CN102349931A - Cockroach extract, preparation method thereof, and application of cockroach extract preparation in treatment of cardiovascular disease - Google Patents
Cockroach extract, preparation method thereof, and application of cockroach extract preparation in treatment of cardiovascular disease Download PDFInfo
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Abstract
The invention provides a cockroach extract, a preparation method thereof, and application of a cockroach extract preparation in treatment of cardiovascular disease. The cockroach extract comprises the following components (based on dry weight): 20-40% of composite nucleoside base, 30-60% of associated amino acid, 2-4% of uracil, 3-6% of hypoxanthine and 5-10% of inosine. The preparation method comprises the following steps of: pulverizing dried cockroach into coarse powder, carrying out percolation or reflux extraction with 90-95% ethanol to obtain dense paste, carrying out oil-water separation, and removing precipitate and oil to obtain water-layer drug liquid; and adsorbing with activated carbon, carrying out secondary alcohol extraction and water extraction, decolorizing, carrying out ultrafiltration and concentrating to obtain the cockroach extract dense paste. Combined with proper pharmaceutical auxiliary materials, the cockroach extract can be prepared into aqueous injection, freeze-dried powder injection, capsules, granules, tablets, oral liquid, dropping pills and other various dosage forms, and the obtained preparation can be used for treating cardiovascular disease, particularly heart failure caused by pulmonary heart disease, cardiomyopathy, rheumatism, coronary disease, high-altitude heart disease and the like.
Description
Technical field
The invention belongs to biological extraction pharmaceutical technology field, further belong to cockroach and extract the pharmaceutical technology field, be specifically related to a kind of cockroach extractive and preparation method thereof and its application on the treatment cardiovascular diseases.
Background technology
Cockroach, having another name called periplaneta americana (being commonly called as Blatta seu periplaneta) is that volume is maximum in the Blattidae, becomes long 29-35 millimeters of polypide.The cockroach epidermis contains sclerotin and chitin; Elements such as bromine, zinc, nickel, manganese, potassium, calcium, titanium, chlorine, sulfur, silicon, aluminum, magnesium; The muscle hydrolysis can get 13 seed amino acids; The plain B1 of also little in store life, B2, nicotinic acid and ascorbic acid etc. in the health contain trehalose, trehalase, glycoprotein, inositol, protocatechuic acid Fructus Vitis viniferae glycoside etc. in the lymph; All contain ergothioneine, 1-methyl-2-pyridinium carboxylate, trigonelline, glycine, betanin, anus alkali, trimethylamine, adenine etc.Modern scientific research shows that cockroach extractive has very strong biological activity, but also there is significant limitation in prior art for the application of cockroach extractive, and the cockroach extractive application potential awaits further development and utilization.
Summary of the invention
First purpose of the present invention is the deficiency to prior art; A kind of cockroach extractive is provided; Second purpose is to provide the method for preparing of this cockroach extractive; The 3rd purpose is to provide the pharmaceutical preparation that contains this cockroach extractive, and the 4th purpose is to provide this to contain the application of the pharmaceutical preparation of cockroach extractive.
First purpose of the present invention is achieved in that described cockroach extractive, and it contains compound nucleoside base 20～40%, combination aminoacid 30～60%, uracil 2～4%, hypoxanthine 3～6% and the inosine 5～10% of weight ratio.
As preferred version: described cockroach extractive contains the compound nucleoside base of weight ratio 25～40%, and 35～60% combine aminoacid, 2.5～4% uracil, the inosine of 3.5～6% hypoxanthine and 6～10 %.
Described cockroach extractive contains the compound nucleoside base of weight ratio 22～36%, and 35～48% combine aminoacid, 2.5～3.5% uracil, 3.5～5% hypoxanthine and 6～8% inosine.
Described cockroach extractive contains the compound nucleoside base of weight ratio 24～38%, and 32～48% combine aminoacid, 2.0～3.2% uracil, 3.4～5% hypoxanthine and 7～10% inosine.
The present invention's second purpose is achieved in that the method for preparing of said cockroach extractive may further comprise the steps: the dried polypide of cockroach is ground into coarse powder, makes thick paste through 90～95% ethanol with percolation or reflux extraction; Add purified water in the thick paste and fully stir, precipitate and oils and fats are removed in oil-water separation, collect the water layer medicinal liquid; Activated carbon adsorption, upper prop separates, n-butyl alcohol eluting or phenol solution eluting; Eluent carries out the secondary ethanol extraction after concentrating, and the cold preservation after-filtration carries out water again after filtrating concentrates and carries; Centrifugal filtration gets the water extract after cold preservation, the water extract concentrate through decolouring, after the ultrafiltration the cockroach extractive thick paste.
Described No. 4 yellow color solutions are according to " appendix XI first method of Chinese pharmacopoeia.
The present invention's the 3rd purpose is achieved in that water needle injection, lyophilized injectable powder, capsule, granule, tablet, oral liquid, the drop pill of serving as reasons described pharmaceutical preparation this cockroach extractive and suitable pharmaceutic adjuvant processing.
The application of the pharmaceutical preparation that the present invention's the 4th purpose is achieved in that any one prepared dosage form on the treatment cardiovascular disease is applied in the heart failure that especially pulmonary heart disease, cardiomyopathy, rheumatism, coronary heart disease and hypertensive cardiopathy etc. is caused.
Cockroach extractive of the present invention and preparation thereof can promote myocardial cell Ca
2+Interior stream, gentleness increases myocardial contraction enduringly, and blood vessel dilating reduces pulmonary artery pressure, PCP; Coronary artery dilating, increase coronary flow, the myocardial damage of inhibition Mediated by Free Radicals; Nephrectasia blood vessel, renal blood flow increasing, diuresis; Microcirculation improvement; The correction neuroendocrine is unbalance.
Description of drawings
Accompanying drawing is the standard finger-print of cockroach extractive extractum of the present invention.
The specific embodiment
Below in conjunction with embodiment the present invention is further described, but never in any form the present invention is limited, any change or improvement based on training centre of the present invention is done all belong to protection scope of the present invention.
Cockroach extractive of the present invention can be obtained by following method:
The dried polypide of cockroach is ground into coarse powder, makes thick paste with percolation or reflux extraction, add purified water in the thick paste and fully stir through 90～95% ethanol; Precipitate and oils and fats are removed in oil-water separation, collect the water layer medicinal liquid, activated carbon adsorption; Upper prop separates, n-butyl alcohol eluting or phenol solution eluting, and eluent carries out the secondary ethanol extraction after concentrating; The cold preservation after-filtration; Carry out water again after filtrating concentrates and carry, centrifugal filtration gets the water extract after cold preservation, the water extract after decolouring, ultrafiltration, concentrate the cockroach extractive thick paste.
Described percolation adds 90～95% alcohol dipping 48～60 hours of 5～10 times of weight ratios in the dried worm coarse powder of cockroach, reuse 90～95% ethanol are done the slow percolation of solvent to filtrating and are little yellow, and filtrating gets thick paste through concentrating under reduced pressure, reclaims ethanol simultaneously.
Described reflux extraction, 90～95% alcohol heat reflux that add 5～10 times of weight ratios at the dried worm coarse powder of cockroach extracted 8～12 hours, and filtrating gets thick paste through concentrating under reduced pressure, reclaims ethanol simultaneously.
The method for preparing of described cockroach extractive further comprises:
With behind the alcohol extraction thick paste importing oil water separator, the purified water that adds 4～6 times of weight portions fully stirs, and leaves standstill to layering obviously, removes bottom precipitate and upper strata oils and fats, collects the brown medicinal liquid in middle level in A, the oil-water separation step;
B, upper prop separating step herb liquid add the medical injection active carbon of particle diameter 200～325 orders; Fully stirred the back static 1～2 hour; Be adsorbed as colourless back to medicinal liquid and adorn post, liquid is drained, adding water-saturated n-butanol or concentration are 5～10% phenol solution eluting; Collect the eluting medicinal liquid, be eluted to the medicinal liquid color and be not deeper than yellow color solution No. 4; The eluting medicinal liquid gets thick paste at 60～80 ℃ of following concentrating under reduced pressure, and reclaims n-butyl alcohol or reclaim phenol solution;
With stirring behind 65～85% ethanol of B step gained thick paste with 8～10 times of weight portions, 4～8 ℃ of following cold preservations 12～24 hours, cross and filter supernatant in C, the secondary alcohol extraction step, the supernatant concentrating under reduced pressure gets thick paste, reclaims ethanol simultaneously;
After D, water are carried in the step C step gained thick paste are fully stirred with the purified water of 8～10 times of weight portions; At 12～24 hours after-filtration of 4～8 ℃ of following cold preservation; Filtering residue is at 2～6 ℃ of following low-temperature centrifugations, and merging filtrate and centrate obtain clear liquid, adds the medical injection active carbon of particle diameter 200～325 orders of liquid measure 0.1～0.5% weight ratio in the clear liquid; The limit edged stirs, and after being heated to 60～80 ℃ static 0.5～1 hour; Decarbonization filtering then, filtered solution again after ultrafiltration concentrating under reduced pressure get the cockroach extractive thick paste; The relative density of described thick paste is 1.2～1.3.
The pharmaceutic adjuvant that can adopt during useful in preparing drug formulations of the present invention comprises: PEG400, sodium chloride, sodium hydroxide, starch, dextrin, Icing Sugar, Pulvis Talci, magnesium stearate, silicon dioxide, polyethylene glycol 6000, methyl-silicone oil, glycerol, sodium benzoate.
Cockroach extractive is the transparent dope of pale brown color, the tool hygroscopicity.Differentiate, check and measure by following method:
1, differentiates
⑴ get these article 0.05g, and thin up is to 5ml, as need testing solution.Other gets cockroach medical material 1g, adds n-butyl alcohol-glacial acetic acid (4:1) mixed liquor 50ml, and supersound process 30min filters, and filtrating evaporate to dryness, residue add water 1ml dissolving as control medicinal material solution.According to thin layer chromatography (" an appendix VI of Chinese pharmacopoeia B) test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica GF254 lamellae; Launch with n-butyl alcohol-glacial acetic acid-water (4:1:1), take out, dry; Put under the uviol lamp (254nm) and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, should show the speckle of 3 same colors.Spray with 2% ninhydrin solution, 105 ℃ to be heated to speckle colour developing clear again, with the corresponding position of control medicinal material chromatograph on, should show the speckle of 1 same color.
⑵ get these article 0.05g, and thin up is to 5ml, through strong alkalinity anion exchange glucosan chromatographic column (QAE-Sephadex A-25 type; Internal diameter 1.5cm, the high 5cm of post), water 20ml eluting; Discard water elution liquid, reuse 0.02mol/L hydrochloric acid eluting discards eluent 8ml just; Collect continuous eluent 20ml, as need testing solution.Detecting according to HPLC (" an appendix VI of Chinese pharmacopoeia D), is filler with the octadecylsilane chemically bonded silica, chromatographic column (column length 150mm, column internal diameter are 4.6mm); With 0.05mol/L sodium-acetate buffer (pH 5.0)-methanol (90:10) is mobile phase, detects with PDAD (DAD) detector, and the detection wavelength is 246nm, and flow velocity is per minute 0.5ml, and column temperature is 30 ℃.The accurate need testing solution 5 μ l that draw inject chromatograph of liquid, measure, and write down the chromatogram in 12 minutes.The DAD ultraviolet detection spectrogram of main peak in the test sample chromatogram (maximum peak area) has absorption maximum in the wavelength of 246nm and 286nm.
2, inspection
Color and luster: get these article 0.05 and restrain, add water 10ml dissolving and process the solution that every 1ml contains 5mg, press (" appendix XI first method of Chinese pharmacopoeia) inspection, compare with yellow No. 7 standard color solutions, must not be darker. in accordance with the law
N-butyl alcohol: the preparation of inner mark solution is smart claims that the isoamyl alcohol reference substance is an amount of, adds no organic water and is mixed with the solution that every 1ml contains 10 μ g, as inner mark solution.
The preparation of reference substance solution is smart claims that the n-butyl alcohol reference substance is an amount of, adds inner mark solution and processes the solution that 1ml contains 10 μ g, as reference substance solution.
Algoscopy: precision takes by weighing these article 0.5g, and the accurate inner mark solution 5ml that adds dissolves, and shakes up, and is need testing solution.Get each 2 μ l of reference substance solution and need testing solution; Inject gas chromatograph; Measure according to organic solvent residual method (" two appendix VIII of Chinese pharmacopoeia P method), with 401 organic carrier chromatographic columns (column length 2m), 170 ℃ of mensuration of column temperature; Press internal standard method with peak area and calculate the n-butyl alcohol residual quantity, must not surpass 0.1%.
Tannin: get these article 0.05g, add water 1ml dissolving after, add 1 of spirit of vinegar, add 4～5 of gelatin sodium chloride solutions (contain gelatin 1%, the aqueous solution of sodium chloride 10% must fresh) again, muddiness and deposition must not appear.
Resin: get these article 0.25g, add water 5ml dissolving after, add 1 of hydrochloric acid, placed 30 minutes, should not have the resin-like thing and separate out.
Oxalates: get these article 0.10g, add water 2ml dissolving after, add 2～3 of 3% calcium chloride test solutions, placed 10 minutes, muddiness or deposition must not appear.
Potassium ion: get these article 0.10g, burn to carbonization with little heated earlier, 500～600 ℃ of blazing ashing extremely fully, add 6% acetic acid and make dissolving again.Put in the 25ml measuring bottle, thin up is to scale, mixing; Measuring 1ml puts in the 10ml nessler colorimetric tube; Add 12 of alkaline formaldehyde solution (get formalin, transfer pH to 8.0～9.0), 2 of 3% EDTA sodium solutions with the 0.1mol/L sodium hydroxide solution; 3% tetraphenyl borate sodium solution 0.5ml adds water to 10ml as the test sample pipe.Other gets standard potassium ion solution (100 μ g/ml) 0.8ml, puts in the 10ml nessler colorimetric tube, and the same operation is got the test sample pipe and estimated than turbid with the contrast QC as the contrast QC, and the test sample pipe must not be more turbid.
Standard potassium ion solution preparation: get an amount of porphyrize of potassium sulfate, be dried to constant weight in 110 ℃, precision takes by weighing 2.330g, adds water and makes in right amount and be dissolved into 1000ml, and the accurate 10ml thin up of drawing becomes 100ml, promptly gets, and every 1ml is equivalent to 100 μ g potassium ions.
Protein: get these article 0.1g, add water 2ml dissolving, add freshly prepared 30% sulfosalicylic acid solution 2ml, mixing was placed 5 minutes, muddiness must not occur.
Loss on drying:, subtract weight loss and must not cross 15.0% 105 ℃ of dryings 3 hours.By (" an appendix IX of Chinese pharmacopoeia G) inspection.
Residue on ignition: get these article 1.0g, press (" an appendix IX of Chinese pharmacopoeia J) inspection, leave over residue and must not cross 1.0%. in accordance with the law
Heavy metal: get residue under the residue on ignition item, press (" an appendix IX of Chinese pharmacopoeia E second method) inspection, contain heavy metal and must not cross 20/1000000ths. in accordance with the law
Arsenic salt: get these article 1.0g, slowly heat carbonization, incomplete like carbonization; After adding a small amount of sulphuric acid moistening, slowly heating makes complete carbonization, again in 500～600 ℃ of blazing ashing; Put coldly, add hydrochloric acid 5ml, make its dissolving in the water-bath; Add water 23ml, press (" an appendix IX of Chinese pharmacopoeia F first method) inspection, contain arsenic salt and must not cross 2/1000000ths. in accordance with the law
Finger printing: get these article 0.05g, thin up is to 5ml, through strong-base anion-exchange resin chromatographic column (Dowex2, Cl-type, 200 orders, internal diameter 1.5cm; High 2.5cm, wet method dress post, 0.5 mol/L sodium hydroxide solution 10ml eluting, reuse water 10ml eluting are adopted in the chromatographic column pretreatment; Subsequent use), water 10ml eluting discards water elution liquid, reuse 0.25mol/L acetum eluting; Discard eluent 5ml just, collect continuous eluent 25ml, shake up, as need testing solution.It is an amount of to get the inosine reference substance, and accurate the title decides, and adds water and is mixed with the solution that every 1ml contains inosine 50 μ g, as reference substance solution.Measuring according to HPLC (" an appendix VI of Chinese pharmacopoeia D), is filler with the octadecylsilane chemically bonded silica, chromatographic column (column length 150mm, column internal diameter are 4.6mm); Gradient elution is a mobile phase A with 0.05 mol/L sodium-acetate buffer (pH 5.0), is Mobile phase B with methanol, and the detection wavelength is 254nm, and flow velocity is per minute 0.6ml, and column temperature is 30 ℃.Number of theoretical plate calculates by the inosine peak should be not less than 5000.
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0～5 | 90 | 10 |
| 5～15 | 90→74 | 10→26 |
| 15～18 | 74 | 26 |
Accurate respectively reference substance solution and each 5 μ l of need testing solution of drawing inject chromatograph of liquid, write down the chromatogram in 25 minutes.The test sample chromatogram should with the standard finger-print basically identical.The test sample chromatogram is imported chromatographic fingerprints of Chinese materia medica similarity evaluation system (2004, the 1.0B version), compare, calculate similarity with standard finger-print.Similarity should be not less than 0.90.
3, assay
4, effective component content is measured in the cockroach extractive
The mensuration of compound nucleoside base: precision is measured cockroach extractive 1ml, puts in the 50ml measuring bottle, with the dissolving of glycine-hydrochloride buffer and be diluted to scale; Shake up, precision is measured 5ml and is put in the 100ml measuring bottle, adds above-mentioned buffer to scale; Shake up, promptly get need testing solution.According to ultraviolet visible spectrophotometry (" appendix VA of Chinese pharmacopoeia), measure trap in the 254nm wavelength, be calculated as follows the compound nucleoside base contents C (mg) of every ml cockroach extractive injection
In the formula: A is the need testing solution trap
490 is that compound nucleoside base class concentration is 1% o'clock average absorption degree in the 254nm wavelength in the cockroach extractive.
N is an extension rate
Measure the result and should be 10.0mg～18.0mg for containing compound nucleoside base among the every 1ml of cockroach extractive.
In conjunction with amino acid whose mensuration:
The preparation standard curve.Precision takes by weighing glutamic acid reference substance 10mg, adds the heat of solution of about 160ml water temperature, puts in the 200ml volumetric flask, adds water to scale, shakes up, and promptly gets the reference substance solution that contains 0.05mg among every 1ml.Get 6 in tool plug test tube, accurate respectively reference substance solution 0.2,0.4,0.6,0.8, the 1.0ml of adding adds water to 1.0ml respectively, is that blank respectively adds 0.2mol/L citrate buffer solution (pH5.0) 1.0ml with 1.0ml water.Ninhydrin reagent 1.0ml shakes up, and heating is 15 minutes in 100 ℃ of water-baths; Take out, use water cooling, placed 5～10 minutes; Each adds 60% ethanol 3.0ml, shakes up, and measures trap according to ultraviolet visible spectrophotometry (" an appendix V of Chinese pharmacopoeia A) in the 570nm wavelength.With the aminoglutaric acid concentration is abscissa, and trap is a vertical coordinate drawing standard curve.
The preparation need testing solution.Precision is measured cockroach extractive 1.0ml, in the 50ml measuring bottle, adds water to scale, shakes up, and is the test sample diluent.Precision is measured 1.0ml test sample diluent, puts in the sky ampoule, adds 1.0ml hydrochloric acid; Seal, in 110 ℃ of hydrolysis 8 hours, taking-up was put cold; All forward content to evaporating dish mid-boiling water bath evaporate to dryness, add water and make in right amount in dissolving and the immigration 10ml measuring bottle, add water to scale; Shake up, be the test sample hydrolyzed solution.
Measure.Precision is measured test sample hydrolyzed solution 0.1ml, adds water to 1.0ml, places test tube A, is test sample hydrolyzed solution not; Getting test sample hydrolyzed solution 1.0ml places test tube B many.With 1.0ml water is blank; According to above-mentioned standard curve prepare the item under method from " respectively adding 0.2mol/L citrate buffer solution (pH5.0) 1.0ml "; Measure the trap of not hydrolysis and hydrolyzed solution respectively, find its amino acid concentration, calculate from standard curve; Promptly get free aminoacid content (A) and total amino acids content (B) in every gram cockroach extractive, by (B-A) promptly gets in every gram cockroach extractive and combines amino acid content.
The every 1ml of these article contains combination aminoacid with glutamic acid (C
5H
9NO
4) meter, should be 15.0 mg～25.0mg.
The mensuration of uracil, hypoxanthine and inosine:
According to " the high effective liquid chromatography for measuring of an appendix VI of Chinese pharmacopoeia D.
Chromatographic condition and system suitability test.With the octadecylsilane chemically bonded silica is filler.Mobile phase A is 0.05mol/L sodium-acetate buffer (pH5.0); Mobile phase B is methanol-mobile phase A (80:20).According to the form below carries out gradient elution; The detection wavelength is 254nm, and flow velocity is 0.5ml/min, and number of theoretical plate calculates by uracil, hypoxanthine and inosine peak respectively all should be not less than 3000.Separating degree between uracil, hypoxanthine and the inosine should meet the requirements.
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0～5 | 90 | 10 |
| 5～15 | 90→70 | 10→30 |
| 15～18 | 70 | 30 |
The preparation of reference substance solution.It is an amount of that precision takes by weighing uracil, hypoxanthine and inosine reference substance, adds mobile phase A and process and contain the solution that uracil, hypoxanthine and inosine are respectively 30 μ g, 40 μ g, 70 μ g among every 1ml, shakes up, and promptly gets.
The preparation of need testing solution.Precision is measured cockroach extractive 1ml, puts in the 50ml measuring bottle, is diluted to scale with mobile phase A, shakes up, and promptly gets.
Measure.Accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject chromatograph of liquid, measure, and promptly get.
Contain uracil (C among the every ml of cockroach extractive
4H
4N
2O
2) should be 1.0mg～1.8mg, hypoxanthine (C
5H
5N
4O) should be 1.5mg～2.5mg, inosine (C
10H
12N
4O
5) should be 3.5mg～5.0mg.
Embodiment 1
Get the dried polypide coarse powder of cockroach 1000g, added 90% ethanol 5L dipping 48 hours, reuse 90%% ethanol is done the slow percolation of solvent to filtrating and is little yellow, and filtrating gets thick paste through concentrating under reduced pressure, reclaims ethanol simultaneously; Behind alcohol extraction thick paste importing oil water separator, the purified water that adds 4 times of weight portions fully stirs, and leaves standstill to layering obviously, removes bottom precipitate and upper strata oils and fats, collects the brown medicinal liquid in middle level; The medical injection active carbon of medicinal liquid adding particle diameter 200～325 orders fully stirred the back static 1 hour, was adsorbed as colourless back to medicinal liquid and adorned post; Liquid is drained; Add the water-saturated n-butanol eluting, collect the eluting medicinal liquid, be eluted to the medicinal liquid color and be not deeper than yellow color solution No. 4; The eluting medicinal liquid gets thick paste at 60 ℃ of following concentrating under reduced pressure, and reclaims n-butyl alcohol or reclaim phenol solution; Stir behind 65% ethanol of gained thick paste with 8 times of weight portions, 4 ℃ of following cold preservations 12 hours, cross and filter supernatant, the supernatant concentrating under reduced pressure gets thick paste, reclaims ethanol simultaneously; After the gained thick paste fully stirs with the purified water of 8 times of weight portions; At 12 hours after-filtration of 4 ℃ of following cold preservation; Filtering residue is at 2 ℃ of following low-temperature centrifugations, and merging filtrate and centrate obtain clear liquid, adds the medical injection active carbon of particle diameter 200 orders of liquid measure 0.1% weight ratio in the clear liquid; The limit edged stirs, and after being heated to 60 ℃ static 0.5 hour; Decarbonization filtering then, filtered solution filters through the film filter of aperture 0.22 μ m, and concentrating under reduced pressure gets the about 5.8g of cockroach extractive thick paste to filtered solution under 60 ℃ of temperature through not being higher than after the ultrafiltration apparatus ultrafiltration again; The relative density of described thick paste is 1.2～1.3.
Consist of through detecting the present embodiment cockroach extractive: compound nucleoside base 36.3%, combine aminoacid 40.2%, uracil (C
4H
4N
2O
2) 2.5%, hypoxanthine (C
5H
5N
4O) 3.3%, inosine (C
10H
12N
4O
5) 6.5%.
Embodiment 2
Get the dried polypide coarse powder of cockroach 1000g, added 95% ethanol 10L dipping 60 hours, reuse 95% ethanol is done the slow percolation of solvent to filtrating and is little yellow, and filtrating gets thick paste through concentrating under reduced pressure, reclaims ethanol simultaneously; Behind alcohol extraction thick paste importing oil water separator, the purified water that adds 6 times of weight portions fully stirs, and leaves standstill to layering obviously, removes bottom precipitate and upper strata oils and fats, collects the brown medicinal liquid in middle level; The medical injection active carbon of medicinal liquid adding particle diameter 325 orders fully stirred the back static 2 hours, was adsorbed as colourless back to medicinal liquid and adorned post; Liquid is drained; Adding concentration is 5% phenol solution eluting, collects the eluting medicinal liquid, is eluted to the medicinal liquid color and is not deeper than yellow color solution No. 4; The eluting medicinal liquid gets thick paste at 80 ℃ of following concentrating under reduced pressure, and reclaims n-butyl alcohol or reclaim phenol solution; Stir behind 85% ethanol of gained thick paste with 10 times of weight portions, 8 ℃ of following cold preservations 24 hours, cross and filter supernatant, the supernatant concentrating under reduced pressure gets thick paste, reclaims ethanol simultaneously; After the gained thick paste fully stirs with the purified water of 10 times of weight portions; At 24 hours after-filtration of 8 ℃ of following cold preservation; Filtering residue is at 6 ℃ of following low-temperature centrifugations, and merging filtrate and centrate obtain clear liquid, adds the medical injection active carbon of particle diameter 325 orders of liquid measure 0.5% weight ratio in the clear liquid; The limit edged stirs, and after being heated to 80 ℃ static 1 hour; Decarbonization filtering then, filtered solution again after ultrafiltration concentrating under reduced pressure get cockroach extractive thick paste 6g; The relative density of described thick paste is 1.2～1.3.
Consist of through detecting the present embodiment cockroach extractive: compound nucleoside base 20.8%, combine aminoacid 49.1%, uracil (C
4H
4N
2O
2) 2.3%, hypoxanthine (C
5H
5N
4O) 4.6%, inosine (C
10H
12N
4O
5) 9.1%.
Embodiment 3
Get the dried polypide coarse powder of cockroach 1000g, added 95% ethanol 8L dipping 50 hours, reuse 95% ethanol is done the slow percolation of solvent to filtrating and is little yellow, and filtrating gets thick paste through concentrating under reduced pressure, reclaims ethanol simultaneously; Behind alcohol extraction thick paste importing oil water separator, the purified water that adds 5 times of weight portions fully stirs, and leaves standstill to layering obviously, removes bottom precipitate and upper strata oils and fats, collects the brown medicinal liquid in middle level; Medicinal liquid adds the medical injection active carbon of particle diameter 250 orders; Fully stirred the back static 1.5 hours; Be adsorbed as colourless back to medicinal liquid and adorn post, liquid is drained, adding water-saturated n-butanol or concentration are 8% phenol solution eluting; Collect the eluting medicinal liquid, be eluted to the medicinal liquid color and be not deeper than yellow color solution No. 4; The eluting medicinal liquid gets thick paste at 70 ℃ of following concentrating under reduced pressure, and reclaims n-butyl alcohol or reclaim phenol solution; Stir behind 75% ethanol of gained thick paste with 9 times of weight portions, 6 ℃ of following cold preservations 18 hours, cross and filter supernatant, the supernatant concentrating under reduced pressure gets thick paste, reclaims ethanol simultaneously; After the gained thick paste fully stirs with the purified water of 9 times of weight portions; At 18 hours after-filtration of 6 ℃ of following cold preservation; Filtering residue is at 4 ℃ of following low-temperature centrifugations, and merging filtrate and centrate obtain clear liquid, adds the medical injection active carbon of particle diameter 250 orders of liquid measure 0.3% weight ratio in the clear liquid; The limit edged stirs, and after being heated to 70 ℃ static 0.8 hour; Decarbonization filtering then, filtered solution filters through the film filter of aperture 0.22 μ m, and concentrating under reduced pressure gets the about 5.6g of cockroach extractive thick paste to filtered solution under 70 ℃ of temperature through not being higher than after the ultrafiltration apparatus ultrafiltration again; The relative density of described thick paste is 1.2～1.3.
Consist of through detecting the present embodiment cockroach extractive: compound nucleoside base 39.3%, combine aminoacid 31.2%, uracil (C
4H
4N
2O
2) 3.6%, hypoxanthine (C
5H
5N
4O) 3.3%, inosine (C
10H
12N
4O
5) 5.5%.
Embodiment 4
Get the dried polypide coarse powder of cockroach 1000g, add 90% ethanol 5L hot reflux and extracted 8 hours, filtrating gets thick paste through concentrating under reduced pressure, reclaims ethanol simultaneously; Behind alcohol extraction thick paste importing oil water separator, the purified water that adds 4 times of weight portions fully stirs, and leaves standstill to layering obviously, removes bottom precipitate and upper strata oils and fats, collects the brown medicinal liquid in middle level; The medical injection active carbon of medicinal liquid adding particle diameter 200 orders fully stirred the back static 1 hour, was adsorbed as colourless back to medicinal liquid and adorned post, and liquid is drained, and added the water-saturated n-butanol eluting, collected the eluting medicinal liquid, was eluted to the medicinal liquid color and was not deeper than yellow color solution No. 4; The eluting medicinal liquid gets thick paste at 60 ℃ of following concentrating under reduced pressure, and reclaims n-butyl alcohol or reclaim phenol solution; Stir behind 65% ethanol of gained thick paste with 8 times of weight portions, 4 ℃ of following cold preservations 12 hours, cross and filter supernatant, the supernatant concentrating under reduced pressure gets thick paste, reclaims ethanol simultaneously; After the gained thick paste fully stirs with the purified water of 8 times of weight portions; At 12 hours after-filtration of 4 ℃ of following cold preservation; Filtering residue is at 2 ℃ of following low-temperature centrifugations, and merging filtrate and centrate obtain clear liquid, adds the medical injection active carbon of particle diameter 200 orders of liquid measure 0.1% weight ratio in the clear liquid; The limit edged stirs, and after being heated to 60 ℃ static 0.5 hour; Decarbonization filtering then, filtered solution filters through the film filter of aperture 0.22 μ m, and concentrating under reduced pressure gets the about 5.2g of cockroach extractive thick paste to filtered solution under 70 ℃ of temperature through not being higher than after the ultrafiltration apparatus ultrafiltration again; The relative density of described thick paste is 1.2～1.3.
Consist of through detecting the present embodiment cockroach extractive: compound nucleoside base 35%, combine aminoacid 43%, uracil (C
4H
4N
2O
2) 3.01%, hypoxanthine (C
5H
5N
4O) 4.2%, inosine (C
10H
12N
4O
5) 8.0%.
Get the dried polypide coarse powder of cockroach 1000g, add 95% ethanol 10L hot reflux and extracted 12 hours, filtrating gets thick paste through concentrating under reduced pressure, reclaims ethanol simultaneously; Behind alcohol extraction thick paste importing oil water separator, the purified water that adds 6 times of weight portions fully stirs, and leaves standstill to layering obviously, removes bottom precipitate and upper strata oils and fats, collects the brown medicinal liquid in middle level; The medical injection active carbon of medicinal liquid adding particle diameter 325 orders fully stirred the back static 2 hours, was adsorbed as colourless back to medicinal liquid and adorned post; Liquid is drained; Adding concentration is 5% phenol solution eluting, collects the eluting medicinal liquid, is eluted to the medicinal liquid color and is not deeper than yellow color solution No. 4; The eluting medicinal liquid gets thick paste at 80 ℃ of following concentrating under reduced pressure, and reclaims n-butyl alcohol or reclaim phenol solution; Stir behind 85% ethanol of gained thick paste with 10 times of weight portions, 8 ℃ of following cold preservations 24 hours, cross and filter supernatant, the supernatant concentrating under reduced pressure gets thick paste, reclaims ethanol simultaneously; After the gained thick paste fully stirs with the purified water of 10 times of weight portions; At 24 hours after-filtration of 8 ℃ of following cold preservation; Filtering residue is at 6 ℃ of following low-temperature centrifugations, and merging filtrate and centrate obtain clear liquid, adds the medical injection active carbon of particle diameter 325 orders of liquid measure 0.5% weight ratio in the clear liquid; The limit edged stirs, and after being heated to 80 ℃ static 1 hour; Decarbonization filtering then, filtered solution filters through the film filter of aperture 0.22 μ m, and concentrating under reduced pressure gets the about 5.4g of cockroach extractive thick paste to filtered solution under 70 ℃ of temperature through not being higher than after the ultrafiltration apparatus ultrafiltration again; The relative density of described thick paste is 1.2～1.3.
Consist of through detecting the present embodiment cockroach extractive: compound nucleoside base 32%, combine aminoacid 45.2%, uracil (C
4H
4N
2O
2) 3.6%, hypoxanthine (C
5H
5N
4O) 4.0%, inosine (C
10H
12N
4O
5) 7.02%.
Get the dried polypide coarse powder of cockroach 1000g, get the dried polypide coarse powder of cockroach 1000g, add 90% ethanol 8L hot reflux and extracted 10 hours, filtrating gets thick paste through concentrating under reduced pressure, reclaims ethanol simultaneously; Behind alcohol extraction thick paste importing oil water separator, the purified water that adds 5 times of weight portions fully stirs, and leaves standstill to layering obviously, removes bottom precipitate and upper strata oils and fats, collects the brown medicinal liquid in middle level; Medicinal liquid adds the medical injection active carbon of particle diameter 250 orders; Fully stirred the back static 1.5 hours; Be adsorbed as colourless back to medicinal liquid and adorn post, liquid is drained, adding water-saturated n-butanol or concentration are 8% phenol solution eluting; Collect the eluting medicinal liquid, be eluted to the medicinal liquid color and be not deeper than yellow color solution No. 4; The eluting medicinal liquid gets thick paste at 70 ℃ of following concentrating under reduced pressure, and reclaims n-butyl alcohol or reclaim phenol solution; Stir behind 75% ethanol of gained thick paste with 9 times of weight portions, 6 ℃ of following cold preservations 18 hours, cross and filter supernatant, the supernatant concentrating under reduced pressure gets thick paste, reclaims ethanol simultaneously; After the gained thick paste fully stirs with the purified water of 9 times of weight portions; At 18 hours after-filtration of 6 ℃ of following cold preservation; Filtering residue is at 4 ℃ of following low-temperature centrifugations, and merging filtrate and centrate obtain clear liquid, adds the medical injection active carbon of particle diameter 250 orders of liquid measure 0.3% weight ratio in the clear liquid; The limit edged stirs, and after being heated to 70 ℃ static 0.8 hour; Decarbonization filtering then, filtered solution filters through the film filter of aperture 0.22 μ m, and concentrating under reduced pressure gets the about 5.8g of cockroach extractive thick paste to filtered solution under 70 ℃ of temperature through not being higher than after the ultrafiltration apparatus ultrafiltration again; The relative density of described thick paste is 1.2～1.3.
Consist of through detecting the present embodiment cockroach extractive: compound nucleoside base 26%, combine aminoacid 39.1%, uracil (C
4H
4N
2O
2) 2.2%, hypoxanthine (C
5H
5N
4O) 3.8%, inosine (C
10H
12N
4O
5) 6.7%.
Get embodiment 1 gained cockroach extractive extractum 50g (in dry product), dissolve in right amount, add 200g Polyethylene Glycol-400,9g sodium chloride, mixing with water for injection; Add the injection water to 1000ml, transfer pH value 5.4～6.4, mixing; Filter, canned, sterilization gets final product injection.
Embodiment 8
Get embodiment 2 gained cockroach extractive extractum 50g (in dry product), dissolve in right amount, add sodium chloride 9g, mixing with water for injection; Add the injection water to 1000ml, transfer pH value 5.4～6.4, mixing, aseptic filtration; Fill, lyophilization promptly obtains lyophilized injectable powder.
Get embodiment 3 gained cockroach extractive extractum 50g (in dry product), add starch 50g, mixing, 80 ℃ of dryings, pulverizing; Cross 120 mesh sieves, get mixed powder, it is an amount of to add ethanol; Mixing is processed granule, add 1% Pulvis Talci and 0.3% magnesium stearate mix homogeneously again after; Incapsulate, promptly obtain capsule.
Get embodiment 4 gained cockroach extractive extractum 50g (in dry product), add an amount of purified water dissolving, add cockroach extractive, the sucrose of 2 times of amounts and the dextrin of 2 times of amounts; Mixing adds an amount of ethanol and granulates as wetting agent, dry, granulate; Mix, granule is processed in packing.
Embodiment 11
Get embodiment 5 gained cockroach extractive extractum 50g (in dry product), add starch 50g, mixing, 120 mesh sieves are crossed in 80 ℃ of dryings, pulverizing; Get mixed powder, it is an amount of to add ethanol, and mixing is processed granule; Add 0.3% magnesium stearate and 1% silicon dioxide again and mix the back tabletting, coating, packing promptly gets.
Embodiment 12
Get embodiment 6 gained cockroach extractive extractum 50g (in dry product), add an amount of purified water dissolving, add 10% glycerol, 0.3% sodium benzoate, mixing adds purified water to 500ml, transfers pH value 6.4～7.4, mixing, and fill, sterilization promptly gets oral solutions.
Embodiment 13
Get embodiment 2 gained cockroach extractive extractum 50g (in dry product), with after the polyethylene glycol 6000 fusion of substrate 120g with cockroach extractive extractum mix homogeneously, 80 ℃ of down insulations; Use internal diameter to be 3.3mm, external diameter is 5.1 mm, dropper; Dripping speed with 60-70/minutes splashes in the methyl-silicone oil; Collect drop pill, remove liquid coolant, promptly get.
The test of pesticide effectiveness result of pharmaceutical preparation of the present invention
Gained preparation of the present invention has benefiting QI for activating blood circulation, activating YANG for promoting diuresis.Be used for gas sun two void, cardiopalmus, tachypnea, edema, dim complexion, lip cyanosis due to the blood-stasis internal-depression; Above-mentioned patient is seen in chronic heart failure.
Select sample to carry out controlled trial at random, injection of the present invention and dobutamine matched group, each 100 example; Other has open trial 208 examples, total 408 examples.
Result of the test shows: injection treatment group obvious effective rate 37% of the present invention, effective percentage 89%; Open trial group obvious effective rate 55.29%, effective percentage 87.5%.Preparation for treating group of the present invention and matched group (dobutamine) compare, statistics has significant difference P ﹤ 0.01.
Preparation of the present invention declines to right heart failure whole-heartedly, cardiac function II level, and III level curative effect is obvious, all is superior to the dobutamine matched group.Two groups of cardiac function onset time no difference of science of statistics P＞0.05. treatment group cardiac function onset time is 3.31 days.
Preparation for treating group of the present invention is respectively 93.35% and 88.46% to Qi deficiency blood stasis type and motive YIN-deficiency type curative effect, with matched group there were significant differences P ﹤ 0.01 and P ﹤ 0.05.
Preparation for treating group of the present invention has obvious curative effects to breathing, heart rate and edema.Heart rate onset time comparison is inhaled in two group callings, and significant difference P ﹤ 0.01 and P ﹤ 0.05 are all arranged.
Traditional Chinese medical science disease integration relatively, preparation for treating group of the present invention is improved cardiopalmus, tachypnea, weak and fear of cold integration and obviously is better than matched group P ﹤ 0.01.Electrocardiogram, rabat, blood pressure, two groups of no difference of science of statistics, security inspection is not seen obvious adverse reaction.
In sum, preparation of the present invention can improve cardiac function, alleviates diseases such as cardiopalmus, tachypnea, edema, and is obvious to Qi deficiency blood stasis type and motive YIN-deficiency type curative effect.Treatment congestive heart failure curative effect is reliable, safe in utilization.
Claims (10)
1. cockroach extractive is characterized in that: its contain weight ratio compound nucleoside base 20～40%, combine aminoacid 30～60%, uracil 2～4%, hypoxanthine 3～6% and inosine 5～10%.
2. cockroach extractive according to claim 1 is characterized in that: it contains the compound nucleoside base of weight ratio 25～40%, and 35～60% combine aminoacid, 2.5～4% uracil, the inosine of 3.5～6% hypoxanthine and 6～10 %.
3. cockroach extractive according to claim 1 is characterized in that: it contains the compound nucleoside base of weight ratio 22～36%, and 35～48% combine aminoacid, 2.5～3.5% uracil, 3.5～5% hypoxanthine and 6～8% inosine.
4. cockroach extractive according to claim 1 is characterized in that: it contains the compound nucleoside base of weight ratio 24～38%, and 32～48% combine aminoacid, 2.0～3.2% uracil, 3.4～5% hypoxanthine and 7～10% inosine.
5. a method for preparing for preparing claim 1,2,3 or 4 said cockroach extractives is characterized in that being may further comprise the steps: the dried polypide of cockroach is ground into coarse powder, makes thick paste through 90～95% ethanol with percolation or reflux extraction; Add purified water in the thick paste and fully stir, precipitate and oils and fats are removed in oil-water separation, collect the water layer medicinal liquid; Activated carbon adsorption, upper prop separates, n-butyl alcohol eluting or phenol solution eluting; Eluent carries out the secondary ethanol extraction after concentrating, and the cold preservation after-filtration carries out water again after filtrating concentrates and carries; Centrifugal filtration gets the water extract after cold preservation, the water extract concentrate through decolouring, after the ultrafiltration the cockroach extractive thick paste.
6. the method for preparing of cockroach extractive according to claim 5; It is characterized in that being described percolation; 90～95% alcohol dipping 48～60 hours that in the dried worm coarse powder of cockroach, add 5～10 times of weight ratios; Reuse 90～95% ethanol are done the slow percolation of solvent to filtrating and are little yellow, and filtrating gets thick paste through concentrating under reduced pressure, reclaims ethanol simultaneously.
7. the method for preparing of cockroach extractive according to claim 5; It is characterized in that being described reflux extraction; 90～95% alcohol heat reflux that add 5～10 times of weight ratios at the dried worm coarse powder of cockroach extracted 8～12 hours, and filtrating gets thick paste through concentrating under reduced pressure, reclaims ethanol simultaneously.
8. the method for preparing of cockroach extractive according to claim 5 is characterized in that being described:
With behind the alcohol extraction thick paste importing oil water separator, the purified water that adds 4～6 times of weight portions fully stirs, and leaves standstill to layering obviously, removes bottom precipitate and upper strata oils and fats, collects the brown medicinal liquid in middle level in A, the oil-water separation step;
B, upper prop separating step herb liquid add the medical injection active carbon of particle diameter 200～325 orders; Fully stirred the back static 1～2 hour; Be adsorbed as colourless back to medicinal liquid and adorn post, liquid is drained, adding water-saturated n-butanol or concentration are 5～10% phenol solution eluting; Collect the eluting medicinal liquid, be eluted to the medicinal liquid color and be not deeper than yellow color solution No. 4; The eluting medicinal liquid gets thick paste at 60～80 ℃ of following concentrating under reduced pressure, and reclaims n-butyl alcohol or reclaim phenol solution;
With stirring behind 65～85% ethanol of B step gained thick paste with 8～10 times of weight portions, 4～8 ℃ of following cold preservations 12～24 hours, cross and filter supernatant in C, the secondary alcohol extraction step, the supernatant concentrating under reduced pressure gets thick paste, reclaims ethanol simultaneously;
After D, water are carried in the step C step gained thick paste are fully stirred with the purified water of 8～10 times of weight portions; At 12～24 hours after-filtration of 4～8 ℃ of following cold preservation; Filtering residue is at 2～6 ℃ of following low-temperature centrifugations, and merging filtrate and centrate obtain clear liquid, adds the medical injection active carbon of particle diameter 200～325 orders of liquid measure 0.1～0.5% weight ratio in the clear liquid; The limit edged stirs, and after being heated to 60～80 ℃ static 0.5～1 hour; Decarbonization filtering then, filtered solution again after ultrafiltration concentrating under reduced pressure get the cockroach extractive thick paste; The relative density of described thick paste is 1.2～1.3.
9. by the preparation of claim 1,2, the preparation of 3 or 4 described cockroach extractives, it is characterized in that: described preparation water needle injection, lyophilized injectable powder, capsule, granule, tablet, oral liquid, the drop pill that this cockroach extractive and suitable pharmaceutic adjuvant are processed of serving as reasons.
10. one kind with the pharmaceutical preparation of any one prepared dosage form of claim 9 application on the treatment cardiovascular disease, is applied in the heart failure that especially pulmonary heart disease, cardiomyopathy, rheumatism, coronary heart disease and hypertensive cardiopathy etc. is caused.
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| CN111925408A (en) * | 2020-08-14 | 2020-11-13 | 云南中医药大学 | Preparation method of periplaneta americana polypeptide extract by using water as medium |
| CN111925408B (en) * | 2020-08-14 | 2023-06-06 | 云南中医药大学 | Preparation method of periplaneta americana polypeptide extract taking water as medium |
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