CN101156875A - Cockroach extractive for treating cardiovascular disease, preparation method and its composition - Google Patents
Cockroach extractive for treating cardiovascular disease, preparation method and its composition Download PDFInfo
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Abstract
The invention relates to extractive taking blattaria used as the medical source and being applied to the angiocarpy field, the preparation method thereof and a composition containing the extractive and pharmaceutically acceptable carrier. The extractive adopts water, alcohol and/or methanol as the extracting dissolvent, wherein, the volume concentration of the aqueous solution of the alcohol and/or methanol is 0 less than or equal to V/V less than or equal to 60 percent. The extractive has the advantages that blood fat is reduced, cholesterin is reduced, angina pectoris is prevented, the heart is tonified, and heart failure is resisted. The extraction process of the invention is simple and highly effective, the specialization is strong, and the obtained product has high effective component content. The extractive of the invention can be produced to be the composition containing the extractive and the pharmaceutically acceptable carrier.
Description
Technical field
The present invention relates to a kind of extract that is applied to cardiovascular field, preparation method with and compositions, relating in particular to the cockroach is the extract that is applied to cardiovascular field, the preparation method of medicament sources and the compositions that contains this extract and pharmaceutically suitable carrier.
Background technology
Cockroach is commonly called as Blatta seu periplaneta, claims stone Rhizoma Zingiberis Recens, negative dish, sliding worm, croton bug, fragrant wife etc. again.Belong to Insecta, Blattaria, nearly 2300 kinds of the whole world is distributed in the torrid zone and area, temperate zone more.Groton bug, oriental cockroach, Australian cockroach, periplaneta americana etc. are arranged in the frequent species.The cockroach body is oblong, dorsoventrally compressed.Little and flexible, be hidden under the shirtfront, and stretch out the oblique back lower place.Mouthpart is typical biting mouthparts, and feeler is thread, and elongated more piece, its length equal body length more or grew its body.Compound eye is a pair of, owing to be looped around the foreign side of feeler, so be kidney-shaped, simple eye two or do not have.The shirtfront prosperity is peltate, covers head.The wing prosperity, the fore wing keratin becomes proala coriacea; Hind wing is membranous.Foot is elongated, is suitable for walking fast.
Cockroach belongs to traditional medical material, medicinal record in the Compendium of Material Medica: cockroach, abnormal smells from the patient: salty, cold, poisonous.Cure mainly: blood stasis, disease heavily fortified point, cold and heat, the therapeutic method to keep the adverse QI flowing downwards, sharp blood vessels.But cockroach self is poisonous, and traditional administering mode can not be brought into play the efficacy of drugs of cockroach to the greatest extent.
Along with modern medicine research development of science and technology, the mankind have seen that cockroach has high medical value, number of patent application is CN94108943.6 (publication number CN1116931, the applying date is on August 14th, 1994), number of patent application is CN03100149.1 (publication number CN1424052, the applying date is on January 7th, 2003), the patent No. is that (publication number is: the CN1548147 applying date is CN03125189.7: on May 12nd, 2003), the patent No. is CN94118839.6 (publication number CN1124141, the applying date is December in 1994 9 days) etc. Chinese patent, the report cockroach extractive has treatment hepatitis activity, treatment alzheimer disease and activity of fighting against senium, treat heart failure in addition, the activity of rising blood pressure.Cockroach becomes the focus of the concern of field of medicaments gradually, but the Study on Preparation of its active substance research and active compound is not developed accordingly, imprecise to the cognition of cockroach active component, the extraction process level is low, technology is numerous and diverse, cause the incompatible big requirement of producing of preparation of the pharmaceutical preparation of cockroach, the pharmaceutical value of cockroach is fully used.
Summary of the invention
The invention provides a kind of cardiovascular field that is applied to, the extract from cockroach obtains can prepare by the following method: A, be extraction solvent with water and ethanol and/or methanol, cockroach pulverized, wherein ethanol and/or methanol aqueous solution volumetric concentration 0≤V/V≤60%;
B, accent pH to 3-5; Homogenate;
C, airtight leaves standstill; Cockroach is fully soaked; 0-15 ℃ of solid-liquid separation, get supernatant;
D, heating liquid are to little boiling, and adjust pH removes deproteinize;
E, the following material of intercepting molecular weight 100KDa.
In the above-mentioned preparation method in the steps A extraction solvent be water and ethanol and/or methanol, the expression extraction solvent is water, ethanol water, methanol aqueous solution or methanol and the blended aqueous solution of ethanol.When V/V=0, the expression extraction solvent is a water.When V/V=60%, the volumetric concentration of expression ethanol water or methanol aqueous solution be 60% or the aqueous solution volumetric concentration of ethanol and methyl alcohol mixed liquor be 60%.
Step C in the above-mentioned preparation method by regulating pH value, makes the protein sol instability in the solution, removes the protein in the extract.This method can not make the less biologically active peptide dissolubility of molecular weight diminish, and can not cause the biologically active peptide precipitation, so contain biologically active peptide in the extract of the present invention.
Cockroach of the present invention can be selected the insecticide of multiple cockroach class for use, the preferred periplaneta americana of the present invention.
Cockroach extractive of the present invention mainly comprises biologically active peptide, acid mucopolysaccharide, compound nucleoside base and sticking propylhomoserin.
Cockroach extractive of the present invention contains ribosidoadenine, and promptly extract contains biologically active peptide, acid mucopolysaccharide, compound nucleoside base, sticking propylhomoserin and ribosidoadenine.
Cockroach extractive of the present invention can also contain Buddhist nun's theobromine, and promptly extract contains biologically active peptide, acid mucopolysaccharide, compound nucleoside base, sticking propylhomoserin and Buddhist nun's theobromine; Perhaps biologically active peptide, acid mucopolysaccharide, compound nucleoside base, sticking propylhomoserin, ribosidoadenine and Buddhist nun's theobromine.
Cockroach extractive of the present invention can also contain glycine, and promptly extract contains biologically active peptide, acid mucopolysaccharide, compound nucleoside base, sticking propylhomoserin and glycine; Perhaps biologically active peptide, acid mucopolysaccharide, compound nucleoside base, sticking propylhomoserin, ribosidoadenine and glycine; Perhaps biologically active peptide, acid mucopolysaccharide, compound nucleoside base, sticking propylhomoserin, ribosidoadenine, Buddhist nun's theobromine and glycine.
Biologically active peptide of the present invention is a series of micromolecule oligopeptides, and by two more than the aminoacid, 50 following polymer that connect by amido link of aminoacid constitute.The molecular weight section is between 0.18--1KDa.
Acid mucopolysaccharide of the present invention is meant protein denaturation is separated, and holds back the following material of 100KDa.It has a kind of repetition disaccharidase structure, contains alduronic acid and amino sugar residue, and carboxyl in the molecule or sulfate link to each other with the oxygen atom of sugar or link to each other with the amino nitrogen atom of aminohexose.
Compound nucleoside base of the present invention is the class product behind the nucleolysis, finds in the experiment that it contains uracil and hypoxanthine.
Sticking propylhomoserin of the present invention is the class material that sugar derivatives forms in conjunction with aminoacid, and under acid condition, the heating hydrolyzable is aminoacid and carbohydrate derivative.
The invention provides a kind of preparation method of cockroach extractive, this preparation method comprises following steps:
A, cockroach is pulverized, add water and ethanol and/or methanol that 2-5 doubly measures, further refinement makes the cockroach cell rupture, wherein ethanol and/or methanol aqueous solution volumetric concentration 0≤V/V≤60%;
B, accent pH to 3-5; Homogenate;
C, airtight leaves standstill; Cockroach is fully soaked; 0-15 ℃ of solid-liquid separation, get supernatant;
D, heating liquid are to little boiling, and adjust pH removes deproteinize;
E, the following material of intercepting molecular weight 100KDa.
The granularity of " pulverizing " in the steps A can reach the millimeter level; " further refinement " is meant that by rubber mill the process that equipment such as tissue mashing machine diminish the material particle diameter can make particle diameter reach nanoscale by said process." refinement " time was at 60-90 minute.Extraction solvent is water and ethanol and/or methanol, and the expression extraction solvent is water, ethanol water, methanol aqueous solution or methanol and the blended aqueous solution of ethanol.When V/V=0, the expression extraction solvent is a water.When V/V=60%, the volumetric concentration of expression ethanol or methanol aqueous solution be 60% or the aqueous solution volumetric concentration of ethanol and methyl alcohol mixed liquor be 60%.
" homogenate " of step B is 0%≤v/v＜40% o'clock in the volumetric concentration of water and ethanol and/or methanol, and temperature need be at 70-90 ℃; The volumetric concentration of water and ethanol and/or methanol is 40%≤v/v≤60% o'clock, and temperature gets final product in room temperature.
Step C, the time of " cockroach is fully soaked " is generally 8--12 hour, and when commercial production, because the cause of working time, standing over night gets final product.The temperature 2-8 of solid-liquid separation ℃ is preferred.
Step D, the temperature difference of different extracting solution " little boiling " is generally at 80-100 ℃.By regulating pH value, make the protein sol instability in the solution, remove the protein in the extract.This method can not make the less biologically active peptide dissolubility of molecular weight diminish, and can not cause the biologically active peptide precipitation, so contain biologically active peptide in the extract of the present invention.Behind the isolating protein, also should transfer the pH4-5 of solution, heating, whether observing protein eliminates repeatedly.
Intercept the material of the following molecular weight of 100KDa then.
Preparation gained extracting solution should be preserved down at 2-8 ℃.
It is extraction solvent that the present invention adopts water and ethanol and/or methanol, and wherein ethanol and/or methanol aqueous solution volumetric concentration 0≤V/V≤60% can be finished goal of the invention; The volumetric concentration of ethanol of the present invention and/or methanol aqueous solution is a preferable range at 0%≤v/v≤40%, is more preferably scope at 0%≤v/v≤10%.
The present invention also provides the compositions of extract as medicinal application, and this extract can be made different products by different disposal technology respectively.Can vacuum concentration, make tablet, capsule, soft capsule, drop pill, extractum, powder, granule or powder preparation to be taken after being infused in boiling water, pill, unguentum and sublimed preparation.Can be after degerming, adjuvants such as adding mannitol are made powder ampoule agent for injection, especially freeze-dried powder injection.Can the degerming fill, obtain the injection of cockroach extractive.
The invention provides cockroach and obtain the application of extract in the medicine of preparation treatment heart failure, myocardial ischemia and hyperlipidemia.
The active constituent content of the extract of technology of the present invention is very high, for the application of this medicine provides the basis.The technology that is adopted has more specificity to the extraction of working substance, has avoided the stripping of impurity such as pigment and oil, thereby has reduced the step of remove impurity in extraction process, has shortened the production cycle, has reduced production cost; Avoided in a large number with an organic solvent having reduced industrial waste exhaust, made production more help environmental conservation.
Because work simplification was saved with the existing technology comparison production time, reduced the production cycle, increased economic efficiency.
Embodiment
Specifically introduce content of the present invention below by embodiment, but content of the present invention is not limited only to the content of embodiment.
Embodiment 1, with water the extraction solvent preparation
1, cockroach is crushed to≤1mm, in jacketed pan, adds 4 times of amount distilled waters, the circulation colloid mill is 60 minutes under frozen water cooling state.
2, transfer pH4.0 with HCL.
3, logical steam to 90 ℃ does not stop to stir, and 90 minutes, covered and enclosed left standstill overnight while hot.
4, keep 10 ℃ of temperature of liquid centrifugal, 4200r/min.Get supernatant.Take out precipitation, add 1 times of amount distilled water, be heated to 90 ℃, 20 minutes, cooling back frozen centrifugation.Merge supernatant.
5, above-mentioned supernatant slowly adds HCL while stirring, transfers pH4.0.In the jacketed pan internal heating to little boiling, stop heating.
6, poured into immediately while hot and covered in the stainless-steel pan, covered completely, left standstill overnight.
7, next day, get supernatant, precipitation is handled as step 4, merges supernatant.
8, step 7 supernatant is transferred pH8.5 with NaOH, is heated to and boils, and has moved to and has covered stainless-steel pan, covers completely, leaves standstill overnight.
9, next day, get supernatant.Lower sediment such as step 4 are handled, and merge supernatant.
10, this supernatant is transferred pH4.5 with HCL.
11, be heated to 90 ℃, 15 minutes.Observe repeatedly: in 30 minutes,, then cover completely, leave standstill overnight as producing precipitation.
12, next day, ultrafiltrate is collected in the 100KDa ultrafiltration.Be stored in refrigerator-freezer and make 2 ℃ of temperature maintenance, deposit to be checked.
Embodiment 2 is the extraction solvent preparation with 60% ethanol
1, cockroach is crushed to≤1mm, adds 3 times of amount 60% alcoholic solution, the circulation colloid mill is 90 minutes under room temperature state.
2, transfer pH4.0 with 2N HCL.
3, under the room temperature, magnetic stirrer does not stop to stir, and keeps 90 minutes, leaves standstill overnight.
4, it is centrifugal to keep 8 ℃ of temperature of liquid, 4200r/min.Get supernatant.In precipitation, add 1 times of amount extraction solvent, centrifugal.Merge supernatant.
5, above-mentioned supernatant slowly adds HCL while stirring, transfers pH4.0.Be heated to little boiling in condensation reflux unit.
6, poured into immediately while hot and covered stainless-steel pan, covered completely, left standstill overnight.
7, next day, get supernatant, precipitation is handled as step 4, merges supernatant.
8, step 7 supernatant is transferred pH8.0 with NaOH, is heated to 75 ℃, has moved to after 15 minutes and has covered stainless-steel pan, covers completely, leaves standstill overnight.
9, next day, get supernatant.Precipitation is handled as step 4, merges supernatant.
10, this supernatant is transferred pH4.5 with HCL.
11, be heated to 75 ℃, 15 minutes.Observe repeatedly: in 15 minutes,, moved to and covered in the stainless-steel pan, covered completely, left standstill overnight as producing precipitation.
12, above-mentioned solution moves into vacuum concentration pot, and 70 ℃ are concentrated into semiliquid thick paste shape, adds distilled water and most concentrates front volume.
13, ultrafiltrate is collected in 100KDa ultrafiltration, and putting refrigerator-freezer, to make temperature be 8 ℃, deposits to be checked.
Embodiment 3 is the extraction solvent preparation with 40% ethanol
1, cockroach is crushed to≤1mm, adds 3 times of amount 40% alcoholic solution, the circulation colloid mill is 70 minutes under room temperature state.
Step 2-4 is with embodiment 2.
5, above-mentioned supernatant is heated to little boiling, holding temperature in condensation reflux unit.Slowly add HCL while stirring, transfer pH4.0.
Step 6-10 is with embodiment 2.
11, be heated to 80 ℃, 15 minutes.Observe repeatedly: in 15 minutes,, moved to and covered in the stainless-steel pan, covered completely, left standstill every spending the night as producing precipitation.
Subsequent processing steps is with embodiment 2, ultrafiltrate, and putting refrigerator-freezer, to make temperature be 15 ℃, deposits to be checked.
The preparation of embodiment 460% methanol
Reaction condition and step be 60% ethanol extraction together, needs the reaction temperature control of heating strict, and whole experiment should be noted the seal of experimental apparatus.
Embodiment 540% methanol extraction
Reaction condition and step be 40% ethanol extraction together, needs the reaction temperature control of heating strict.Whole experiment should be noted the seal of experimental apparatus.
Embodiment 6 is the extraction solvent preparation with 30% ethanol
1, cockroach is crushed to≤1mm, adds 3 times of amount 30% alcoholic solution, the circulation colloid mill is 80 minutes under the frozen water cooling state.
2, transfer pH4.0 with HCL.
3, be heated to 80 ℃ in condensation reflux unit and do not stop to stir, kept 90 minutes, covered and enclosed leaves standstill overnight while hot.
4, it is centrifugal to keep 0 ℃ of temperature of liquid, 4200r/min.Get supernatant.In precipitation, add 1 times of amount extraction solvent, centrifugal.Merge supernatant.
5, above-mentioned supernatant slowly adds HCL while stirring, transfers pH4.0.Be heated to little boiling in condensation reflux unit.
6, poured into immediately while hot and covered stainless-steel pan, covered overnight leaving standstill completely.
7, next day, get supernatant, lower sediment such as step 4 are handled, and merge supernatant.
8, step 7 supernatant is transferred pH8.5 with NaOH, is heated to 80 ℃, has moved to after 10 minutes and has covered in the stainless-steel pan, covers completely, leaves standstill overnight.
9, next day, get supernatant.Lower sediment such as step 4 are handled, and merge supernatant.
10, this supernatant is transferred pH4.5 with HCL.
11, be heated to 80 ℃, 15 minutes.Observe repeatedly: in 15 minutes,, moved to and covered in the stainless-steel pan, covered completely, left standstill overnight as producing precipitation.
12, above-mentioned solution moves in the vacuum concentration pot, and 70 ℃ are concentrated into semiliquid thick paste shape, adds distilled water and most concentrates front volume.
13, ultrafiltrate is collected in 100KDa ultrafiltration, and putting refrigerator-freezer, to make temperature be 6 ℃, deposits to be checked.
Embodiment 7 is the extraction solvent preparation with 10% ethanol
1, cockroach is crushed to≤1mm, adds 3 times of amount 10% alcoholic solution, the circulation colloid mill is 80 minutes under room temperature state.
Step 2-4 is with embodiment 2.
5, above-mentioned supernatant slowly adds HCL while stirring, transfers pH4.0.Be heated to little boiling in condensation reflux unit.
Step 6-10 is with embodiment 6.
11, be heated to 80 ℃, 15 minutes.Observe repeatedly: in 15 minutes,, moved to and covered in the stainless-steel pan, covered completely, left standstill overnight as producing precipitation.
Subsequent processing steps is with embodiment 6.
Embodiment 8 is the extraction solvent preparation with 5% ethanol
1, cockroach is crushed to≤1mm, adds 3 times of amount 5% alcoholic solution, the circulation colloid mill is 80 minutes under room temperature state.
Step 2-4 is with embodiment 7.
5, above-mentioned supernatant slowly adds HCL while stirring, transfers pH4.0.In the jacketed pan internal heating to little boiling.
Step 6-10 is with embodiment 7.
11, be heated to 80 ℃, 15 minutes.Observe repeatedly: in 15 minutes,, moved to and covered in the stainless-steel pan, covered completely, left standstill overnight as producing precipitation.
Subsequent processing steps is with embodiment 7.
Embodiment 9 is the extraction solvent preparation with 30% methanol
Reaction condition and step be 30% ethanol extraction together, and whole experiment should be noted the seal of experimental apparatus.
Embodiment 10 is the extraction solvent preparation with 10% methanol
Reaction condition and step be 10% ethanol extraction together, and whole experiment should be noted the seal of experimental apparatus.
Embodiment 11 is the extraction solvent preparation with 5% methanol
Reaction condition and step be 5% ethanol extraction together, and whole experiment should be noted the seal of experimental apparatus.
Embodiment 12 is the extraction solvent preparation with the aqueous solution of ethanol and methanol mixed (volume ratio 1: 1), and the volumetric concentration of ethanol and methanol is 60%
Reaction condition and step be 60% ethanol extraction together, and whole experiment should be noted the seal of experimental apparatus.
Embodiment 13 is the extraction solvent preparation with the aqueous solution of ethanol and methanol mixed (volume ratio 1: 3), and the volumetric concentration of ethanol and methanol is 60%
Reaction condition and step be 60% ethanol extraction together, and whole experiment should be noted the seal of experimental apparatus.
Embodiment 14 is the extraction solvent preparation with the aqueous solution of ethanol and methanol mixed (volume ratio 1: 1), and the volumetric concentration of ethanol and methanol is 30%
Reaction condition and step be 30% ethanol extraction together, and whole experiment should be noted the seal of experimental apparatus.
Embodiment 13 is the extraction solvent preparation with the aqueous solution of ethanol and methanol mixed (volume ratio 3: 1), and the volumetric concentration of ethanol and methanol is 30%
Reaction condition and step be 30% ethanol extraction together, and whole experiment should be noted the seal of experimental apparatus.
Embodiment 14 freeze-dried powder injections:
The mannitol of adding 3% in the liquid of extract, adjusting pH is 4.5, under the state that air cleaning is local 100 grades, the 0.22um mocromembrane filters, sterile filling.Lyophilizing promptly.
Embodiment 15 injection
Add sodium chloride accent etc. in the liquid of extract and ooze, transfer pH4.5, under the aseptic condition, 0.22um filters embedding, promptly.
Embodiment 16 tablets
Every content
Extract: 40mg
Microcrystalline Cellulose: 55mg
Crosslinked carboxymethyl fecula sodium: 5mg
Magnesium stearate: 1mg
Technology: resultant extract liquid vacuum is condensed into extractum.Each adjuvant is crossed 80 mesh sieves, and mixing adds starch slurry (15-17%) system soft material, after granulating with 14 mesh sieves, puts 60 ℃ of dryings, and 2 mesh sieve granulate add crosslinked carboxymethyl fecula sodium and magnesium stearate mixing, tabletting, promptly.
Embodiment 17 soft capsules
Unit quantity (g/ grain)
Extract: 0.025
PEG：0.225
Gelatin: 0.16
Glycerol: 0.09
Hydroxyethyl-cellulose: 0.007
Citric acid: 0.008
Sorbic acid methyl ester: 0.001
Cera Flava: 0.01
Water: 0.25
Technology: resultant extract liquid vacuum is condensed into extractum.Take by weighing gelatin and add 100L gelatin retort, under stirring, add suitable quantity of water, airtight, treat that gelatin dissolves fully after, add glycerol, amount of colorant again, stir, vacuumize degassing 2 hours adds in the gelatin heat-preserving container, the insulation standing over night is stand-by.Suspending agent is added in the diluent, and mixing takes by weighing medicine, with mixing diluents, treats to add antioxidant, antiseptic behind the complete mixing, stirs, and room temperature leaves standstill.The gelatin heat-preserving container is suspended from certain altitude, the medicinal liquid for preparing is poured in the drug bucket, change mould and begin to press soft capsule, adjust the soft capsule device to scope of design, dry 24 hours, reject the soft gelatin capsule of presentation quality difference, with the soft gelatin capsule washing, dry 24 hours, get the soft capsule semi-finished product, the qualified back packing of quality inspection, labeling, packing get product.
Embodiment 18 small-molecule peptides qualitative
Adopt the polypeptide biuret reaction, get the 2.5ml sample solution, add trichloroacetic acid (TCA) aqueous solution of 2.5ml10% (W/V), mix homogeneously, leave standstill 10min, filter then, get filtrate 1ml, add 2 of 10% sodium hydroxide solution, fully shake up, add copper sulfate test solution gradually, shake up with adding, solution presents aubergine.
Through above-mentioned processing, prove and contain polypeptide in the sample.
Embodiment 19 small-molecular peptides quantitatively
Biuret method: get the 2.5ml sample solution, trichloroacetic acid (TCA) aqueous solution that adds 2.5ml10% (W/V), with mix homogeneously on the whirlpool mixed instrument, leave standstill 10min, centrifugal 15min under 4000r/min then, supernatant is all transferred in the 50ml volumetric flask, and be settled to scale, shake up with 5%TCA.Getting the above-mentioned solution of 6.0ml then puts in another test tube, add biuret reagent 4.0ml (sample liquid: biuret reagent=3: 2, v/v), with mix homogeneously on the whirlpool mixed instrument, leave standstill 10min, the centrifugal 10min of 2000r/min gets supernatant and measures the OD value down in 540nm, the reference standard curve is tried to achieve the peptide concentration C (mg/ml) in the sample, calculates the content of polypeptide in the sample.
The making of standard curve: the volumetric flask of getting 10 10ml, prepare 0.0,0.2,0.4,0.6,0.8,1.0,1.2,1.4,1.6 and the Gly-Gly-Tyr-Arg tetrapeptide standard solution of 1.8mg/ml successively with 5%TCA, get the 6.0ml standard solution then respectively, the biuret reagent that adds 4.0ml, mix homogeneously on the whirlpool mixed instrument, leave standstill 10min, the centrifugal 10min of 2000r/min gets supernatant and measures OD value (doing blank with first pipe) down in 540nm.Concentration with peptide is abscissa X (mg/ml), and the OD value is vertical coordinate Y, the production standard curve.
Through measuring the content such as the following table of each batch sample small-molecular peptides:
Embodiment 20 hypoxanthine, uracil, inosine qualitative
With octadecyl silane is filler, and it is an amount of that precision takes by weighing hypoxanthine, uracil, inosine reference substance, and each is mixed with the solution that every 1ml contains 0.12mg with mobile phase A, promptly gets reference substance solution with 1: 1: 1 volume mixture again.Precision takes by weighing sample 0.05g, adds mobile phase A 50ml dissolving and promptly gets need testing solution.Get each 20 μ l of reference substance and test sample, inject chromatograph of liquid.Mobile phase A: PH=5.3, the 0.05mol/L sodium acetate buffer; Mobile phase B: with mobile phase A prepare 80% methanol.Detect wavelength 260nm, the 0.6ml/min that puts up a guest for the night, balance B%=0 starts that B% is the 0-35% linear gradient in the 15min of back, and 35% returns B%=0 after keeping 2min, writes down about 20min chromatogram.Theoretical cam curve by uracil calculate should be not less than 1000, three peaks get retention time should be with hypoxanthine, uracil in the reference substance solution, that three peaks of inosine get retention time is consistent, the retention time at three peaks is about 10.895,7.655,14.273 successively.
Embodiment 21 hypoxanthine, uracil quantitatively
Chromatographic condition and system suitability octadecyl silane are filler; Mobile phase A: sodium acetate buffer (PH5.0); Mobile phase B: with 80% methanol of A preparation.Detect wavelength 260nm, flow velocity 0.6ml/min uses the 3%B balance, starts back mobile phase and keeps 3 minutes with 3%, changes 30%B into then in 2 minutes, keeps after 7 minutes and returns 3%B.Write down 14 minutes chromatogram.Number of theoretical plate calculates by uracil and hypoxanthine peak respectively all should be not less than 1000, and the separating degree between uracil and the hypoxanthine should meet the requirements.
Algoscopy is got the about 50mg of this product, and accurate the title decides, and puts in the 50ml measuring bottle, adds the mobile phase A dissolving and is diluted to scale, shakes up, and filters, as need testing solution; Get 20 μ l and inject chromatograph of liquid, the record chromatogram; Other gets uracil and the hypoxanthine reference substance is an amount of, with mobile phase A dissolving and be diluted to the mixing reference substance solution that every 1ml contains uracil and hypoxanthine 25 μ l respectively, measure with method, by external standard method with calculated by peak area.
Through measuring each batch sample uracil and Determination of Hypoxanthine such as following table:
Embodiment 22 compound nucleoside bases qualitative
Precision takes by weighing this product 0.05g, in the 50ml measuring bottle, with glycinate acid buffer dissolving and be diluted to scale, shakes up, and precision is measured 5ml in the 100ml volumetric flask, adds above-mentioned buffer to scale, shakes up, and promptly gets need testing solution.Measure according to spectrophotography (two appendix IV of Chinese Pharmacopoeia version in 2005), this product has absorption maximum at 260 ± 3nm wavelength place, and there is minimal absorption at the place at 236 ± 2nm wavelength, illustrates to contain compound nucleoside base.
Embodiment 23 compound nucleoside bases quantitatively
Precision takes by weighing this product 0.05g, in the 50ml measuring bottle, with glycinate acid buffer dissolving and be diluted to scale, shakes up, and precision is measured 5ml in the 100ml volumetric flask, adds above-mentioned buffer to scale, shakes up, and promptly gets need testing solution.Measure according to spectrophotography (two appendix IV of Chinese Pharmacopoeia version in 2005), measure trap at 260 ± 3nm wavelength place, calculate the content of compound nucleoside base.
Through measuring the content such as the following table of the compound nucleoside base of each batch sample:
Embodiment 24 acid mucopolysaccharide qualitative
1) toluidine blue test: get 1 of this product solution, point is on chromatography filter paper, after to be dried, drip toluidine blue solution and [get toluidine blue 60mg, add acetum (1 → 200) 100ml and make dissolving] dyeed 1～2 minute, use the unnecessary toluidine blue solution of acetum (1 → 200) flush away then, occur hepatic speckle near the point sample.
2) sulfate radical is differentiated: get two parts of need testing solutions, put respectively in the 50ml nessler colorimetric tube, portion adds 25% barium chloride solution 5ml, shakes up, place 10min, filter repeatedly, clarify fully, add an amount of potassium sulfate solution and water to 50ml to filtrate, shake up, place 10min, solution in contrast.Add 25% barium chloride solution 5ml in another part and make into 50ml in right amount with water, shake up, place 10ml, with putting on the black background, observe downwards from the color comparison tube top, relatively, test sample is positive.
More than reaction shows positive simultaneously, contains acid mucopolysaccharide in the interpret sample.
Embodiment 25 acid mucopolysaccharide quantitatively
The preparation of standard solution accurately takes by weighing 60 ℃ of glucuronic acid standard substance 15mg that are dried to constant weight, place the 25ml measuring bottle, the adding distil water dissolving, and be diluted to scale, shake up, precision is measured 1ml and is put in the 25ml measuring bottle thin up to scale (containing glucuronic acid 24 μ g among the every ml of this solution).
The preparation precision of need testing solution takes by weighing 60 ℃ of sample powder 60mg that are dried to constant weight, places 25ml measuring bottle adding distil water to make dissolving, and is diluted to scale, shakes up; Precision is measured 2ml, places the 25ml measuring bottle, and thin up shakes up to scale (containing this product 192 μ g among every ml), filters, promptly.
The algoscopy precision is measured need testing solution and each 1ml of standard solution, put respectively in the 10ml tool plug test tube, all accurate adding sodium tetraborate-sulfuric acid solution (takes by weighing sodium tetraborate 1.91g, put adding distil water 5ml in the 500ml beaker, adding sulphuric acid 195ml. cools in the rearmounted reagent bottle promptly) 6ml, shake up room temperature and place 20min, add 0.2% carbazole solution 0.2ml, shake up, heat 10min in the boiling water bath, after room temperature is placed 20min, according to spectrophotography (two appendix IV of Chinese Pharmacopoeia version in 2005), measure absorbance, result of calculation at 520nm wavelength place.
Experimental result is as follows
Embodiment 26 sticking propylhomoserin analyses
Adopt the method for CN941018943 to detect, sample shows bluish violet under long wave ultraviolet light, and hydrolysis goes out ammonia, aminoacid, sulfate, glucuronic acid and glucosamine under strong acid and hot conditions, proves to contain sticking propylhomoserin.
Earlier the small-molecule peptide material is removed, generally can be adopted solvent precipitation, as high concentration ethanol, trichloroacetic acid etc.And then according to " Study on Several Problems in the grand injection quality standard of heart arteries and veins " (sharp DaLi Medicine Academy journal; 2000,9 (3): 7-8) Bao Dao sticking propylhomoserin analytical, sticking histidine content is as follows:
Embodiment 27 prevents the myocardial ischemia pharmacodynamics test
1, laboratory animal SD rat is 40, body weight (210 ± 20) g, male and female half and half.
Rat by male and female half and half, is divided into 4 groups at random.Be respectively the blank group, ischemia model group, positive controls (ethanol extraction 4mg/kg), sample sets (4mg/kg).4 rats of blank group, all the other each groups are 12.Each group all adopts intraperitoneal injection.Blank group and model group wait the normal saline of capacity, and the administration volume is the 2mg/kg body weight.Wherein " ethanol extraction " is that (publication number is CN94118839.6: the CN1124141) method of record preparation according to number of patent application.
2. experimental technique: after each organized rat administration 30min, with 20% urethane intraperitoneal injection of anesthesia rat, record standard II lead electrocardiogram 1min was as normal value before the myocardial ischemia.Sample sets, positive controls and ischemia model group respectively in 8s at the uniform velocity sublingual vein inject pituitrin 0.5 μ g/kg, the preparation Model Rats with Acute Myocardial Ischemia, the blank group waits the normal saline of capacity.Write down 5s behind the quiet notes Pit respectively, 10s, 15s, 30s, 1min, 2min, J point and T crest value are measured in Electrocardiographic variation when 5min and 10min.With the electrocardiogram J point, T wave amplitude and heart rate are as degree of myocardial ischemia and drug effect Evaluation on effect index.
Rat heart is cut, wash away blood stains with normal saline, in-20 ℃ of refrigerators behind the freezing 15min from the apex of the heart entad the end direction heart evenly is cut to 8, be placed in 0.1% the N-BT solution, 37 ℃ of gas bath jolting 10-15min, blot surface moisture with absorbent paper, cut infarcted region and non-infarcted region respectively and weigh calculating heart infarction index.Heart infarction index=infarcted region wets, and quality/quality whole-heartedly wets.
3. statistical procedures
3.1 calculate J point change in displacement value behind each group injection Pit respectively, the value that slows down of T wave-amplitude changing value and heart rate is calculated the average and the standard deviation of each index with Excel, and and the ischemia model group organize a t and check.
Each group of table one is to rat electrocardiogram J point change in displacement value behind the pituitrin
Annotate: compare with the ischemia model group, P≤0.05,
Each group of table two is to rat ECG T wave amplitude variations value behind the pituitrin
Annotate: compare P≤0.05 with the ischemia model group
3.2 Blatta seu periplaneta extract is to the exponential influence of acute myocardial ischemia rat heart infarction:
| Group | N (only) | Dosage (mg/kg/d) | Infarction weight/heart gross weight (g) |
| The blank group | 4 | - | 0 |
| The ischemia model group | 12 | - | 0.302±0.095 |
| Sample sets | 12 | 4 | 0.226±0.065 |
| Positive controls (ethanol extraction) | 12 | 4 | 0.243±0.084 |
Annotate: compare P＜0.01 with model group
3.3 Blatta seu periplaneta extract is to the influence of acute myocardial ischemia rat blood serum CK, LDH content
| Group | N (only) | Dosage (mg/kg/d) | LDH(U.L -1) | CK(U.L -1) |
| The blank group | 4 | - | 2132.9±810.1 | 525.8±205.1 |
| The ischemia model group | 12 | - | 2357.4±620.3 | 549.9±245.2 |
| Sample sets | 12 | 4 | 2098.9±799.3 | 520.9±135.5 |
| Positive controls (ethanol extraction) | 12 | 4 | 2157.3±469.7 | 525.1±125.2 |
4. result
4.1 as can be seen from the above table: Blatta seu periplaneta extract causes the obvious suppression effect of having raised (comparing P＜0.05 or P＜0.01 with model group) of Acute Myocardial Ischemia in Rats electrocardiogram J point and T ripple to pituitrin.
4.2 the morphology result shows that Blatta seu periplaneta extract causes the rat of acute myocardial infarction that the obvious suppression effect is arranged to Pit.
4.3 zymetology result shows that Blatta seu periplaneta extract can obviously reduce CK, LDH level, and ischemic myocardial is had protective effect.
The test of embodiment 28 blood fat reducing drug effects
1. laboratory animal SD male rat is 30, body weight (190 ± 20) g
2. experimental technique is fed normal diet with rat under experimental situation, observes 5-10 days, and caudal vein is got blood, from going out serum, measures every blood lipids index normal value.Divide 3 groups of rats at random, 10 every group, each treated animal is used high cholesterol diet instead and is fed, first group of not administration; Irritate stomach for second group and give sample (4mg/kg); Irritate stomach for the 3rd group and give ethanol extraction (4mg/kg).Eyeball is got the every blood lipids index of hematometry after 6 weeks.Wherein " ethanol extraction " is that (publication number is CN94118839.6: the CN1124141) method of record preparation according to number of patent application.
3. date processing
| Group | Dosage (mg/kg) | TC(mmol/l) | TG(mmol/l) | LDL-C(mmol/l) | HDL-C(mmol/l) |
| The normal control group | - | 1.54±0.22 | 0.50±0.24 | 0.98±0.15 | 0.27±0.15 |
| The hyperlipidemia model group | - | 10.14±4.21 | 0.93±0.45 | 9.63±4.32 | 0.17±0.56 |
| Sample sets | 4 | 2.51±0.98 | 0.35±0.04 | 3.11±0.15 | 0.35±0.25 |
| Positive controls | 4 | 4.22±0.25 | 0.59±0.36 | 5.44±3.21 | 0.29±0.33 |
Annotate: compare P＜0.01 with model group, compare P≤0.05 with positive controls.
4, the result compares with hyperlipidemia model, and the TC of sample sets, LDL-C and TG have descended 75.8%, 67.4%, 61.3% respectively, and HDL-C has raise 84.2%; The TC of positive controls, LDL-C and TG have descended 58.3%, 43.0%, 55.9% respectively, and HDL-C has raise 52.6%.Sample and positive control have significant difference, have significant blood fat reducing function.
The embodiment 29 cardiotonic tests of pesticide effectiveness
1. laboratory animal: 20 of Bufo siccuss, male and female have concurrently, body weight (80 ± 20) g
2. experimental technique: sample and positive control drug (ethanol extraction) are diluted to 0.4mg/ml with ringer solution respectively.
Bufo siccus is divided into 2 groups (sample sets, positive controls) at random, gets the frog heart for 10 every group and adopt the Si Shi frog heart cannula, change clothes liquid in the sleeve pipe continuously with ringer solution, to there not being color.Clamp the apex of the heart with the frog heart clip that has long line, long line links to each other with tonotransducer, with the activity of balance recorder record heart contraction.Record in the 15min stablize normal waveform after, adding concentration to sample sets is the sample solution 1ml of 0.4mg/ml, observes the myocardial contraction situation in the record 10min also; Positive controls adopts same experimental technique.Wherein " ethanol extraction " is that (publication number is CN94118839.6: the CN1124141) method of record preparation according to number of patent application.Write down of the influence of every group of medicine respectively, change percentage rate according to following formula myocardial contraction rate to the cardiac muscular tension of isolated frog heart:
Myocardium shrink tension before rate of change=(the preceding myocardium shrink tension of myocardial contraction tension force-administration after the administration)/administration.
3. date processing:
| Group | N (only) | Dosage (0.4mg/ml) | Myocardial contraction tension force (g) | Rate of change |
| Sample sets | 10 | 1ml | 0.82±0.007 | 95.2±0.07％ |
| Positive controls | 10 | 1ml | 0.72±0.005 | 71.8％±0.07％ |
| Blank (administration preceding 0 | - | - | 0.42±0.20 | - |
4. result of the test
The ringer solution of Blatta seu periplaneta extract has tangible excitation to isolated frog heart, with positive controls significant difference is arranged.
Claims (10)
1. one kind is applied to cardiovascular field, and the extract from cockroach obtains is characterized by: prepared by the method that comprises following steps:
A, be extraction solvent, cockroach pulverized, wherein ethanol and/or methanol aqueous solution volumetric concentration 0≤V/V≤60% with water and ethanol and/or methanol;
B, accent pH to 3-5; Homogenate;
C, airtight leaves standstill; Cockroach is fully soaked; 0-15 ℃ of solid-liquid separation, get supernatant;
D, heating liquid are to little boiling, and adjust pH removes deproteinize;
E, the following material of intercepting molecular weight 100KDa.
2. extract mainly contains biologically active peptide, acid mucopolysaccharide, compound nucleoside base and sticking propylhomoserin according to claim 1.
3. contain hypoxanthine, uracil, inosine, ribosidoadenine, nicotinic acid or glycine as extract as described in the claim 2.
4. method for preparing extractive according to claim 1 comprises following steps:
A, cockroach is pulverized, add water and ethanol and/or methanol that 2-5 doubly measures, further refinement makes the cockroach cell rupture, wherein ethanol and/or methanol aqueous solution volumetric concentration 0≤V/V≤60%;
B, accent pH to 3-5; Homogenate;
C, airtight leaves standstill; Cockroach is fully soaked; 0-15 ℃ of solid-liquid separation, get supernatant;
D, heating liquid are to little boiling, and adjust pH removes deproteinize;
E, the following material of intercepting molecular weight 100KDa.
5. method as claimed in claim 4, wherein the volumetric concentration of ethanol and/or methanol aqueous solution is 0≤V/V≤40%.
6. as claim 4 or 5 described methods, wherein the volumetric concentration of ethanol and/or methanol aqueous solution is 0≤V/V≤10%.
7. a pharmaceutical composition for the treatment of cardiovascular disease contains extract as claimed in claim 1 or 2 and pharmaceutically suitable carrier.
8. pharmaceutical composition as claimed in claim 7 is tablet, capsule, soft capsule, drop pill, extractum, powder, granule or powder preparation to be taken after being infused in boiling water, pill, unguentum or sublimed preparation.
9. pharmaceutical composition as claimed in claim 7 is lyophilized injectable powder or injection.
10. as the application of claim 1-5 or 8 or 9 any described cockroach extractives at the medicine of preparation treatment heart failure, myocardial ischemia or hyperlipidemia.
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| CN102293785A (en) * | 2011-08-29 | 2011-12-28 | 河南科技大学 | Method for extracting secondary metabolites of insects or arthropods |
| CN102319268A (en) * | 2011-08-12 | 2012-01-18 | 云南省腾冲制药厂 | A kind of injection that chronic heart failure levies and preparation method thereof of treating |
| CN102349931A (en) * | 2011-08-12 | 2012-02-15 | 云南省腾冲制药厂 | Cockroach extract, preparation method thereof, and application of cockroach extract preparation in treatment of cardiovascular disease |
| CN102973610A (en) * | 2012-12-22 | 2013-03-20 | 昆明赛诺制药有限公司 | Method for preparing phthisic adjuvant with extractum of Periplaneta americana |
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| CN1010828B (en) * | 1987-01-27 | 1990-12-19 | 大理医学院 | Process for preparing drugs of treating traumatic wound |
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| CN102319268A (en) * | 2011-08-12 | 2012-01-18 | 云南省腾冲制药厂 | A kind of injection that chronic heart failure levies and preparation method thereof of treating |
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| CN102319268B (en) * | 2011-08-12 | 2013-01-23 | 云南省腾冲制药厂 | Preparation method of injection used for treating chronic congestive cardiac failure |
| CN102293785A (en) * | 2011-08-29 | 2011-12-28 | 河南科技大学 | Method for extracting secondary metabolites of insects or arthropods |
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| CN114306389A (en) * | 2022-01-20 | 2022-04-12 | 西南大学 | Application of periplaneta americana extract in preparation of product for treating stroke recovery |
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