reduced pressure to the control substance of Rutin 25mg of constant weight, put in 50ml measuring bottle, add appropriate amount of ethanol, supersound process makes dissolving, let cool, with ethanol dilution to scale, shake up, precision measures 20ml, put in 50ml measuring bottle, add water to scale, shake up, obtain (every 1ml is containing anhydrous rutin 0.20mg).
The preparation precision of standard curve measures reference substance solution 1ml, 2ml, 3ml, 4ml, 5ml, 6ml, put in 25ml measuring bottle respectively, respectively add water to 6ml, add 5% sodium nitrite solution 1ml, make mixing, place 6 minutes, add 10% aluminum nitrate solution 1ml, shake up, place 6 minutes, hydro-oxidation sodium test solution 10ml, add water to scale again, shake up, place 15 minutes, with corresponding reagent for blank, according to ultraviolet visible spectrophotometry (" Chinese Pharmacopoeia " annex VA in 2010), absorbance is measured with the wavelength place of 500nn, take absorbance as vertical coordinate, concentration is abscissa, drawing standard curve.
Algoscopy gets this product 0.15g, accurately weighed, and put in tool plug conical flask, precision adds Diluted Alcohol 25ml, close plug, shake up, supersound process 5 minutes, place more than 3 hours, filter, precision measures subsequent filtrate 2ml, put in 25ml measuring bottle, be diluted with water to scale, shake up, as need testing solution.Precision measures need testing solution 2ml, puts in 25ml measuring bottle, the method under the preparation of sighting target directrix curve, from " adding water to 6ml ", measure absorbance, precision measures need testing solution 2ml simultaneously, puts in 25ml measuring bottle in accordance with the law, add water to scale, shake up, as blank solution.Read the amount of rutin in need testing solution from standard curve, calculate, to obtain final product.
Vitexin rhamnoside
Chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filler; With oxolane-methanol-acetonitrile-acetic acid-water (38:3:3:4:152) for mobile phase; Determined wavelength is 330nm.Number of theoretical plate calculates should be not less than 2500 by vitexin rhamnoside peak.
The preparation precision of reference substance solution takes at the vacuum drying apparatus vitexin rhamnoside reference substance of dry 12 hours appropriate, adds Diluted Alcohol and is diluted to scale and is mixed with every 1mL containing 50 μ g vitexin rhamnoside reference substance solution, to obtain final product.
This product under weight differential item is got in the preparation of need testing solution, and porphyrize, gets about 0.25g, accurately weighed, put 50m] in measuring bottle, add Diluted Alcohol 40ml, supersound process 30 minutes, lets cool, is diluted to scale with Diluted Alcohol, shake up, filter, get subsequent filtrate 5ml, be placed in 10mL volumetric flask, add Diluted Alcohol and be diluted to scale, shake up, to obtain final product..
Algoscopy is accurate respectively draws reference substance solution and each 20 μ L of need testing solution, injects hplc determination, to obtain final product.
By above-mentioned technique, obtained three batches of Folium Crataegi extract yield and assay the results are shown in Table 1 respectively.
Table 1 Folium Crataegi extract yield and assay result
Batch Yield (%) General flavone content (%) Vitexin rhamnoside content (%)
1 25.24 28.28 2.98
2 25.56 29.42 2.72
3 25.85 28.02 2.85
On average 25.55 28.57 2.85
Embodiment 2: the preparation of Rhizoma Polygonati extract
Take Rhizoma Polygonati 1 part, under 100 DEG C of conditions, first time adds 10 times amount soak by water 2 hours, and second time adds 8 times amount soak by water 1 hour, collecting decoction, filtrate is concentrated into 1:1, adds ethanol and makes alcohol content be 55%, centrifugal, get supernatant, filter, Distillation recovery ethanol, continue distillation and concentration to appropriate, dry, pulverize, to obtain final product.
The assay of Rhizoma Polygonati extract
The preparation of reference substance solution is learnt from else's experience 105 DEG C and is dried to the anhydrous glucose reference substance 33mg of constant weight, accurately weighed, puts in 100ml measuring bottle, is dissolved in water and is diluted to scale, shaking up, and obtains (containing anhydrous glucose 0.33mg in every 1ml).
The preparation precision of standard curve measures reference substance solution 0.1ml, 0.2ml, 0.3ml, 0.4ml, 0.5ml, 0.6ml, put in 10ml tool plug scale test tube respectively, respectively add water to 2.0ml, shake up, in ice-water bath, slowly drip 0.2% By Anthrone Sulphuric acid solution to scale, mixing, let cool in rearmounted water-bath and be incubated 10 minutes, take out, put immediately in ice-water bath and cool 10 minutes, taking out, take corresponding reagent as blank.According to ultraviolet visible spectrophotometry (" Chinese Pharmacopoeia " annex V A in 2010), measure absorbance at 582nm wavelength place, take absorbance as vertical coordinate, concentration is abscissa, drawing standard curve.
Algoscopy is got this product fine powder and is about 0.5g, accurately weighed, put in round-bottomed flask, add 80% ethanol 150ml, to put in water-bath reflux 1 hour, filter while hot, residue 80% hot ethanol washs 3 times, each 10ml, residue and filter paper are put in flask, add water 150ml, to put in boiling water bath reflux 1 hour, filter while hot, residue and flask hot wash 4 times, each 10ml, merging filtrate and washing liquid, let cool, be transferred in 250ml measuring bottle, add water to scale, shake up, precision measures 1ml, put in 10ml tool plug dry test-tube, method under the preparation of sighting target directrix curve, from " adding water to 2.0ml ", measure absorbance in accordance with the law, the weight (mg) containing anhydrous glucose in need testing solution is read from standard curve, calculate, obtain.
By above-mentioned technique, obtained three batches of Rhizoma Polygonati extract yield and assay the results are shown in Table 2 respectively.
Table 2 Rhizoma Polygonati extract yield and assay result
Batch Yield (%) Polyoses content (%)
1 45.75 6.21
2 45.61 6.35
3 45.89 6.41
On average 45.75 6.32
Embodiment 3: the preparation of composition tablet
Prescription
Method for making: Folium Crataegi is extracted to obtain Folium Crataegi extract according to embodiment 1 method; Rhizoma Polygonati extracts to obtain Rhizoma Polygonati extract according to embodiment 2.Take extract and adjuvant according to recipe quantity, pulverize and cross 100 mesh sieves respectively, for subsequent use.By Folium Crataegi extract, Rhizoma Polygonati extract, starch, pregelatinized Starch, microcrystalline Cellulose mix homogeneously, stir, and makes suitable soft material.Cross 18 mesh sieve granules.Granule is dried under the condition of 60 DEG C.Dried granule adds carboxymethyl starch sodium and magnesium stearate, crosses 18 mesh sieve granulate, mix homogeneously.Sampling, semi-finished product are chemically examined.According to chemically examining the sheet weight sheet determined.Finished product is examined entirely, packaging warehouse-in.
Embodiment 4: the preparation of composition capsule
Prescription
Method for making: Folium Crataegi is extracted to obtain Folium Crataegi extract according to embodiment 1 method; Rhizoma Polygonati extracts to obtain Rhizoma Polygonati extract according to embodiment 2.Take extract and adjuvant according to recipe quantity, pulverize and cross 100 mesh sieves respectively, for subsequent use.By Folium Crataegi extract, Rhizoma Polygonati extract, starch mix homogeneously, stirs, and makes suitable soft material.Cross 18 mesh sieve granules.Granule is dried under the condition of 60 DEG C.Cross 18 mesh sieve granulate, mix homogeneously.Sampling, semi-finished product are chemically examined.According to chemically examining, the weight determined is encapsulated.Finished product is examined entirely, packaging warehouse-in.
Embodiment 5: the preparation of composition granule
Prescription
Method for making: Folium Crataegi is extracted to obtain Folium Crataegi extract according to embodiment 1 method; Rhizoma Polygonati extracts to obtain Rhizoma Polygonati extract according to embodiment 2.Take extract and adjuvant according to recipe quantity, pulverize and cross 100 mesh sieves respectively, for subsequent use.By Folium Crataegi extract, Rhizoma Polygonati extract, the method mix homogeneously that lactose powder is progressively increased with equivalent, adds 2%HPMC60% alcoholic solution in right amount, stirs, make suitable soft material.Cross 18 mesh sieve granules.Granule is dried under the condition of 55 DEG C.Dry granule crosses 18 mesh sieve granulate.Sampling, in semi-finished product chemical examination granule, the content of principal agent, determines loading amount.Packaging, finished product is examined entirely, packaging warehouse-in.
Embodiment 6: the preparation of composition soft agent
Prescription
Method for making: Folium Crataegi is extracted to obtain Folium Crataegi extract according to embodiment 1 method; Rhizoma Polygonati extracts to obtain Rhizoma Polygonati extract according to embodiment 2.Take extract according to recipe quantity, pulverize and cross 100 mesh sieves respectively, for subsequent use.By the soybean oil of recipe quantity and soybean phospholipid, Cera Flava heating and melting, mixing, let cool, add Folium Crataegi extract, Rhizoma Polygonati extract grinds well, and is pressed into soft capsule.
Embodiment 7: the preparation of Folium Crataegi total flavones extract
The Folium Crataegi extract of Example 1, after defat with petroleum ether by 1/2 amount, discard petroleum ether liquid, be extracted with ethyl acetate again, extract reclaim under reduced pressure ethyl acetate is also concentrated into dry, add suitable quantity of water and make dissolving, be added on (granularity: 30 ~ 60 orders on processed good polyamide column, with 95% ethanol wet method dress post, first use 95% ethanol elution of 3 times of column volumes, use the water elution of 3 times of column volumes extremely without alcohol taste afterwards, for subsequent use), first use the water elution of 2 times of column volumes, discard water lotion, then 80% ethanol elution of 3 times of column volumes is used, collect eluent, reclaim ethanol and be concentrated into the concentrated solution of relative density about 1.02 ~ 1.08 (60 DEG C), spraying dry, obtain Folium Crataegi total flavones extract.
By above-mentioned technique, obtained three batches of Folium Crataegi total flavones extract yields and assay the results are shown in Table 3 respectively.
Table 3 Folium Crataegi total flavones extract yield and assay result
Batch Yield (%) General flavone content (%) Vitexin rhamnoside content (%)
1 19.17 85.70 9.14
2 19.24 85.88 9.31
3 19.10 85.91 9.30
On average 19.17 85.83 9.25
Embodiment 8: the preparation of coarse solomon's seal polysaccharide extract
The Rhizoma Polygonati extract of Example 2, the alcohol settling of amass with diploid 95%, carries out 4 times repeatedly.Fall small-molecule substance, again precipitate with ethanol with dialyzer dialysis, finally use the absolute ether of equal volume and appropriate washing with acetone precipitate, vacuum drying obtains coarse solomon's seal polysaccharide extract.
By above-mentioned technique, obtained three batches of coarse solomon's seal polysaccharide extract yields and assay the results are shown in Table 4 respectively.
Table 4 coarse solomon's seal polysaccharide extract yield and assay result
Batch Yield (%) Polyoses content (%)
1 15.32 21.43
2 15.16 21.58
3 15.23 21.03
On average 15.24 21.35
Embodiment 9: the preparation of compositions aqueous injection
Prescription
Method for making: the Folium Crataegi total flavones extract that the Folium Crataegi taking recipe quantity is prepared according to embodiment 7 and the coarse solomon's seal polysaccharide extract that Rhizoma Polygonati is prepared according to embodiment 8.Carry the pipeline and container etc. that process dosing the previous day, rinse with fresh water for injection more before use.Folium Crataegi total flavones extract and coarse solomon's seal polysaccharide extract are added heated and stirred in the water for injection of dosing amount 70% to dissolve completely.Benefit adds to the full amount of water for injection.Add the needle-use activated carbon of dosing amount 0.05%, heated and stirred 15 minutes.Through sand stick filtering decarbonization.Measure and regulate the pH value of solution.Through the microporous filter membrane fine straining of 0.45um.Check the clarity of solution, semi-finished product are chemically examined.By solution embedding in glass ampule.100 DEG C of flowing steam sterilizations 30 minutes.The methylene blue solution while hot sample being put into 0.01% is hunted leak.Lamp inspection, finished product is examined entirely, packaging warehouse-in.
Embodiment 10: the preparation of composition powder injection
Prescription
Method for making: the Folium Crataegi total flavones extract that the Folium Crataegi taking recipe quantity is prepared according to embodiment 7 and the coarse solomon's seal polysaccharide extract that Rhizoma Polygonati is prepared according to embodiment 8.First by the container tool of dosing and antibiotic glass bottle, plug etc. carry out aseptic process.Folium Crataegi total flavones extract and coarse solomon's seal polysaccharide extract are added heated and stirred in the sterile water for injection of dosing amount 40% to dissolve completely.The sterile water for injection heated and stirred that mannitol adds 30% of dosing amount is dissolved completely, merges above-mentioned solution, adds sterile water for injection to full dose.Add the needle-use activated carbon of dosing amount 0.05%, heated and stirred 15 minutes.Through sand stick filtering decarbonization.Measure and regulate the pH value of solution.Through the microporous filter membrane fine straining of 0.22um.Check the clarity of solution, semi-finished product are chemically examined.Be sub-packed in antibiotic glass bottle, half tamponade.Sample is put into freeze dryer lyophilization.Carry out lyophilizing by following freeze-dry process: 1. pre-freeze: be cooled to-35 DEG C, keep temperature 5 hours; 2. low-temperature distillation :-35 DEG C of insulations, open vacuum pump evacuation and keep 2 hours, then slowly heat up, temperature is risen to 0 DEG C by 30 hours; 3. high temperature drying: be warming up to 25 DEG C in 2 hours, is incubated to 2 hours; 4. shut down lyophilizing to terminate.Tamponade, rolls lid.Finished product is examined entirely, packaging warehouse-in.
Embodiment 11: the preparation of compositions sodium chloride injection
Prescription
Method for making: the Folium Crataegi total flavones extract that the Folium Crataegi taking recipe quantity is prepared according to embodiment 7 and the coarse solomon's seal polysaccharide extract that Rhizoma Polygonati is prepared according to embodiment 8.Carry the pipeline and container etc. that process dosing the previous day, rinse with fresh water for injection more before use.Folium Crataegi total flavones extract and coarse solomon's seal polysaccharide extract being added heated and stirred in the water for injection of dosing amount 40% dissolves completely, is dissolved completely by the water for injection of sodium chloride by dosing amount 20%.Merge above-mentioned solution, benefit adds to the full amount of water for injection.Add the needle-use activated carbon of dosing amount 0.05%, heated and stirred 15 minutes.Through sand stick filtering decarbonization.Measure and regulate the pH value of solution.Through the microporous filter membrane fine straining of 0.45um.Check the clarity of solution, semi-finished product are chemically examined.Fill is in the infusion bottle of 250ml.Become 5 DEG C of pressure sterilizings 30 minutes.Lamp inspection, finished product is examined entirely, packaging warehouse-in.
Embodiment 12: the preparation of compositions glucose injection
Prescription
Method for making: the Folium Crataegi total flavones extract that the Folium Crataegi taking recipe quantity is prepared according to embodiment 7 and the coarse solomon's seal polysaccharide extract that Rhizoma Polygonati is prepared according to embodiment 8.Carry the pipeline and container etc. that process dosing the previous day, rinse with fresh water for injection more before use.Folium Crataegi total flavones extract and coarse solomon's seal polysaccharide extract being added heated and stirred in the water for injection of dosing amount 40% dissolves completely, is dissolved completely by the water for injection of glucose by dosing amount 20%.Merge above-mentioned solution, benefit adds to the full amount of water for injection.Add the needle-use activated carbon of dosing amount 0.05%, heated and stirred 15 minutes.Through sand stick filtering decarbonization.Measure and regulate the pH value of solution.Through the microporous filter membrane fine straining of 0.45um.Check the clarity of solution, semi-finished product are chemically examined.Fill is in the infusion bottle of 250ml.115 DEG C of pressure sterilizings 30 minutes.Lamp inspection, finished product is examined entirely, packaging warehouse-in.
Test sample study on the stability
Composition capsule, prescription and preparation method are see the preparation of embodiment 4 capsule.
Investigation project: character, moisture, content.
Accelerated test and long-term stable experiment method and result: place 6 months under this product being put the condition of temperature 40 DEG C ± 2 DEG C, relative humidity 75% ± 5% and temperature 25 DEG C ± 2 DEG C, relative humidity 60% ± 10% condition under place 24 months, indices has no significant change, and experimental result shows that composition capsule is placed basicly stable for a long time.
Observation of curative effect
The research of test example 1 present composition acute toxicity test
Get Kun Ming mice 20, male and female half and half.Fasting 12 hours, every gavages 40% (g/mL) present composition medicated powder (preparing by purified water) by 0.4ml/10g.Reach Cmax, maximum administration volume.Gavage 1 time in one day, mice active situation and dead quantity in Continuous Observation 7 days, result mice is by after above-mentioned dosage, and all without death in 7 days, freely, normally, hair is glossy, without loose stool etc. for diet in activity.The maximum tolerated dose of mice is 16g/Kg as calculated, is 356 times of clinical adult's consumption.
The research of test example 2 present composition general pharmacology
1) on the impact of Kunming mouse general behavior performance
After gastric infusion, each administration treated animal compares with blank group, movable normal, walk with a steady step, without the phenomenon such as sialorrhea, amyostasia, have no generation untoward reaction.
2) on the impact of Kunming mouse spontaneous activity
After gastric infusion, animal is put in mice activity count instrument active box by 0.5h, records the animal activity number of times in 10min after adapting to 5min; Each dosage group compares animal activity number of times with blank group.After result administration, each treated animal compares with blank group, and significant difference does not appear in movable number of times, and data are in table 5.
Table 5 medicine is on the impact of mice autonomic activities
Group Dosage (mg/kg) Number of animals Movable number of times
Blank group - 10 236.5±21.89
High dose group 2500 10 233.5+15.91
Middle dosage group 1250 10 239.5+18.74
Low dose group 410 10 231.3±18.59
Note: * P<0.05 (comparing with model group).
3) sub-threshold dose pentobarbital sodium is brought out to the impact of Kunming mouse sleep
1h after each treated animal gastric infusion, lumbar injection pentobarbital sodium 32mg/kg (for the maximum threshold dose that animal 90% ~ 100% mice righting reflex measured in advance does not disappear), the Mus number of more than righting reflex loss 1min within the rear 15min of record pentobarbital sodium injection.Result: each group laboratory animal, after giving corresponding medicine, is brought out mice sleep to sub-threshold dose pentobarbital sodium and do not detected difference, and display sub-threshold dose pentobarbital sodium brings out mice and falls asleep uninfluenced, and data are in table 6.
Table 6 brings out the impact of mice sleep to sub-threshold dose pentobarbital sodium
Group Dosage (mg/kg) Number of animals Sleep number of elements Male and female Sleep rate (%)
Blank group - 10 6 ♀2♂4 60
High dose group 2500 10 8 ♀5♂3 80
Middle dosage group 1250 10 6 ♀3♂3 60
Low dose group 410 10 7 ♀3♂4 70
4) on the impact of the pentobarbital sodium length of one's sleep
Each group of Kunming mouse gavage is to 1h after gastric infusion, lumbar injection pentobarbital sodium 40mg/kg (for the minimum dose that the animal 100% measured in advance falls asleep), taking righting reflex loss as time for falling asleep, is sleep time (min) from righting reflex loss to recovery time.After result administration, each treated animal compares with blank group, and significant difference does not appear in the length of one's sleep, and data are in table 7.
5) on the impact of mice coordination exercise
Get surface and twine white glue cloth, diameter 1cm, long 60cm iron staff, vertically fix.1h after gastric infusion, is positioned over top by mice, makes it naturally creep, and observes mice coordination exercise ability, and records it and climb to the bottom time.The each treated animal of result compares with blank group, creeps all steadily, and slide and drop phenomenon, and significant difference does not appear in the pole-climbing time, and data are in table 8.
Table 7 is on the impact of the pentobarbital sodium in mice length of one's sleep
Group Dosage (mg/kg) Number of animals Sleep number of elements
Blank group - 10 30.91+1.31
High dose group 2500 10 31.45+2.23
Middle dosage group 1250 10 29.05±2.75
Low dose group 410 10 30.76±2.46
Note: * P < 0.05 (comparing with model group).
Table 8 is on the impact of mice pole-climbing time
Group Dosage (mg/kg) Number of animals Pole-climbing time (s)
Blank group - 10 11.67±0.64
High dose group 2500 10 12.32±0.45
Middle dosage group 1250 10 12.43+0.59
Low dose group 410 10 11.79±0.83
Note: * P<0.05 (comparing with model group).
6) on the impact of Wistar Rat Cardiovascular system
With pentobarbital sodium intraperitoneal injection of anesthesia animal, be separated common carotid artery and with System of organism signal linkage record blood pressure and heart rate; Electrocardiogram is traced with limb lead (II leads).Record once respectively before gastric infusion, then record These parameters after gastric infusion.After result administration, each treated animal compares with blank group, and significant difference does not appear in electrocardio and the every numerical value of blood pressure, and data are in table 9 and table 10.
Table 9 is on the cardiac electrical impact of rat
Group Dosage (mg/kg) Number of animals Heart rate (beat/min) Maximum (mV) Minima (mV)
Blank group - 8 367.96±21.76 0.42+0.09 -0.26±0.12
High dose group 1700 8 365.58+14.78 0.48±0.10 -0.30+0.14
Middle dosage group 850 8 365.01+43.31 0.48±0.01 -0.33±0.19
Low dose group 285 8 365.04±56.39 0.38+0.10 -0.30±0.19
Note: * P<0.05 (comparing with model group).
Table 10 is on the impact of rat blood pressure
Group Dosage (mg/kg) Number of animals Systolic pressure (mmHg) Diastolic pressure (mmHg)
Blank group - 8 73.20+13.00 45.87±13.37
High dose group 1700 8 68.89±9.20 44.80+9.46
Middle dosage group 850 8 70.00±21.19 44.33+21.25
Low dose group 285 8 86.96+17.49 59.77±15.26
Note: * P<0.05 (comparing with model group).
7) on the impact of wistar rats respiratory system
The respiratory frequency of System of organism signal record animal, average expiration peak pressure and average suction paddy pressure.After result administration, each treated animal compares with blank group, and respiratory frequency, expiration peak value and air-breathing valley all do not occur significant difference, and data are in table 11.
Table 11 is on the impact of respiratory system in rats
Group Dosage (mg/kg) Number of animals Frequency (beat/min) Maximum (ml/s) Minima (ml/s)
Blank group - 8 123.22±12.02 0.13±0.13 0.02±0.12
High dose group 1700 8 119.25±6.91 0.25±0.13 0.15±0.15
Middle dosage group 850 8 117.28±17.37 0.23±0.08 0.15±0.08
Low dose group 285 8 113.84±1.92 0.26±0.16 0.17±0.18
Note: * P<0.05 (comparing with model group).
The present composition is in general pharmacology indices detects, and each administration group compares with blank group, unknown significance difference.Illustrate that the present composition is to nervous system, cardiovascular and respiratory system do not produce harmful effect, can be applicable to clinical.
The pharmacodynamics test of the research one compositions treatment rats with myocardial ischemia of test example 3 present composition pharmacodynamics.
Myocardial infarction model is copied according to literature method.Adopt intraperitoneal injection of anesthesia with 1% pentobarbital sodium (0.04g/kg), dorsal position is fixed on operating-table.Record standard II lead electrocardiogram, row tracheotomy, be connected with artificial respirator, open breast and expose heart, prop up through the left room of arteria coronaria with surgical thread between aorta circular cone and left auricle, ligation left coronary artery (a sham operated rats threading not ligation), heart resets, close thoracic cavity rapidly after drum lung, penicillin is embrocated in wound, prevention traumatic infection.Observe rats breathing recovery situation after closing breast, off line can put back to mouse cage if any autonomous respiration.Occur that the back of a bow is raised as myocardial ischemia modeling success with ECG ST section.Chronic myocardial ischemia model is formed after surrounding.
1) on the impact of rats with myocardial ischemia S-T section after coronary artery ligation
Rats with myocardial ischemia after screening is evenly divided into 5 groups at random, sham operated rats, model group, positive drug control group, high dose group, middle dosage group, low dose group 6 groups except sham operated rats, often organizes 12, totally 72, raise in the lump.Administration group gavage give corresponding dosage containing drug solns, blank group, model group gavage gives the distilled water of equivalent, once a day, totally 10 days.After last administration, 1% pentobarbital sodium (40mg/kg) intraperitoneal anesthesia of each group rat, connects biological functional system, and monitoring limb lead electrocardiogram, measures S-T section.Compare between group with t inspection represent, result with represent.The each administration group of result compares with model group can be improved S-T section and raise, and data are in table 12.
Impact that table 12 changes ECG ST section ( )
Group Number of animals (only) Dosage (mg/kg) ST section changes (mv)
Blank group 12 - 0.08+0.03
Model group 12 - 0.24±0.02*
High dose group 12 1700 0.12±0.01#
Middle dosage group 12 850 0.13±0.02#
Low dose group 12 285 0.19±0.01#
Positive control 12 325 0.13±0.01#
Note: compare with blank group, * P<0.05; Compare with model group, #P<0.05.
2) on the impact of Serum fibrosis markers
Laboratory animal grouping and administration the same.In last administration 1 hour, eyeball gets blood, by the operation of test kit description, and detect serum creatine kinase (CK), lactic acid dehydrogenase (LDH), aspartate amino transferase (AST) activity with semi-automatic biochemical analyzer, record data.Compare between group and represent with t inspection, result represents with X ± S.The each administration group of result compares with model group, and the present composition obviously can reduce creatine kinase in serum (CK), lactic acid dehydrogenase (LDH), aspartate amino transferase (AST), and data are in table 13.
Table 13 on the impact of Serum fibrosis markers ( )
Note: compare with blank group, * P<0.05; Compare with model group, #P<0.05.
3) on the impact of myocardial metabolism product
Laboratory animal grouping and administration the same.In last administration 1 hour, eyeball got blood, by the operation of test kit description, and detected serum lactic (LD), free fatty (NEFA), Na-KATP enzyme index with 756PC type ultraviolet-uisible spectrophotometer.Record data.Compare between group with t inspection represent, result with represent.The each administration group of result compares with model group group, and present composition atpase activity reduces, and lactic acid and free fatty acid content reduce, and data are in table 14.
4) on the impact of Plasma free radical damage criterion
Laboratory animal grouping and administration the same.In last administration 1 hour, eyeball gets blood, by the operation of test kit description, and detect malonaldehyde (MDA), total number born (SOD), glutathion peroxidase (GSH-Px) index with 756PC type ultraviolet-uisible spectrophotometer.Record data.Compare between group and represent with t inspection, result represents with X ± S.The each administration group of result compares with model group, the each administration group of the present composition can make malonaldehyde (MDA) content reduce, the activity of total number born (SOD), glutathion peroxidase (GSH-Px) raises, and data are in table 15.
5) on the impact of hemorheology index
Laboratory animal grouping and administration the same.In last administration 1 hour, abdominal aortic blood, sent and checks with clinical laboratory.Compare between group and represent with t inspection, result represents with X ± S.The each administration group of result compares with model group, each administration of the present composition
Table 14 on the impact of myocardial metabolism product index ( )
Note: compare with blank group, * P<0.05; Compare with model group, #P<0.05.
Table 15 is on the impact of myocardial metabolism product index
Note: compare with blank group, * P<0.05; Compare with model group, #P<0.05.
Organize whole blood viscosity reduction, plasma viscosity reduction, packed cell volume reduction, erythrocyte sedimentation rate reduction, its energy inhibiting erythrocyte aggregation is described, reduction erythrocyte fragility, strengthens its morphotropism, in table 16.
6) on the impact of hemodynamic index
Laboratory animal grouping and administration the same.In last administration 1 hour, being lain on the back by rat is fixed on operating-table, cut skin of neck, be separated right carotid, row arterial cannulation, the conduit containing 0.1% heparin sodium liquid is inserted right carotid artery, left ventricle is inserted through right common carotid artery, according to display, the change of pressure figure judges whether intubate enters ventricle, and cardiac catheter Bonding pressure transducer, inputs signal in physiograph.And recorded heart rate (HR), left ventricular systolic pressure (LVSP), maximal ascending rate of internal pressure of left ventricle (+dp/dtmax), left ventricular diastolic pressure (LVDp), left ventricular end-diastolic pressure (LVDEp), maximal descending rate of internal (-dP/dtmax).Record data.Compare between group with t inspection represent, result with represent.The each administration group of result compares with model group, the present composition each administration group can make that heart rate is tending towards normally, left ventricular systolic pressure raises, left room diastolic pressure reduces, the maximum climbing speed in left room raises, the maximum fall off rate in left room raises, last diastolic pressure reduction eventually, in table 17.
Table 17 is on hemorheological impact
Note: compare with blank group, * P<0.05; Compare with model group, #P<0.05.
Table 16 on hemorheological impact ( )
Note: compare with blank group, * P<0.05; Compare with model group, #P<0.05.
7) on the morphologic impact of myocardial histopathology
Laboratory animal grouping and administration the same.In last administration 1 hour, take out heart, send and check in Pathology Deparment.The results are shown in Table 18.
8) to myocardial necrosis area estimation
Laboratory animal grouping and administration the same.In last administration 1 hour, put to death rat, presternum is fixed with tweezers, cut off breast bone, cut whole for heart, with the normal saline flushing removing blood stains of pre-cooling, filter paper suck dry moisture, weigh whole-heartedly,-30 DEG C of freezing half an hour, under heart ligature, the thick myocardium sheet of 1 ~ 2mm is cut into along left room longer axis parallel, evenly be cut into 4 ~ 5, be placed in the TTC solution (the PBS buffer of pH value 7.4) of 1% concentration, 37 DEG C of temperature incubate 15min, unnecessary dyestuff is rinsed with water, filter paper suck dry moisture, visible normal myocardium is red, ischemic myocardium is canescence, cut off infarcted myocardium, weigh, calculate infarction size (infarcted region accounts for percentage ratio heavy whole-heartedly).Record data, compare between group and represent with t inspection, result represents with X ± S.The each administration group of result compares with model group, and each administration group of the present composition can make myocardial infarction area obviously reduce, and difference has significance, in table 19.
Table 18 is on the morphologic impact of myocardial histopathology
The shadow of table 19 pair myocardial infarction area
Group Number of animals (only) Dosage (mg/kg) Myocardial infarction area (%)
Model group 12 - 13.49+0.61
High dose group 12 1700 7.13±0.84#
Middle dosage group 12 8500 5.92±0.40#
Low dose group 12 284 9.86±1.25#
Positive control 12 325 7.30±0.70#
Note: compare with blank group, * P<0.05; Compare with model group, #P<0.05.
Conclusion (of pressure testing): the improvement that the change of the present composition to myocardial infarction and ischemia model rat indices has and therapeutical effect.
The pharmacodynamics test of the research ~ compositions treatment hyperlipidemia rats of test example 4 present composition pharmacodynamics.
1) on the impact of hyperlipidemia rats lipid metabolism level
High lipid food composition and preparation: 3% cholesterol, 10% Adeps Sus domestica, 5% dried hen egg yolk, 0.3% sodium cholate, 0.2% methylthiouracil, 81.6% normal feedstuff.10kg high lipid food adds 1 ~ 2kg white sugar.In proportion first by cholesterol, dried hen egg yolk, sodium cholate, propylthiouracil, white sugar ground and mixed, then adds in warm Adeps Sus domestica, then mixes thoroughly with normal feedstuff, to obtain final product.
Modeling: 72 SD rat normal diets are fed one week.Random selection 12 is as blank group Mus, and labelling of weighing, gives normal diet and feed.All the other 60 Mus are weighed labelling, give high lipid food and feed, all freely drink water, feed the foundation that 4 weeks carry out hyperlipemia model altogether.Record starts the rat body weight of surrounding after modeling respectively, and gets blood 1.5 ~ 2ml in centrifuge tube in 4th week end eyeground vein clump, leaves standstill, 3500rmin -1centrifugal 15min, separation of serum, measure the content of T-CHOL (TC) in its Diagnostic Value of Fasting Serum, triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), HDL-C (HDL-C) with semi-automatic biochemical analyzer, detection method is in accordance with the description of corresponding reagent box.When T-CHOL (TC), triglyceride (TG) content are all apparently higher than blank group in serum, illustrate that hyperlipidemia animal model is successfully prepared.Next day starts gastric infusion, and positive drug is with 0.325g/kg gastric infusion, and the present composition is high, in, low dose group respectively with 1.70g/kg, 0.850g/kg, 0.285g/kg gastric infusion, totally 10 days.Continue during administration to give high lipid food.In last administration after 1 hour, socket of the eye angular vein clump gets blood 2.5ml, and 4 DEG C leave standstill, 3500rmin -1centrifugal 15min, separation of serum, in accordance with corresponding reagent box description, detects T-CHOL (TC), triglyceride (TG) level and ApoA with semi-automatic biochemical analyzer 1(ApoA 1) and apolipoprotein B (ApoB) level.Compare between group and represent with t inspection, result represents with X ± S.The each administration group of result obviously can reduce the content of T-CHOL in serum (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and apolipoprotein B (ApoB) compared with model group, obviously increases HDL-C (HDL-C) and ApoA 1(ApoA 1) content, in table 20.
Table 20 is on the impact of hyperlipemia lipid metabolism level
Note: compare with blank group, * P<0.05; Compare with model group, #P<0.05.
2) on the impact of hyperlipidemia rats level of lipid
Laboratory animal grouping and administration the same.In last administration 1 hour, socket of the eye angular vein clump got blood, and 4 DEG C leave standstill, 3500rmin -1centrifugal 15min, separation of serum, in accordance with corresponding reagent box description, total number born (SOD) is detected with ultraviolet-uisible spectrophotometer, malonaldehyde (MDA), nitric oxide (NO) and glutathion peroxidase (GSH-Px) level.Compare between group and represent with t inspection, result represents with X ± S.The each administration group of result compares with model group obviously can reduce malonaldehyde (MDA) content, the content of obvious increase nitric oxide (NO), raise total number born (SOD), glutathion peroxidase (GSH-Px) lives life, in table 21.
Table 21 is on the impact of hyperlipemia level of lipid
Note: compare with blank group, * P<0.05; Compare with model group, #P<0.05.
3) on the impact of hyperlipidemia rats hemorheology index
Laboratory animal grouping and administration the same.In last administration 1 hour, abdominal aortic blood, sent and checks with clinical laboratory.Compare between group with t inspection represent, result with represent.The each administration group of result compares with model group that whole blood viscosity reduces, plasma viscosity reduces, packed cell volume reduces, erythrocyte sedimentation rate reduces, and illustrates that it can inhibiting erythrocyte aggregation, reduces erythrocyte fragility, strengthen its morphotropism, in table 22.
Table 22 is on the impact of hyperlipemia lectin from hemolymph
Note: compare with blank group, * P<0.05; Compare with model group, #P<0.05.
4) on the impact of hyperlipidemia rats liver organization pathomorphism
Laboratory animal grouping and administration the same.After last administration 1h, carry out routine to hepatic tissue and draw materials, 10% formalin solution is fixed, and conventional H E dyes.Optical microphotograph Microscopic observation liver organization form.This send checks in Pathology Deparment.The results are shown in Table 23.
Conclusion (of pressure testing): the improvement have the change of Hyperlipemia model rat indices and therapeutical effect.
Table 23 is on the morphologic impact of myocardial histopathology
The research of test example 5 present composition long term toxicity test.
Experiment purpose: observation repetition per os gives the toxic reaction that the present composition produces rat, the symptom occurred and the order of severity, and the target organ of toxicity and the degree of reversibility of infringement thereof are provided, determine nontoxic crude protein, to evaluate the safety of present composition long-term prescription, for drafting clinical trial dosage and observation index provides reference.
Experimental animal: cleaning agent SD rat, male and female half and half, quantity 144, body weight 80 grams ~ 110 grams (6 ~ 9 week age).
Animal divides into groups: first animal normally raises 1 week at laboratory, rejects disease Mus and abnormal Mus, gets rat 120, is divided into 4 groups at random by body weight, often organizes 30, male and female half and half.Dividing separately is Normal group, high, medium and low three the dosage groups of the present composition.
Dosage: the maximum tolerated dose of mice is 16g/Kg, does not occur toxic reaction, therefore, in the case, should be greater than the principle design dosage of clinical more than 50 times according to the administration of rat high dose group.Present composition high dose group rat presses 2.7g/kg administration (60 times of clinical plan dosage), middle dosage group rat presses 1.35g/kg gastric infusion (30 times of clinical plan dosage), low dose group rat presses 0.45g/kg gastric infusion (10 times of clinical plan dosage), volume is 2ml/100g, and Normal group gives same volume distilled water.
Administration time: on every Mondays to about the 8:30 administration in the morning 1 time of Saturday, not administration on Sunday.Successive administration 6 months.
Observation index
1) general observation of symptoms
1. observing time: before administration, each cage is observed with every morning convalescent period, and during administration, the upper and lower noon observes simultaneously.
2. number of cases is observed: before administration, all buy animal, total Test animal after administration.
3. frequency is observed: terminate from buying day to convalescent period, once a day.
4. observed content: observe the outward appearance of animal, by hair, gait, behavioral activity, to the reaction of sound, have atremia, spasm, whether steadily breathe, have without exception.During administration and after administration, except above-mentioned reaction is observed in attention, also want special survey with or without drowsiness, vomiting, feces shape and color etc.Administration also will observe reactivity when arresting animal simultaneously, the state around eye, nose, mouth, hypogastric region, anus place with or without manure contamination, skin color, with or without wound, tumor etc., the state of thorax abdomen, muscle tonus degree etc.
2) body weight determination
1. assay method: adopt electronic balance to measure.
2. number of cases is measured: before administration, all buy animal, total Test animal after administration.
3. measure frequency: buy day and grouping day (before administration) is respectively surveyed once, during administration and convalescent period claim weekly a body weight, in addition, carrying out when dissected and first measuring the body weight of animal.
3) food ration measures
1. assay method: often group altogether supply deduct surplus altogether and be and often organize food ration every day, often organize every day food ration and be divided by every treated animal number and often organize every animal food ration every day.
2. measure number of times: measure once before administration, measure weekly once with convalescent period during administration.
3) hematology and blood biochemical analysis detect
1. period is detected: terminate each detection once in administration 3 months, 6 months (administration terminates) and convalescent period.
2. number of cases is detected: administration 3 months, administration 6 months (administration terminates) and convalescent period terminate each group and detect 10 respectively.
3. blood-sampling method: Rat Fast more than 14 hours before blood sampling, urethane 0.1g/100g body weight intraperitoneal injection of anesthesia, special blood-drawing pipe and blood taking needle are taken a blood sample from ventral aorta.Peripheral blood cell counts project and method are in table 24.
Table 24 peripheral blood cell counts project and method
Project Anticoagulant Algoscopy Use instrument
Red blood cell count(RBC) (RBC) EDTA-K2 Electric-resistivity method Animal blood analyser
Hemoglobin (HGB) EDTA-K2 Photoelectric colorimetry Animal blood analyser
Hematid specific volume (HCT) EDTA-K2 Histogram calculation method Animal blood analyser
Mean corpuscular volume (MCV) (MCV) EDTA-K2 Histogram calculation method Animal blood analyser
Average hemoglobin amount (MCH) EDTA-K2 Histogram calculation method Animal blood analyser
Mean corpuscular hemoglobin concentration (MCHC) EDTA-K2 Histogram calculation method Animal blood analyser
Numeration of leukocyte (WBC) EDTA-K2 Electric-resistivity method Animal blood analyser
Neutrophilic granulocyte percentage ratio (GRAN%) EDTA-K2 Histogram calculation method Animal blood analyser
Cent lymphocytes (LYMPH%) EDTA-K2 Histogram calculation method Animal blood analyser
Mononuclear cell percentage ratio (MONO%) EDTA-K2 Histogram calculation method Animal blood analyser
Platelet count (PLT) EDTA-K2 Histogram calculation method Animal blood analyser
Note: when find on hemopoietic system have affect time, the inspection of bone marrow should be carried out further.
4. biochemistry detection blood sample treatments: after ventral aorta blood sampling, room temperature leaves standstill 1 hours, centrifugal 3000rpm, 10min, separation of serum.Serum, as do not detected the same day, is put into cryopreservation tube by serum, and-80 DEG C frozen.Concrete test item and method are see current edition novel technique guideline.Concrete test item and method are in table 25.
Table 25 blood parameters test item and method
Project Algoscopy Use instrument
Glutamate pyruvate transaminase (ALT) Continuous monitoring method Automatic clinical chemistry analyzer
Glutamic oxaloacetic transaminase, GOT (AST) Continuous monitoring method Automatic clinical chemistry analyzer
Alkali phosphatase (ALP) Continuous monitoring method Automatic clinical chemistry analyzer
Creatine phosphokinase (CK) Continuous monitoring method Automatic clinical chemistry analyzer
Blood urea nitrogen (BUN) Dynamic method Automatic clinical chemistry analyzer
Creatinine (CREA) Dynamic method Automatic clinical chemistry analyzer
Total protein (TP) End-point method Automatic clinical chemistry analyzer
Albumin (ALB) End-point method Automatic clinical chemistry analyzer
Globulin (GIB) End-point method Automatic clinical chemistry analyzer
Blood glucose (GLU) End-point method Automatic clinical chemistry analyzer
Total bilirubin (TBIL) End-point method Automatic clinical chemistry analyzer
T-CHOL (CHOL) End-point method Automatic clinical chemistry analyzer
Triglyceride (TG) End-point method Automatic clinical chemistry analyzer
Potassium ion (K +) concentration Electrode method Electrolyte analyser
Sodium ion (Na +) concentration Electrode method Electrolyte analyser
Chloride ion (Cl -) concentration Electrode method Electrolyte analyser
5) process of death or moribund animals
1. processing requirements: find as early as possible, dissects in time, seeks reason as possible.
2. moribund animals: record dying time, body weight, symptom.
3. dead animal: the time of the dead discovery of record, body weight, postmortem finding of naked eye.
4. biochemical analysis: moribund animals is got blood and carried out hematology and blood biochemistry detection before putting to death.
5. pathologic finding: except confirming animal dead caused by gavage, other dying and dead animal all will carry out histopathologic examination.
6) dissect
1. anatomic method: anaesthetize with urethane 0.1g/100g body weight lumbar injection.Before cuing open inspection, the situation of each animal is checked comprehensively; When dissected carries out one by one by system, observes between internal organs color and luster, form, position relationship and internal organs with or without being adhered; Organ surface and digestive tube inner membrance are with or without congestion, hyperemia, petechia, edema, ulcer; Thoracic cavity, abdominal cavity, pericardial cavity with or without pathological changes such as hydrops, as noted abnormalities, its position of accurate recording and pathological change type in the remarks column of dissecting finding record.
2. between when dissected: same to detection time
3. number of cases is dissected: administration 3 months, administration 6 months (administration terminates) and 1 month convalescent period terminate each group and dissect 10 respectively.
7) organ weights measures
Assay method: adopt electronic balance to weigh the absolute weight (simultaneously calculating organ coefficient) of 13 internal organs (following table) respectively.
1. period is measured: when dissected measures
2. number of cases is measured: number of cases is identical with dissecting
3. internal organs table:
Sequence number 1 2 3 4 5 6 7
Internal organs Brain (brain, cerebellum) Heart Liver Spleen Lungs Kidney Adrenal gland
Internal organs Thymus Uterus Ovary Testis Epididymis Prostate
8) histopathologic examination
All animals all takes out required organs and tissues by current edition novel technique guideline, and abnormal tissue and internal organs are thought in perusal.Specimen to be fixed in 10% formalin fixative one week, again fixes 12 hours after repairing block, and tissue (the first decalcification of skeletal tissue) is with gradient alcohol dehydration, dimethylbenzene is transparent, paraffin embedding, and paraffin slicing machine is cut into slices, H.E. dye, neutral gum sealing, light microscopy checking.The kind of record pathological changes and degree.As pathological change occurs a certain tissue, other this tissue of dosage treated animal also should carry out histopathological examination to determine dose-response relationship.
Observe organs and tissues table:
Experimental result
1) ordinary circumstance: each treated animal is during administration and surrounding convalescent period, and outward appearance sign, behavioral activity are normal, urine, excrement character are without difference, and diet is normal, and each treated animal body weight increase and decrease is without significant difference, and group difference is not obvious.Food-intake is in table 26, table 27 and table 28.29 be the results are shown in Table on the impact of body weight, table 30 and table 31.
Table 26 medication 3 months rats eating amounts (g/ days /)
Table 27 medication 3 ~ 6 months rats eating amounts (g/ days /)
The change of table 28 convalescent period rats eating amount (g/ days/only)
2) hematological indices check result
Rat administration after 90 days and 180 days 24 hours, each group each 10 of rat, ventral aorta was taken a blood sample, and collects anticoagulated whole blood, detects hematology's indices with blood counting instrument.After administration 24 hours the last time, 10 rats that each group rat retains after living and killing 10 stopped administrations, normally raise, continue to observe surrounding more all work kill.With under the identical condition of situation during medication 6 months, the index of detection is also identical.
Administration after 90 days result show: compare with blank group, high dose group Erythrocytes (RBC) significantly reduces, and mean corpuscular volume (MCV) enlarges markedly, and mononuclear cell percentage ratio (MONO%) significantly declines; Low dose group cent lymphocytes (LYMPH%) significantly declines.All the other indexs: as packed cell volume (HCT), average hemoglobin amount (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), hemoglobin (HGB) content, total white blood cells (wBC) and neutrophil cell percentage ratio (GRAN%) differential counting thereof, each administration group compares with blank group, without significant difference, the results are shown in Table 32 and table 33.
The table 32 medication change to rat serum routine in 3 months ( )
Group Matched group High dose group Middle dosage group Low dose group
Number of animals (only) 10 10 10 10
WBC(×10 9／L) 11.64+2.36 13.32±4.93 13.09±3.86 9.66±1.89
RBC(×10 12／L) 8.68±1.03 7.39±1.09* 8.65±1.03 8.48±1.42
HGB(g／L) 152.4±13.27 135.7+21.68 147.0±13.97 146.1±17.22
HCT(％) 42.12±3.99 37.66±5.50 41.62±4.84 40.71±6.96
MCV(fL) 48.51±2.81 51.66±2.20* 48.14±1.89 48.60±1.90
MCH(pg) 17.96±0.69 18.19±0.94 17.26±0.86 17.99±1.34
MCHC(g／L) 361.06±14.20 357.93±19.46 355.30±18.67 361.84±26.57
PLT(×10 9／L) 545.42±59.05 570.24±111.99 564.57±72.24 542.71±84.01
Note: compare with blank group, * P<0.05.
The table 33 medication change to rat WBC differential counting (%) in 3 months
Group Matched group High dose group Middle dosage group Low dose group
Number of animals (only) 10 10 10 10
GRAN％ 19.10±9.01 29.21±20.40 23.83±12.43 23.44±13.07
LYMPH％ 58.77±10.22 48.10±21.83 47.81±18.79 40.78±21.86*
MONO％ 219±1.66 12.3±6.08*** 18.2±7.61 16.3±9.07
Note: compare with blank group, * P<0.05; * * P<0.001.
Administration after 180 days result show: compare with blank group, high and low dose group leukocyte (WBC) sum obviously reduces (P<0.05), and wherein high dose group reduces the most obvious; In, low dose group mononuclear cell percentage ratio (MONO%) significantly raises; High, medium and low dosage group erythrocyte (RBC) sum, hemoglobin (HGB) content and packed cell volume (HCT) significantly reduce, wherein: in, low dose group reduces the most obvious; Middle dosage group average hemoglobin amount (MCH) and mean corpuscular hemoglobin concentration (MCHC) obviously raise; All the other indexs: as mean corpuscular volume (MCV), platelet count (PLT), total white blood cells (WBC) and differential counting [cent lymphocytes (LYMPH%) and neutrophil cell percentage ratio (GRAN%)] thereof, each administration group compares with blank group, no significant difference between group, the results are shown in Table 34 and table 35.
The table 34 medication change to rat serum routine in 6 months
Group Matched group High dose group Middle dosage group Low dose group
Number of animals (only) 10 10 10 10
WBC(×10 9／L) 14.68±2.64 8.89±2.79*** 12.49±4.03 10.79±3.60*
RBC(×10 12／L) 10.69+1.44 8.52+1.67** 8.45±0.69*** 8.03±0.79***
HGB(g／L) 176.3±14.71 97.6±68.78** 152.6±6.93*** 126.6±25.79***
HCT(％) 52.56±4.50 42.22±8.03** 40.85±3.64* 39.80±2.38***
MCV(fL) 49.31+2.62 49.86±2.35 48.75±2.85 49.40±3.59
MCH(pg) 16.831.48± 13.42±6.17 18.16+1.33 15.85±3.11
MCHC(g／L) 339.48+139.44 266.61+121.37 377.36+19.65*** 319.16±62.52
PLT(×10 9／L) 843.98+141.98 726.90+135.23 854.69±110.71 813.79±150.26
Note: compare with blank group, * P<0.05; * P<0.01; * * P<0.001.
The table 35 medication change to rat wBC differential counting (%) in 6 months
Group Matched group High dose group Middle dosage group Low dose group
Number of animals (only) 10 10 10 10
GRAN％ 22.45±10.71 16.20±12.07 22.28±7.69 21.39±11.22
LYMPH％ 62.04±17.01 61.13±23.89 61.86±8.47 63.62±11.42
MONO％ 12.09±2.31 12.52±5.41 15.75±3.03* 15.31±2.75*
Note: compare with blank group, * P<0.05.
Surrounding convalescent period result shows: each administration treated animal hematological indices compares with blank group, and no significant difference between group, the results are shown in Table 36 and table 37.
The change of table 36 convalescent period rat serum routine
Group Matched group High dose group Middle dosage group Low dose group
Number of animals (only) 10 10 10 10
WBC(×10 9／L) 14.23±4.91 10.16±4.16* 13.12±3.98 11.98±2.88
RBC(×10 12／L) 9.03±1.25 8.58±0.58 8.77±0.70 8.49±1.15
HGB(g／L) 164.1+15.03 156.8±6.85 159.4±7.33 161.0±15.70
HCT(％) 43.47±4.46 41.82±2.15 42.41±2.52 41.70±4.62
MCV(fL) 48.24+4.26 49.41±3.12 48.76±1.93 49.34±3.18
MCH(pg) 18.21+1.20 18.46±1.36 18.29±0.83 19.05+1.62
MCHC(g／L) 378.25+10.18 374.21±11.29 375.16+9.91 385.83+12.55
PLT(×10 9／L) 820.98±86.06 776.76±145.18 819.49±165.06 794.65±120.80
Note: compare with blank group, * P<0.05.
The change of table 37 convalescent period rat WBC differential counting (%)
Group Matched group High dose group Middle dosage group Low dose group
Number of animals (only) 10 10 10 10
GRAN％ 26.67±12.33 31.85+10.73 22.06±9.12 23.21+10.34
LYMPH％ 61.45±11.32 55.76±11.69 65.46±8.80 63.62±9.50
MONO％ 12.19±3.45 12.32±1.54 12.38±1.14 13.00±1.60
3) blood parameters testing result
Through rat administration above-mentioned each group of 10 rats after 90 days and 180 days of getting anticoagulated whole blood, continue to collect blood, separation of serum, with automatic clinical chemistry analyzer, detects every biochemical indicator.After administration 24 hours the last time, 10 rats that each group rat retains after living and killing 10 stopped administrations, normally raise, continue to observe surrounding more all work kill.With under the identical condition of situation during medication 6 months, the index of detection is also identical.
Administration after 90 days result show: compare with blank group, in, low dose group total protein (TP) content significantly raises, wherein: low dose group raises obviously; Low dose group albumin content (ALB) significantly raises; High, medium and low dosage Histaglobin (GIB) content significantly raises, wherein: low dose group raises the most obvious; High, middle dosage group sodium ion (Na +) concentration significantly raises; High dose potassium ion (K +) concentration significantly raises.All the other indexs: as glutamate pyruvate transaminase (ALT), glutamic oxaloacetic transaminase, GOT (AST), blood urea nitrogen (BUN), creatinine (CREA), blood glucose (GLU), total bilirubin (TBIL), T-CHOL (CHOL), triglyceride (TG), chloride ion (Cl -) to compare group difference not obvious for concentration each administration group and blank group, do not make significant difference, the results are shown in Table 38.
The table 38 medication impact on rat blood biochemical indicator in 3 months
Group Matched group High dose group Middle dosage group Low dose group
Number of animals (only) 10 10 10 10
ALT(U／L) 80.28±53.80 70.57±18.58 57.59±16.36 67.25±18.95
AST(U／L) 218.05±143.76 148.54±36.16 151.80+16.38 174.36±38.31
ALP(U／L) 116.62±46.48 186.02±52.14 128.06±64.60 150.59±61.07
CK(U／L) 724.14±521.28 420.93±127.30 753.74±751.61 835.02+623.75
TP(g／L) 74.38±4.05 80.14±9.00 80.27±5.59* 84.47±6.41***
ALB(g／L) 32.63±2.28 32.27+5.54 34.21±2.35 35.01±1.62*
GIB(g／L) 41.32±2.76 48.04±8.91* 45.96±4.98* 49.31±5.64***
BUN(mmol／L) 6.40±1.25 7.70±2.49 7.21±1.42 6.55±1.64
CREA(μmol／L) 39.01±3.90 37.25±6.75 35.54±3.82 39.74±6.08
GLU(mmol／L) 5.16±0.70 4.46±0.98 4.63±0.87 5.07±0.69
TG(mmol／L) 0.57±0.20 0.89±0.49 0.78±0.82 0.68±0.21
CHOL(mmol／L) 1.72±0.33 1.69±0.37 1.91±0.33 1.87±0.22
TBIL(μmol／L) 0.61±0.34 0.63±0.36 0.67±0.32 0.47±0.21
K +(mmol／L) 4.54±0.45 5.80±0.58 5.05±1.00 5.41±1.35
Na +(mmol／L) 142.72±1.77 145.40±2.44* 145.63±4.67* 145.07±4.87
Cl -(mmol/L) 101.46±1.90 101.27±2.56 101.66±2.67 101.90±2.32
Note: compare with blank group, * P<0.05; * * P<0.001.
Administration after 180 days result show: compare with blank group, in, low dosage aspartate amino transferase (GOT), creatinine (CREA) content significantly reduces, wherein: low dose group reduces the most obvious; In, low dose group total protein (TP) content significantly raises, in, low dose group albumin content (ALB) significantly raises; High and low dose group sodium ion (Na +), chloride ion (Cl -) concentration significantly raises, wherein: low dose group raises the most obvious; All the other indexs: as alanine aminotransferase (ALT), alkali phosphatase (ALP), blood urea nitrogen (BUN), blood glucose (GLU), total bilirubin (TBIL), T-CHOL (CHOL), that triglyceride (TG) each administration group and blank group compare group difference is not obvious, do not make significant difference, the results are shown in Table 39.
The table 39 medication impact on rat blood biochemical indicator in 6 months
Group Matched group High dose group Middle dosage group Low dose group
Number of animals (only) 10 10 10 10
GPT(U／L) 111.64±86.90 100.52±42.18 83.31±35.09 72.84±13.47
GOT(U／L) 278.32±124.50 243.13±62.91 168.35±36.27 161.57±28.20**
AKP(U／L) 214.63±105.46 197.17±92.75 238.12±112.01 265.06±109.57
CK(U／L) 2009.26±1301.80 1694.03±941.99 1449.29±712.35 1163.93±402.47
BUN(mmol／L) 6.59±1.12 5.90±1.47 5.71±0.38 5.59±1.20
CR(μmol／L) 28.23±10.11 36.93±23.75 15.79±6.57** 18.86±8.19*
GLU(mmol／L) 10.16±5.32 10.81±3.99 5.29±0.52 5.09±0.50
TPR(g／L) 67.87±4.06 63.61±4.11 81.14±6.84 77.24±4.27***
ALB(g／L) 31.86±2.50 31.90±10.52 36.15±3.56 35.30±1.97**
TBIL(μmol／L) 0.88±0.59 0.53±0.99 1.24±0.63 1.10±0.36
CHO(mmol／L) 1.36±0.37 1.30±0.12 1.74±0.48 1.53±0.36
TG(mmol／L) 1.09±0.34 1.05±0.44 1.22±0.55 0.81±0.22
K +(mmol／L) 5.17±0.68 4.92±0.15 5.03±0.48 4.67±0.31
Na +(mmol／L) 137.12±1.73 140.95±2.19* 139.00±1.90 141.07±3.68**
Cl -(mmol／L) 99.62±1.50 100.06±2.52* 99.35±1.74 101.59±3.05**
Note: compare with sky day matched group, * P < 0.05; * P<0.01; * * P<0.001.
Surrounding convalescent period result shows: compare with blank group, and middle dosage group alkali phosphatase (ALP) content significantly reduces; High and low dose group total protein (TP) content significantly raises; High and low dose group albumin (ALB) content significantly raises; High and low dose Histaglobin (GIB) content significantly raises, wherein: low dose group raises the most obvious; High dose group sodium ion (Na +) concentration significantly raises; All the other indexs: as glutamate pyruvate transaminase (ALT), glutamic oxaloacetic transaminase, GOT (AST), blood urea nitrogen (BUN), creatinine (CREA), creatine phosphokinase (CK), blood glucose (GLU), total bilirubin (TBIL), T-CHOL (CHOL), triglyceride (TG), potassium ion (K +), chloride ion (Cl -) to compare group difference not obvious for concentration each administration group and blank group, do not make significant difference, the results are shown in Table 40.
The change of table 40 convalescent period rat biochemical indicator
Group Matched group High dose group Middle dosage group Low dose group
Number of animals (only) 10 10 10 10
ALT(U／L) 88.76±49.77 67.39±15.01 112.04±10.90 63.56±11.56
AST(U／L) 149.20±34.32 133.12±19.51 208.04±80.71 142.44±37.86
ALP(U／L) 211.26±82.10 184.47±35.68 128.97±29.53** 248.77±99.16
CK(U／L) 635.44±196.80 873.58±292.11 838.30±305.35 926.53±729.45
TP(g／L) 70.89±3.87 76.39±2.47*** 67.29±7.76 80.51±3.17
ALB(g／L) 31.23±1.78 33.69±2.03*** 30.96±3.34 34.66±2.57*
GIB(g／L) 38.70±3.05 42.48±2.72* 37.04±5.37 45.68±3.97***
BUN(mmol／L) 7.69+1.32 7.63±0.96 6.71±1.45 7.29±0.96
CREA(μmol／L) 29.61±3.96 28.78±4.71 30.86±3.73 33.68±5.86
GLU(mmol／L) 6.23±1.14 5.09±0.30 6.43±1.51 5.38±0.76
TG(mmol／L) 0.70±0.26 0.98±0.48 0.73±0.41 1.00±0.47
CHOL(mmol／L) 1.40±0.38 1.44±0.27 1.47±0.35 1.67±0.30
TBIL(μmol／L) 0.23±0.11 0.28±0.18 0.25±0.37 0.34±0.20
K +(mmol／L) 5.74±0.93 6.07±0.57 5.44±0.75 6.41±0.55
Na +(mmol／L) 142.75+2.07 145.19±0.86* 140.52±1.26 145.97±3.29
Cl -(mmol／L) 101.06±1.13 102.05±1.20 99.51±1.87 101.24±2.16
Note: compare with blank group, * P<0.05; * * P<0.001.
4) system postmortem and histopathologic examination's result
Administration after 90 days result show: each administration group organ coefficient is substantially close with blank group, illustrates that medicine has no significant effect organ weights, in table 41.The internal organs histopathology microscopy of blank group and each administration group rat, by the statistical result of sxemiquantitative scoring.Result shows: respectively organize minority example except distilled water matched group and medication and see have the slight heart, lung, renal interstitial, little enteremia regulating liver-QI steatosis, and the spleen congestive enlargement of minority example, lymph node chronic inflammation, all do not find the pathological changes relevant with drug toxicity, and each internal organs of the animal changed are dispersed in each group, do not have statistical significance.Result of the test is in table 42.
Table 41 medication 3 months is on the impact on Rats Organs and Tissues coefficient
Group Matched group High dose group Middle dosage group Low dose group
Number of animals (only) 10 10 10 10
Heart percentage of liveweight percent (%) 0.38±0.05 0.36±0.10 0.36±0.06 0.35±0.07
Liver percentage of liveweight percent (%) 3.24±0.61 2.94±0.59 3.35±0.74 3.04±0.44
Spleen percentage of liveweight percent (%) 0.16±0.10 0.19±0.03 0.19±0.05 0.18±0.02
Lung percentage of liveweight percent (%) 0.61+0.29 0.59±0.13 0.58±0.08 0.61±0.18
Kidney percentage of liveweight percent (%) 0.32±0.04 0.30±0.06 0.27±0.07 0.25±0.05
Adrenal gland's percentage of liveweight percent (%) 0.0132±0.0053 0.0116±0.0051 0.0116±0.0033 0.0128±0.0028
Thymus percentage of liveweight percent (%) 0.0719±0.0513 0.0686±0.0330 0.0845±0.0206 0.0776±0.0277
Brain percentage of liveweight percent (%) 0.6745±0.0917 0.6742±0.1307 0.6772±0.1230 0.6932±0.0978
Testis percentage of liveweight percent (%) 0.47±0.09 0.40±0.15 0.42±0.12 0.43±0.05
Epididymis percentage of liveweight percent (%) 0.23±0.07 0.23±0.02 0.23±0.03 0.21±0.03
Prostate percentage of liveweight percent (%) 0.15±0.04 0.14±0.04 0.13±0.04 0.14±0.03
Uterus percentage of liveweight percent (%) 0.32±0.070 0.30±0.100 0.30±0.128 0.27±0.150
Ovary percentage of liveweight percent (%) 0.031±0.016 0.032±0.014 0.033±0.011 0.031±0.014
Table 42 administration 3 months rat pathological examination results
Note: in table: "+" represents that internal organs have Minimal change; "-" represents that internal organs do not have obvious pathological change; Numeral (1 ~ 10) represents the number of elements of animal.
Administration after 180 days result show: organ coefficient and the blank group of three dosage groups are basically identical, illustrate that medicine has no significant effect organ weights, in table 43.The internal organs histopathology microscopy of blank group and each administration group rat, by the statistical result of sxemiquantitative scoring.Result shows: respectively organize minority example except distilled water matched group and medication and see have the slight heart, liver, bladder, lung and the congestive enlargement of minority example spleen, lymph node chronic inflammation, all do not find the pathological changes relevant with drug toxicity, and each internal organs of the animal changed are dispersed in each group, do not have statistical significance.Experimental result is in table 44.
Table 43 medication 6 months is on the impact on Rats Organs and Tissues coefficient
Group Matched group High dose group Middle dosage group Low dose group
Number of animals (only) 10 10 10 10
Heart percentage of liveweight percent (%) 0.40±0.07 0.37±0.05 0.37±0.09 0.36±0.06
Liver percentage of liveweight percent (%) 3.24±0.59 3.34+0.55 3.24+0.71 3.22±0.50
Spleen percentage of liveweight percent (%) 0.16±0.06 0.15±0.04 0.15±0.02 0.18±0.06
Lung percentage of liveweight percent (%) 0.48±0.07 0.48±0.06 0.49±0.08 0.54±0.08
Kidney percentage of liveweight percent (%) 0.29±0.05 0.30±0.06 0.29±0.04 0.28±0.04
Adrenal gland's percentage of liveweight percent (%) 0.0124±0.0046 0.0126±0.0051 0.0108±0.0047 0.0119±0.0058
Thymus percentage of liveweight percent (%) 0.0948±0.0336 0.0928±0.0300 0.0907±0.0276 0.1016±0.0412
Brain percentage of liveweight percent (%) 0.5835±0.0918 0.5920±0.0490 0.5706±0.0796 0.5841±0.0984
Testis percentage of liveweight percent (%) 0.50±0.09 0.51±0.12 0.46±0.05 0.52±0.10
Epididymis percentage of liveweight percent (%) 0.16+0.06 0.17±0.03 0.12±0.01 0.19+0.01
Prostate percentage of liveweight percent (%) 0.16±0.03 0.16±0.03 0.13±0.06 0.15±0.03
Uterus percentage of liveweight percent (%) 0.21±0.04 0.18±0.08 0.21±0.01 0.22±0.11
Ovary percentage of liveweight percent (%) 0.02±0.05 0.02±0.01 0.02±0.01 0.03±0.00
Table 44 administration 6 months rat pathological examination results
Note: in table: "+" represents that internal organs have Minimal change; "-" represents that internal organs do not have obvious pathological change; Number (1 ~ 10) represents the number of elements of animal.
Surrounding convalescent period result shows: carry out comprehensively gross necropsy meticulously to internal organs, no abnormal organ-tissue.The organ coefficient of 10 organs and tissues such as each treated animal internal organs heart, liver, spleen, lung, kidney, adrenal gland, uterus, brain, ovary, thymus is substantially close with matched group, has no significant effect, in table 45 to organ weights.The internal organs histopathology microscopy of blank group and each administration group rat, by the statistical result of sxemiquantitative scoring.Result shows: respectively organize minority example except distilled water matched group and medication and see have the slight heart, small intestinal, liver, kidney, lung and the congestive enlargement of minority example spleen, lymph node, stomach, large intestine chronic inflammation, all do not find the pathological changes relevant with drug toxicity, and each internal organs of the animal changed are dispersed in each group, do not have statistical significance.Experimental result is in table 46.
The present composition is with 10 times of clinical plan dosage (0.45g/kg), 30 times of clinical plan dosage (1.35g/kg), 60 times of clinical plan dosage (2.7g/kg), to the continuous gavage of rat 180 days, and carry out experiment mid-term (administration 90 days) and 1 month convalescent period laboratory observation.Result is:
(1) rat growthing development and general status are not had a significant effect, only have some impact when the medicine feed initial stage, but recovered normal after 2 weeks.
(2) on the impact of rat blood index
The change of table 45 convalescent period Rats Organs and Tissues weight
Group Matched group High dose group Middle dosage group Low dose group
Number of animals (only) 10 10 10 10
Heart percentage of liveweight percent (%) 0.33±0.07 0.34±0.03 0.35±0.06 0.34±0.04
Liver percentage of liveweight percent (%) 3.07±0.90 3.21±0.50 3.05±0.58 3.18±0.43
Spleen percentage of liveweight percent (%) 0.16±0.05 0.19±0.04 0.20±0.08 0.17±0.07
Lung percentage of liveweight percent (%) 0.54±0.07 0.54±0.10 0.53±0.12 0.53±0.07
Kidney percentage of liveweight percent (%) 0.30±0.05 0.33±0.05 0.28±0.05 0.30±0.05
Adrenal gland's percentage of liveweight percent (%) 0.0109±0.0034 0.0095±0.0031 0.0102±0.0029 0.0107±0.0033
Thymus percentage of liveweight percent (%) 0.0721±0.0324 0.0760±0.0159 0.0772±0.0220 0.0697±0.0417
Brain percentage of liveweight percent (%) 0.5509+0.0543 0.5728±0.0483 0.5676±0.0586 0.5571±0.0718
Testis percentage of liveweight percent (%) 0.4208±0.05 0.3937±0.02 0.3981±0.15 0.4274±0.09
Epididymis percentage of liveweight percent (%) 0.1640±0.09 0.1424±0.04 0.1636±0.02 0.1522±0.04
Prostate percentage of liveweight percent (%) 0.1452±0.05 0.1347±0.06 0.1602±0.07 0.1381±0.09
Uterus percentage of liveweight percent (%) 0.3619±0.05 0.3903±0.05 0.3531±0.09 0.3378±0.09
Ovary percentage of liveweight percent (%) 0.0219±0.016 0.0266±0.017 0.0243±0.004 0.0195±0.010
Table 46 rat long term toxication convalescent period puts to death animal pathological examination results
Note: in table: "+" represents that internal organs have Minimal change; "-" represents that internal organs do not have obvious disease to bury change; The number of elements of numeral (1 ~ 10) table toy.
Successive administration 3 months, compares with blank group, and the present composition all has appreciable impact to erythrocyte, leukocyte in rat blood.Wherein: high dose group obviously can reduce mononuclear cell percentage ratio (MONO%) content in Rat Erythrocytes and leukocyte, can enlarge markedly Rat Erythrocytes average external volume (MCV); Low dose group significantly reduces the content of rat leukocyte medium-sized lymphocyte percentage ratio (LYMPH%).
Successive administration 6 months, compares with blank group, and the present composition all has appreciable impact to erythrocyte, leukocyte, hemoglobin in rat blood.Wherein: leukocyte in rat blood (wBC) content obviously can fall in high and low dose group; In, low dose group can the remarkable content of mononuclear cell percentage ratio (MONO%) in leukocyte increasing; High, medium and low dosage group all significantly can reduce erythrocyte (RBC), hemoglobin (HGB) content and packed cell volume (HCT);
After drug withdrawal January, whole blood index all recovers normal.
(3) on the impact of rat blood biochemical indicator
Successive administration 3 months, compares with blank group, and the present composition affects more obvious on albumen in rat blood and electrolytical content.Wherein: in, low dose group significantly can raise total protein in rat blood (TPR) content; Low dose group energy significantly raising albumin content (ALB); High, medium and low dosage group all significantly raises globulin (GIB) content; The remarkable increasing of Na ion of high, middle dosage group (Na +) concentration and high dose group can also significantly raise potassium ion (K +) concentration.
Successive administration 6 months, compare with blank group, the present composition, except on except albumen in rat blood and the impact comparatively obviously of electrolytical content, also affects larger on aspartate amino transferase (GOT) and creatinine (CREA) content.Wherein: in, low dose group significantly can raise total protein in rat blood (TP), albumin content (ALB) content; High and low dose group can remarkable increasing of Na ion (Na +), chloride ion (Cl -) concentration.In, low dose group significantly can reduce aspartate amino transferase (GOT), creatinine (CREA) content.
Drug withdrawal is after 1 month, and rat blood Mid-Heaven Gate winter histidine amino group transferring enzyme (GOT), creatinine (CREA) recover normal; In high and low dose group, total protein (TPR), albumin (ALB) and globulin (GIB) content still significantly raise; Middle dosage group significantly can reduce the content of alkali phosphatase (ALP); High dose group sodium ion (Na +) concentration still significantly raises.
(4) have no significant effect Rats Organs and Tissues weight (heart, liver, spleen, lung, kidney, adrenal gland, thymus, uterus, ovary, testis, epididymis, prostate and brain be totally 13 internal organs), each group organ coefficient is substantially close.To organs and tissues (except above-mentioned 13 internal organs, also have pancreas, stomach, ileum, colon, hypophysis cerebri, spinal cord, bone marrow, lymph node, bladder, sciatic nerve, thyroid, parathyroid gland, aorta, submaxillary gland, aorta) histopathologic examination, do not find significantly relevant with drug toxicity pathomorphology change.

Claims (12)

1. treat a herbal mixture for cardiovascular and cerebrovascular disease, it is characterized in that: this Chinese medicine is made up of the raw material of Chinese medicine comprising following composition and parts by weight:
Folium Crataegi 1 ~ 100 part of Rhizoma Polygonati 1 ~ 100 part.
2. a kind of herbal mixture for the treatment of cardiovascular and cerebrovascular disease according to claim 1, is characterized in that, the active raw materials of this Chinese medicine is primarily of Folium Crataegi and Rhizoma Polygonati composition.
3. a kind of herbal mixture for the treatment of cardiovascular and cerebrovascular disease according to claim 1 and 2, is characterized in that, the active raw materials of this Chinese medicine is made up of Folium Crataegi and Rhizoma Polygonati, and the parts by weight of this raw material of Chinese medicine are:
Folium Crataegi 1 ~ 10 part of Rhizoma Polygonati 1 ~ 5 part.
4. a kind of herbal mixture for the treatment of cardiovascular and cerebrovascular disease according to claim 3, is characterized in that, the parts by weight of raw material of Chinese medicine are:
Folium Crataegi 5 parts of Rhizoma Polygonatis 1 part.
5. a kind of herbal mixture for the treatment of cardiovascular and cerebrovascular disease according to claim 4, is characterized in that: described dosage form be any one clinically or pharmaceutically acceptable dosage form.
6. a kind of herbal mixture for the treatment of cardiovascular and cerebrovascular disease according to claim 5, is characterized in that: described dosage form is drop pill, tablet, capsule, granule, soft capsule, oral liquid or injection.
7. a kind of preparation method for the treatment of the herbal mixture of cardiovascular and cerebrovascular disease according to claim 6, is characterized in that comprising the following steps:
With water or any solvent that other is suitable for carrying out traditional Chinese medicine extyaction, each of Folium Crataegi and Rhizoma Polygonati is carried out separately to the extracts active ingredients of one or many;
Or the extracts active ingredients of one or many is carried out with water or any solvent that other is suitable for the carrying out traditional Chinese medicine extyaction mixture to Folium Crataegi and Rhizoma Polygonati.
8. a kind of preparation method for the treatment of the herbal mixture of heart disease according to claim 7, is characterized in that:
(1) Folium Crataegi is taken by proportional quantity, after 0.1 ~ 10 hour solvent soaking time by Folium Crataegi weight 2 times ~ 40 times, extract 0.1 ~ 10 hour under the condition of 30 DEG C ~ 100 DEG C, described solvent soaking and extraction operation can be carried out 1 time or repeat nearly 6 times, merge extractive liquid, distillating recovering solvent, distillate is centrifugal, with the solvent wash of Folium Crataegi medical material amount 0.1 times ~ 100 times of volumes precipitation once or repeated washing reach five times, merge centrifugal after supernatant and the supernatant of washing precipitation, distillation and concentration near dry after, dry, pulverize, to obtain final product; Described solvent is that water or any other are suitable for carrying out the solvent of traditional Chinese medicine extyaction.
(2) Rhizoma Polygonati is taken by proportional quantity, under 50 DEG C ~ 100 DEG C conditions, first time adds Rhizoma Polygonati weight 2 times ~ 40 times solvent extractions 0.1 ~ 10 hour, leaching process can carry out once or nearly 5 times, united extraction gained decocting liquid, and distillation and concentration becomes thick paste, dry, pulverize, obtain Rhizoma Polygonati extract; Described solvent is that water or any other are suitable for carrying out the solvent of traditional Chinese medicine extyaction;
(3) after the Rhizoma Polygonati extract mix homogeneously that the Folium Crataegi extract obtained according to above-mentioned steps (1) and above-mentioned steps (2) obtain, add suitable additives, make injection, oral solid formulation or oral liquid, described oral solid formulation is tablet, capsule, granule, soft capsule, described oral liquid is oral liquid or syrup, and described additives are one or more in filler, excipient, correctives or antiseptic.
9. a kind of preparation method for the treatment of the herbal mixture of cardiovascular and cerebrovascular disease according to claim 8, is characterized in that: step (2) is undertaken by following:
Take Rhizoma Polygonati by proportional quantity, under 50 DEG C ~ 100 DEG C conditions, first time adds the water extraction 0.1 ~ 10 hour of Rhizoma Polygonati weight 2 times ~ 40 times, and leaching process can carry out once or nearly 5 times, united extraction gained decocting liquid, filters and filtrate is concentrated 0.1 times ~ 20 times of Rhizoma Polygonati weight, adding ethanol and make alcohol content be 20% ~ 90%, static or centrifugal, get supernatant, filter, Distillation recovery ethanol, continue distillation and concentration and become thick paste, dry, pulverize, obtain Rhizoma Polygonati extract.
10. a kind of preparation method for the treatment of heart disease herbal mixture according to claim 8, is characterized in that:
(1) Folium Crataegi 5 parts is taken, after the 45% soak with ethanol time 2 h of 8 times for the first time by Folium Crataegi weight, extract 2 hours under the condition of 80 DEG C, 45% ethanol of 5 times of second time Folium Crataegi weight extracts 1 hour the condition of 80 DEG C, third time, Folium Crataegi weight 4 times amount 45% ethanol extracted 0.5 hour the condition of 80 DEG C, 0.5 hour is extracted the condition of 80 DEG C 4th time with Folium Crataegi weight 3 times amount 45% ethanol, merge extractive liquid, Distillation recovery ethanol, distillate centrifuge is centrifugal 30min under the rotating speed of 3000R per minute, with the purified water washing precipitation three times of medical material amount 2 times of volumes, merge centrifugal after supernatant and the supernatant of washing precipitation, distillation and concentration is near dry rear dry, pulverize, obtain.
(2) take Rhizoma Polygonati 1 part, under 100 DEG C of conditions, first time adds 10 times of soak by water 2 hours of Bulbus Allii Macrostemonis and Rhizoma Polygonati gross weight, and second time adds Bulbus Allii Macrostemonis and Rhizoma Polygonati gross weight 8 times of soak by water 1 hour, collecting decoction, filtrate is concentrated into medical material gross weight 1 times, adds ethanol and makes alcohol content be 55%, static or centrifugal, get supernatant, filter, Distillation recovery ethanol, continue distillation and concentration and become thick paste, dry, pulverize, to obtain final product.
(3) after the Rhizoma Polygonati extract mix homogeneously that Folium Crataegi extract step (1) obtained and step (2) obtain, add suitable additives, make 10 parts of oral solid formulations, soft capsule or oral liquids, described additives are: starch, lactose, sodium carboxymethyl cellulose, magnesium stearate, one or more in sucrose, stevioside, sodium benzoate, soybean oil or soybean phospholipid.
11. a kind of preparation methoies for the treatment of heart disease herbal mixture according to claim 8, is characterized in that:
(1) by Folium Crataegi extract that claim 8 step (1) obtains, after defat with petroleum ether by 1/2 amount, discard petroleum ether liquid, be extracted with ethyl acetate again, extract reclaim under reduced pressure ethyl acetate is also concentrated into dry, add suitable quantity of water and make dissolving, be added on polyamide column, first use the water elution of 2 times of column volumes, discard water lotion, then use 80% ethanol elution of 3 times of column volumes, collect eluent, reclaim ethanol and be concentrated into the concentrated solution of 60 DEG C of relative densities about 1.02 ~ 1.08, dry, obtain Folium Crataegi total flavones extract.
(2) by Rhizoma Polygonati extract that claim 8 step (2) obtains, the alcohol settling of amass with diploid 95%, repeatedly carry out 4 times, small-molecule substance is fallen with dialyzer dialysis, precipitate with ethanol again, finally use the absolute ether of equal volume and appropriate washing with acetone precipitate, dry coarse solomon's seal polysaccharide extract.
(3) after the coarse solomon's seal polysaccharide extract mix homogeneously that Folium Crataegi total flavones extract above-mentioned steps (1) obtained and above-mentioned steps (2) obtain, add suitable additives, make 10 parts of injectable powder, small-volume injection or bulk capacity injections.
A kind of preparation method for the treatment of heart disease herbal mixture described in 12. according to Claim 8 ~ 11, is characterized in that, when making injection, in order to increase its dissolubility, adds tween 80 solubilizing agent; Add excipient in injectable powder, described excipient is mannitol or glucose; The isoosmotic adjusting agent for regulating osmotic pressure is added in infusion preparation, described isoosmotic adjusting agent is one or more in sodium chloride, potassium chloride, magnesium chloride, calcium chloride, sodium lactate, glucose, xylitol, sorbitol and dextran, preferred sodium chloride or glucose.
CN201310464242.1A 2013-10-08 2013-10-08 Compound traditional Chinese medicine preparation for treating cardiovascular and cerebrovascular diseases Withdrawn CN104510902A (en)

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