CN109966319A - A kind of raw material of Kangfuxin Liquid and its preparation method and application - Google Patents
A kind of raw material of Kangfuxin Liquid and its preparation method and application Download PDFInfo
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Abstract
The invention discloses raw materials of a kind of Kangfuxin Liquid and its preparation method and application.The raw material of the Kangfuxin Liquid is American cockroach extract, preparation method are as follows: (1) American cockroach carries out refluxing extraction by ethyl alcohol and water mixed solvent；(2) ethyl alcohol is recovered under reduced pressure；(3) purified water is added, flocculation clarification mixes, and stands removal precipitating and grease；(4) it carries out eluting to obtain American cockroach crude extract by macroreticular resin；(5) American cockroach extract is purified to obtain using alcohol precipitation；Content by peptides in the raw material American cockroach extract of the obtained Kangfuxin Liquid of the above method is 70~90%, the American cockroach extract, which prepares endo-medicine, can be used for gastritis, gastrorrhagia, alimentary canal wound repair etc., and preparing external drug can be used for wound, ulcer, burn etc..
Description
Technical field
The present invention relates to pharmaceutical technology field, in particular to a kind of raw material of Kangfuxin Liquid and preparation method thereof and answer
With.
Background technique
American cockroach Periplaneta americana L. is Blattaria Blattidae Periplaneta insect, and main component has
Polyalcohols, polypeptide and glutinous sugared propylhomoserin class etc., have effects that apparent promoting blood circulation, nourishing yin and promoting granulation, are clinically chiefly used in
Accelerate the reparation of its surface of a wound, accelerates falling off for surface of a wound necrotic tissue, promote the growth of granulation tissue, improve the microcirculation of the surface of a wound
Deng, it can be used for treating disease of digestive system, such as gastritis, ulcer, upper gastrointestinal bleeding, gastric cancer, esophagitis etc..
Macroporous absorbent resin is a kind of organic polymer adsorbent, and suction-operated is by adsorption, surface electrical behavior
Or form what hydrogen bond etc. reached, have selectivity good, adsorption capacity is big, high mechanical strength, and regeneration treatment is convenient, adsorption rate
Fastly, the advantages that parsing is easy, used high molecular polymer does not dissolve in any solvent, sufficiently stable at normal temperature, uses
Any substance release is not had in journey to come out, remaining certain impurity can also be cleaned out before use.
American cockroach belongs to animal drugs, and drug efficacy study is complicated, selects effective and reasonable analysis and research method, is conducive to sieve
Select effective substance, enrichment active principle, enhance curative effect of medication and follow-up study, and macroreticular resin isolate and purify polypeptide,
Have the advantages that selectivity is good, mild condition, easy to operate and cost is relatively low when the bioactive substances such as protein, passes through macropore
The method of resin, can effectively obtain the polypeptide isoreactivity substance in American cockroach, and obtained active material is that rehabilitation is new
The main component of liquid.
It is lower for the peptides extraction efficiency of American cockroach in method disclosed in the prior art, about 30~40% or so, and
The present invention can be improved the peptides content in gained American cockroach extract to 70~90%, significantly by improving preparation process
The recovery rate for improving American cockroach peptides, improve the drug action of its wound repair, increase American cockroach resource
Utilization rate.
Summary of the invention
It is an object of the invention to provide a kind of raw material American cockroach extract of Kangfuxin Liquid, the American cockroach essence is mentioned
Peptide constituents content is 70~90% in object.
The invention also discloses a kind of preparation method of the raw material American cockroach extract of Kangfuxin Liquid, specific steps are as follows:
1. the fresh worm of American cockroach or dry worm, refluxing extraction, obtain extracting solution A；
2. ethyl alcohol is recovered under reduced pressure in extracting solution A, medicinal extract B is obtained；
3. purified water is added into medicinal extract B, flocculation solution is added, is mixed, is stood, removal precipitating and grease obtain degreasing
Liquid C；
4. degreaser C collects to obtain ethanol eluate D, through being recovered under reduced pressure by nonpolarity and low pole macroporous absorbent resin
Ethyl alcohol is simultaneously concentrated to get medicinal extract；
5. a certain amount of ethyl alcohol is added, stand, filtration, recycle ethyl alcohol and be concentrated into thick medicinal extract, is dried under reduced pressure, crushes beautiful
The big Lian extract in continent.
Further, a kind of raw material American cockroach extract of Kangfuxin Liquid the preparation method comprises the following steps:
1. the fresh worm of American cockroach or dry worm carry out refluxing extraction using 0~90% ethyl alcohol and water mixed solvent, must extract
Liquid A；
2. ethyl alcohol is recovered under reduced pressure at 50~75 DEG C or less in extracting solution A, medicinal extract B is obtained；
3. the purified water of 6~14 times of medicinal extract weight is added into medicinal extract B, add total volume 1%~3% 4~7% are bright
Sol solution mixes, and stands 16~48 hours at 0~10 DEG C, and removal precipitating and grease obtain degreaser C；
4. degreaser C is by nonpolarity and low pole macroporous absorbent resin, first with the pure water rinsing of 2~6 times of bed volumes
Resin column, then with 40%~80% alcohol flushing resin column, collect to obtain ethanol eluate D, ethyl alcohol is recovered under reduced pressure and is concentrated
The medicinal extract that its relative density is 1.10~1.20 is detected to 60 DEG C；
5. a certain amount of ethyl alcohol is added, 16~48 hours are stood, filtration recycles ethyl alcohol and is concentrated into thick medicinal extract, and 55~75
It DEG C is dried under reduced pressure, crushes to obtain American cockroach extract.
Further, a kind of raw material American cockroach extract of Kangfuxin Liquid the preparation method comprises the following steps:
1. the fresh worm of American cockroach or dry worm carry out refluxing extraction using 0~90% ethyl alcohol and water mixed solvent, must extract
Liquid A；
2. ethyl alcohol is recovered under reduced pressure at 50~75 DEG C in extracting solution A, medicinal extract B is obtained；
3. the purified water of 8~12 times of medicinal extract weight is added into medicinal extract B, add total volume 1%~3% 4~7% are bright
Sol solution mixes, and stands 20~40 hours at 3~7 DEG C, and removal precipitating and grease obtain degreaser C；
4. degreaser C is by nonpolarity and low pole macroporous absorbent resin, first with the pure water rinsing of 4~6 times of bed volumes
Resin column, then with 50~75% alcohol flushing resin column, collect to obtain ethanol eluate D, ethyl alcohol is recovered under reduced pressure and is concentrated into
60 DEG C are detected the medicinal extract that its relative density is 1.10~1.20；
5. a certain amount of ethyl alcohol is added, 16~48 hours are stood, filtration recycles ethyl alcohol and is concentrated into thick medicinal extract, and 60~72
It DEG C is dried under reduced pressure, crushes to obtain American cockroach extract.
Further, a kind of raw material American cockroach extract of Kangfuxin Liquid the preparation method comprises the following steps:
1. the fresh worm of American cockroach or dry worm carry out refluxing extraction using 70% ethyl alcohol and water mixed solvent, obtain extracting solution A；
2. ethyl alcohol is recovered under reduced pressure at 70 DEG C in extracting solution A, medicinal extract B is obtained；
3. the purified water of 8 times of medicinal extract weight is added into medicinal extract B, mix, stands 24 hours at 5 DEG C, removal precipitating and oil
Rouge obtains degreaser C；
4. degreaser C is by nonpolarity and low pole macroporous absorbent resin, first with the pure water rinsing resin of 4 times of bed volumes
Column, then with 70% alcohol flushing resin column, collect to obtain ethanol eluate D, ethyl alcohol is recovered under reduced pressure and is concentrated into 60 DEG C of detections
The medicinal extract that its relative density is 1.10~1.20；
5. a certain amount of ethyl alcohol is added, 16~48 hours are stood, filtration recycles ethyl alcohol and is concentrated into thick medicinal extract, and 70 DEG C subtract
Pressure dry, pulverize to obtain American cockroach extract, i.e. American cockroach extract.
Further, in the American cockroach extract preparation method step 4. described in macroporous absorbent resin its diameter it is high
Ratio be diameter: height=1:8~12, the macroreticular resin include D101, AB-8, HPD100, HPD400 and HP20 type in
Any one；Step 5. described in a certain amount of ethyl alcohol of addition, the amount of the ethyl alcohol are as follows: be added ethyl alcohol to alcohol content reach 80
~90%.
Further, in the preparation method of the American cockroach extract step 4. described in macroporous absorbent resin its diameter
High ratio is diameter: height=1:10；Step 5. described in a certain amount of ethyl alcohol of addition, the amount of the ethyl alcohol are as follows: be added ethyl alcohol extremely
Alcohol content reaches 85%
Further, a kind of raw material American cockroach extract of Kangfuxin Liquid has preparation treatment wound repair medicine
The application of object, health care product, food, daily chemical products.
Further, a kind of raw material American cockroach extract of Kangfuxin Liquid is for oral administration can be used for preparing treatment hemostasis resistance
It is stagnant, have a stomachache bleeding, taste-blindness rate and deficiency of Yin tuberculosis, pulmonary tuberculosis；Diabetic foot ulcer, gastroesophageal reflux, gastritis
And the alimentary canals wound repair drug such as ulcerative colitis；External application can be used for preparing treatment metal-inflicted wound, wound, ulcer, fistula, burning
The surface of a wound drug of wound, scald, bedsore.
Further, the raw material American cockroach extract of a kind of Kangfuxin Liquid, which is characterized in that the America is big
Lian extract is plus pharmaceutically preparation is prepared in common auxiliary material or complementary ingredient.
Beneficial effects of the present invention:
The present invention carries out the peptides in American-cockroach-extract using flocculation clarification, alcohol precipitation binding resin purifying process pure
Change obtains the raw material American cockroach extract of Kangfuxin Liquid.It is purified using macroporous absorbent resin, by free amino acid, nucleosides
Base and monosaccharide have carried out preferable separation with peptide constituents；The present invention passes through on the basis of existing technology, to American cockroach
Crude extract carries out further alcohol precipitation and obtains American cockroach extract, thus by peptides content in American-cockroach-extract from 42~
54% increases to 70% or more, solves filtration difficulty, the rate of filtration in the heavy technique of raw water and holds slowly and in purifying resin technique
Susceptible to plugging problem, improves production efficiency, while the drug action of obtained American cockroach extract its wound repair
It greatly promotes.
Specific embodiment
Below by way of specific embodiment to the raw material American cockroach extract and its system of Kangfuxin Liquid of the present invention
Detailed elaboration is made in Preparation Method and application, and only several embodiments of the present invention are expressed for embodiments discussed below, Bu Nengli
Solution is limitations on the scope of the patent of the present invention, and the range that the invention patent is protected is subject to range described in claim.
Embodiment 1
The dry worm 25kg of American cockroach is taken, 70% alcohol reflux is added and extracts 3 times, 250 liters are added for the first time, extracts 1 hour；
Second of 200 liters of addition, is extracted 1 hour；3rd 200 liters of addition, is extracted 1 hour.Combined extract, filtration, 70 DEG C depressurize back
Ethyl alcohol is received to no alcohol taste, 200 liters of purified waters are added, add 5% gelatin solution of 4.5kg, is mixed, 5 DEG C standing 24 hours,
Removal grease and lower sediment obtain degreaser, filter, the AB-8 type macropore for being 25 liters by the total bed volume handled well
Adsorption resin column (diameter: height=1:10) is received with 100 liters of pure water rinsing resin columns, then with 80 liter 70% of ethanol-eluting resin column
Collect 60 liters of eluents, it is 1.15 (60 DEG C of surveys) that 70 DEG C of eluent, which are recovered under reduced pressure ethyl alcohol and are concentrated into relative density, takes half medicinal extract
70 DEG C are dried under reduced pressure to obtain American cockroach crude extract 405.1g.The other half medicinal extract is added ethyl alcohol and stands 24 to alcohol content up to 85%, 5 DEG C
Hour, filtration, 65 DEG C of recycling ethyl alcohol of filtrate are simultaneously concentrated into thick medicinal extract, and 65 DEG C are dried under reduced pressure, and crush, obtain American cockroach extract
220.5g.Using peptide constituents in HPLC measurement extract, peptide constituents content is 52.6% in American cockroach crude extract, and beautiful
The content of peptide constituents is 90.0% in the big Lian extract in continent.
Embodiment 2
The fresh worm 40kg of American cockroach is taken, 75% alcohol reflux is added and extracts 3 times, 320 liters are added for the first time, extracts 2 hours；
Second of 240 liters of addition, is extracted 1 hour；3rd 240 liters of addition, is extracted 1 hour.Combined extract, filtration, 70 DEG C depressurize back
Ethyl alcohol is received to no alcohol taste, 320 liters of purified waters are added, add 7% gelatin solution of 3.5kg, is mixed, heats and boils 0.5 hour,
36 hours are stood at 2 DEG C, grease is removed and lower sediment obtains degreaser, filter, be by the total bed volume handled well
30 liters of AB-8 type large pore resin absorption column (diameter: height=1:10), with 150 liters of pure water rinsing resin columns, then with 120 liter 75%
Ethanol-eluting resin column, collects 90 liters of elutions, and it is 1.17 (60 DEG C that 70 DEG C of eluent, which are recovered under reduced pressure ethyl alcohol and are concentrated into relative density,
Survey), take 70 DEG C of half medicinal extract to be dried under reduced pressure to obtain American cockroach coarse extraction 442.8g.The other half medicinal extract is added ethyl alcohol to alcohol content and reaches
85%, 1 DEG C stands 24 hours, and filtration, 65 DEG C of recycling ethyl alcohol of filtrate are simultaneously concentrated into thick medicinal extract, and 65 DEG C are dried under reduced pressure, and crushes, obtains beautiful
The big Lian extract 271.2g in continent.Peptide constituents in extract, peptide constituents content in American cockroach crude extract are measured using HPLC
It is 54.0%, and the content of peptide constituents is 87.1% in American cockroach extract.
Embodiment 3
The fresh worm 10kg of American cockroach is taken, 90% alcohol reflux is added and extracts 3 times, 100 liters are added for the first time, extracts 2 hours；
Second of 80 liters of addition, is extracted 1.5 hours；3rd 60 liters of addition, is extracted 1 hour.Combined extract, filtration, 75 DEG C depressurize back
Ethyl alcohol is received to no alcohol taste, 140 liters of purified waters are added, add 4% gelatin solution of 4.5kg, is mixed, 0 DEG C standing 16 hours,
Removal grease and lower sediment obtain degreaser, filter, and are inhaled by the AB-8 type macropore that the total bed volume handled well is 8 liters
Attached resin column (diameter: height=1:8), with 48 liters of pure water rinsing resin columns, then with 48 liter 80% of ethanol-eluting resin column, collection is washed
De- liquid is closely colourless to efflux；It is 1.2 (60 DEG C of surveys) that 75 DEG C of eluent, which are recovered under reduced pressure ethyl alcohol and are concentrated into relative density, takes half
70 DEG C of medicinal extract are dried under reduced pressure to obtain American cockroach crude extract 66.7g.Ethyl alcohol is added to alcohol content up to 80% in the other half medicinal extract, stands 48
Hour, filtration, 70 DEG C of recycling ethyl alcohol of filtrate are simultaneously concentrated into thick medicinal extract, and 70 DEG C are dried under reduced pressure, and crush, obtain American cockroach extract
42.5g.Using peptide constituents in HPLC measurement extract, peptide constituents content is 49.3% in American cockroach crude extract, and beautiful
The content of peptide constituents is 70.0% in the big Lian extract in continent.
Embodiment 4
The fresh worm 10kg of American cockroach is taken, 50% alcohol reflux is added and extracts 3 times, 80 liters are added for the first time, it is small to extract 1.5
When；Second of 75 liters of addition, is extracted 1.5 hours；3rd 40 liters of addition, is extracted 1 hour.Combined extract, filtration, 60 DEG C subtract
It pushes back and receives ethyl alcohol to no alcohol taste, 120 liters of purified waters are added, add 5% gelatin solution of 2.4kg, mix, stand 24 at 10 DEG C
Hour, it removes grease and lower sediment obtains degreaser, filter, the HPD100 for being 10 liters by the total bed volume handled well
Type large pore resin absorption column (diameter: height=1:12), with 20 liters of pure water rinsing resin columns, then with 60 liter 40% of ethanol elution resin
It is closely colourless to efflux to collect eluent for column；It is 1.15 (60 DEG C that 70 DEG C of eluent, which are recovered under reduced pressure ethyl alcohol and are concentrated into relative density,
Survey), take 65 DEG C of half medicinal extract to be dried under reduced pressure to obtain American cockroach crude extract 81.5g.The other half medicinal extract is added ethyl alcohol to alcohol content and reaches
90%, 16 hours are stood, filtration, 65 DEG C of recycling ethyl alcohol of filtrate are simultaneously concentrated into thick medicinal extract, and 70 DEG C are dried under reduced pressure, and crush, obtain America
Big Lian extract 46.1g.Using peptide constituents in HPLC measurement extract, peptide constituents content is in American cockroach crude extract
50.4%, and the content of peptide constituents is 80.9% in American cockroach extract.
Embodiment 5
The dry worm 10kg of American cockroach is taken, is added purified water refluxing extraction 3 times, 120 liters are added for the first time, is extracted 2 hours；The
It is secondary to be added 100 liters, it extracts 1.5 hours；3rd 80 liters of addition, is extracted 1 hour.Combined extract, filtration, 70 DEG C of decompressions are dense
Contracting, is added 60 liters of purified waters, 4.5% gelatin solution of 2kg is added, is mixed, 24 hours is stood at 3 DEG C, supernatant is taken to filter
It crosses, the D101 type large pore resin absorption column (diameter: height=1:10) for being 12 liters by the total bed volume handled well, with 50 liters
Pure water rinsing resin column, then with 50 liter 70% of ethanol-eluting resin column, it is closely colourless to efflux to collect eluent；Eluent 60
DEG C ethyl alcohol being recovered under reduced pressure and being concentrated into relative density is 1.18 (60 DEG C of surveys), takes 65 DEG C of half medicinal extract to be dried under reduced pressure to obtain American cockroach
Crude extract 275.1g.Ethyl alcohol is added to alcohol content up to 85% in the other half medicinal extract, stands 36 hours, filtration, 68 DEG C of recycling second of filtrate
Alcohol is simultaneously concentrated into thick medicinal extract, and 65 DEG C are dried under reduced pressure, and crushes, obtains American cockroach extract 171.3g.Extract is measured using HPLC
Middle peptide constituents, peptide constituents content is 50.6% in American cockroach crude extract, and peptide constituents in American cockroach extract
Content is 75.8%.
Embodiment 6
The dry worm 15kg of American cockroach is taken, 75% alcohol reflux is added and extracts 2 times, 170 liters are added for the first time, extracts 3 hours；
Second of 150 liters of addition, is extracted 2 hours, combined extract, filtration, and 62 DEG C are recovered under reduced pressure ethyl alcohol to no alcohol taste, and 90 liters of addition is pure
Change water, add 4% gelatin solution of 3kg, mix, stand 16 hours at 5 DEG C, removes grease and lower sediment obtains degreaser,
Filtration, the HPD400 type large pore resin absorption column (diameter: height=1:10) for being 15 liters by the total bed volume handled well, is used
90 liters of pure water rinsing resin columns, then with 60 liter 80% of ethanol-eluting resin column, it is closely colourless to efflux to collect eluent；Elution
It is 1.10 (60 DEG C of surveys) that 75 DEG C of liquid, which are recovered under reduced pressure ethyl alcohol and are concentrated into relative density, takes 70 DEG C of half medicinal extract to be dried under reduced pressure to obtain America
Big Lian crude extract 231.4g,.The other half medicinal extract is filtered with 200 mesh gauzes, and ethyl alcohol is added to alcohol content up to 85%, it is small to stand 36
When, filtration, 55 DEG C of recycling ethyl alcohol of filtrate are simultaneously concentrated into thick medicinal extract, and 60 DEG C are dried under reduced pressure, and crush, obtain American cockroach extract
122.7g.Using peptide constituents in HPLC measurement extract, peptide constituents content is 46.6% in American cockroach crude extract, and beautiful
The content of peptide constituents is 76.3% in the big Lian extract in continent.
Embodiment 7
The dry worm 20kg of American cockroach is taken, 30% alcohol reflux is added and extracts 3 times, 200 liters are added for the first time, extracts 2 hours；
Second of 180 liters of addition, is extracted 1.5 hours；3rd 160 liters of addition, is extracted 0.5 hour.Combined extract, filtration, 73 DEG C subtract
It pushes back and receives ethyl alcohol to no alcohol taste, 200 liters of purified waters are added, add 6% gelatin solution of 2.1kg, mix, heating boils 0.5
Hour, 30 hours are stood at 2 DEG C, grease is removed and lower sediment obtains degreaser, filter, pass through the total column bed body handled well
Product is 25 liters of HP20 type large pore resin absorption column (diameter: height=1:10.5), with 80 liters of pure water rinsing resin columns, then with 70 liters
It is closely colourless to efflux to collect eluent for 55% ethanol-eluting resin column；72 DEG C of eluent are recovered under reduced pressure ethyl alcohol and are concentrated into
Relative density is 1.13 (60 DEG C of surveys), takes 68 DEG C of half medicinal extract to be dried under reduced pressure to obtain American cockroach coarse extraction 323.5g.The other half leaching
Ethyl alcohol is added to alcohol content up to 86% in cream, stands 30 hours, and filtration, 55 DEG C of recycling ethyl alcohol of filtrate are simultaneously concentrated into thick medicinal extract, and 62 DEG C
It is dried under reduced pressure, crushes, obtain American cockroach extract 163.5g.Using peptide constituents in HPLC measurement extract, American cockroach is thick
Peptide constituents content is 42.0% in extract, and the content of peptide constituents is 83.5% in American cockroach extract.
The flocculation clarifying effect verification test of embodiment 8
The dry worm 20kg of American cockroach is taken, 60% alcohol reflux is added and extracts 3 times, 200 liters are added for the first time, extracts 2 hours；
Second of 180 liters of addition, is extracted 1.5 hours；3rd 160 liters of addition, is extracted 0.5 hour.Combined extract, filtration, 70 DEG C subtract
It pushes back and receives ethyl alcohol to no alcohol taste, 200 liters of purified waters are added, mixes, is divided into two parts, a copy of it heating is boiled 0.5 hour, 2
DEG C 30 hours are stood, removes grease and lower sediment and obtain degreaser, filter；Another adds 5% gelatin solution of 1kg, mixes
Even, heating is boiled 0.5 hour, stands 30 hours at 2 DEG C, removes grease and lower sediment obtains degreaser, filter, and compares filtration speed
The clarity of degree and filtrate, the results are shown in Table 1.
1 gelatin solution of table flocculation clearance compliance test result result
From above-mentioned test result can be seen that gelatin solution flocculation clarifying effect it is obvious, solve water sink liquid filtration difficulty,
The rate of filtration is slow and the muddy drawbacks such as unstable of filtrate, improves production efficiency, and lay a good foundation for subsequent purification technique.
The measurement of 9 American-cockroach-extract peptide constituents of embodiment
1. American cockroach extract ultrafiltration analysis
Take 1~7 American cockroach extract of above-described embodiment of identical weight appropriate respectively, pure water, which is added, to be made to dissolve, it filters,
Filtrate is divided into three parts, wherein the poly- maple ultrafiltration membrane of two parts of 1000 dalton of difference and the poly- maple ultrafiltration membrane filtration of 1500 dalton,
Filtrate is collected, the total solid of filtrate is measured, as a result see the table below.
2 American cockroach extract peptide constituents ultrafiltration analysis of table
From above-mentioned test result, it can be concluded that, 95% and 96% or more chemical component can pass through in American cockroach extract
The ultrafiltration membrane of 1kDa and 1.5kDa, illustrate in extract be mostly 1.5kDa, even 1kDa chemical component.
2. the detection of peptide constituents in American-cockroach-extract
The content of total peptide in the big Lian coarse extraction in 1~7 Central America of above-described embodiment and extract is measured using HPLC-ELSD,
Total hydrolysis amino acid and free amino acid are first measured, two differences are exactly total peptide content, and the specific method is as follows.
Chromatographic condition and system suitability using octadecylsilane chemically bonded silica as filler (moon rising sun LP-C18,
4.6mm × 250mm, 5 μm)；Using 1% hyptafluorobutyric acid aqueous solution as mobile phase A, using methanol as Mobile phase B, by the regulation in table 3
Carry out gradient elution；Evaporative light scattering detector, column temperature are 25 DEG C.Theoretical cam curve should be not less than by the calculating of phenylalanine peak
5000。
3 gradient elution table of table
The preparation of reference substance solution takes 17 kinds of amino acid mixed standard solutions to cross 0.2 μm of miillpore filter, takes subsequent filtrate to obtain the final product
Reference substance solution.
The preparation of test solution takes this product about 1g, accurately weighed, sets in stuffed conical flask, and water 50ml is added in precision, claims
Determine weight, be ultrasonically treated (power 200W, frequency 40kHz) 60min, let cool, then weighed weight, the weight of less loss is supplied with water,
It shakes up, is centrifuged (8 000r/min) 30min, precision pipettes centrifugate 10ml, is evaporated, accurate in residue that 0.1mol/L hydrochloric acid is added
Solution 2ml dissolution, mix, filtration, take subsequent filtrate to get.
Measuring method is accurate respectively to draw 5 μ l of reference substance solution, 10 μ l, and 10 μ l of test solution injects liquid chromatograph, surveys
It is fixed, it is calculated with external standard two-point method logarithmic equation to get free and hydrolysis amino acid total amount.It is slightly mentioned by the way that American cockroach is calculated
Take and extract in total peptide content, specific test data see the table below.
Total peptide content measurement result in 4 American-cockroach-extract of table
It is obtained from above-mentioned test result, mentions the content of total peptide in American-cockroach-extract from 49.4% by alcohol precipitation process
To 80.5%, and in extract, the overwhelming majority can pass through the poly- maple ultrafiltration membrane of 1kDa to height, illustrate in American cockroach extract
Peptide constituents are small-molecular peptides.
Comparative example 1
The preparation of American cockroach liquid extract: dry American cockroach polypide is crushed, and crosses 24 meshes, and it is thick that American cockroach is made
Powder；It is impregnated with the ethyl alcohol of mass concentration 95%, ethyl alcohol liquid level is made to be higher by the closed preservation of 5~10cm of the American cockroach coarse powder, made
Obtain coarse powder mixed liquor；In 75~80 DEG C of at a temperature of 3 refluxing extractions of progress, when extract for the first time to coarse powder mixed liquor,
The ethyl alcohol of 6 times of overstriking powder weight amount mass concentrations 92%, is extracted 5 hours；Second of overstriking powder weight, 4 times of amount mass concentrations 92%
Ethyl alcohol, extract 4 hours；The ethyl alcohol of 4 times of weight of third time overstriking powder amount mass concentrations 92%, is extracted 4 hours；Merging mentions three times
Liquid is taken, ethanol extract is filtered to obtain；It is concentrated under reduced pressure under conditions of vacuum degree -0.05~-0.06MPa, temperature 60 C, until specific gravity
When being 1.05, ethyl alcohol concentrate is obtained；The distilled water of 8 times of ethyl alcohol concentrate weight amounts is added, warming while stirring is to 80~90 DEG C
Afterwards, it stirs 40 minutes, mixing speed is 40 revs/min, and then heat preservation, which stands 4 hours, is layered grease；Lower layer's aqueous is released, is passed through
After filter paper filtering, filtrate is concentrated under reduced pressure under the conditions of temperature 60 C, vacuum degree -0.05~-0.06MPa, is concentrated into specific gravity and is
When 1.15, American cockroach liquid extract is made.10g American cockroach liquid extract is taken to continue to be concentrated to specific gravity under this condition to be 1.23,
It is dry, American cockroach dry extract about 23g can be obtained；By liquid extract through AB-8 type macroporous adsorbent resin column chromatography, washing, then 90%
Alcohol-water elution, finally uses anhydrous ethanol elution, collects eluent respectively, be concentrated under reduced pressure into no alcohol taste, vacuum decompression at 60 DEG C
It dry, pulverize to obtain American-cockroach-extract G.
Comparative example 2
50 DEG C~60 DEG C of hot water of adult by blattaria culturing time at 5 months or more scalds after death, through 60~70 DEG C of temperature
Spend lower crushed after being dried；Gained powder is placed in extractor, alcoholic solvent is added and carries out refluxing extraction, is extracted three times, first time 3
~5 hours, second and third time 2~4 hours, reflux temperature was controlled at 75~80 DEG C, was the filter cloth mistake of 200~500 mesh with aperture
Filter, merging filtrate.Cold soaking extracts: it extracts three times, impregnate 72 hours every time or more, it is primary at interval of stirring in 6~12 hours therebetween,
It is the filter-cloth filtering of 200~500 mesh, merging filtrate with aperture.Above-mentioned filtrate decompression is concentrated, temperature is 50~70 DEG C, concentration
To medicinal extract without alcohol taste, relative density is 1.05~1.20 to get blattaria alcohol extract at this time.Stream leaching is added in oily-water seperating equipment
The hot water of 3 times of 60~75 DEG C of amounts of cream and medicinal extract volume, is slowly stirred along a direction, and mixing speed is 30~50 turns/
Minute, while 65~75 DEG C are heated to, keep the temperature 1 hour, stand 12 hours cooling, releases water layer after grease separation, with 200~
500 mesh filter-cloth filterings collect filtrate, filtrate decompression concentration, and temperature is 55~65 DEG C, and being concentrated into relative density is 1.1~1.3,
Up to blattaria degreasing extract.AB-8 macroporous absorbent resin segmentation purification: by the ethanol extract of blattaria 50~95% through degreasing
Upper large pore resin absorption column afterwards, and adsorbing 2~3 hours, is washed with water colourless to eluent, with the ethanol elution of various concentration, receives
The ethanol elution of collection 60~95%, filtering, filtrate decompression concentration, temperature are 60 DEG C, and freeze-drying or spray drying crush, i.e.,
Obtain American-cockroach-extract H.
1 American cockroach extract of experimental example is tested for rat reflux esophagitis
1 experimental material
1.1 experimental drug
Control group drug: esomeprazole magnesium enteric coatel tablets (AstraZeneca pharmaceutical Co. Ltd)
1 drug of comparative experiments group: American-cockroach-extract G is prepared according to 1 the method for comparative example；
2 drug of comparative experiments group: American-cockroach-extract H is prepared according to 2 the method for comparative example；
American cockroach crude extract, American cockroach extract: it is prepared according to method described in embodiment 1；
1.2 experimental animal
4 180~220g of week old SD, 70 rats of SPF grade, female are fifty-fifty.It is mentioned by Chengdu up to large Biotechnology Co., Ltd
For credit number (river): 2013-24.
2 experimental methods
2.1 experimental group
70 healthy rats are randomly divided into sham-operation group and operation group, operation group be divided into model group, esomeprazole group,
Comparative experiments group 1, comparative experiments group 2, American cockroach crude extract group, American cockroach extract group, totally 7 groups, every group each 10.
2.2 modeling method
It, will be big after intraperitoneal injection 10% chloraldurate (3ml/kg) anesthesia after operation group rat pre-operative anxiety can't help drink for 24 hours
Mouse, which lies on the back, is fixed on operating table, row part this myectomy+external pylorus part ligation.Under sterile working lower abdomen just
In take 2.5cm notch into abdomen, after exposure distal esophagus and stomach oesophagus junctional area, cardia flesh longitudinal incision 5mm is tied before separation
Left gastric artery branch.Row pylorus half ligatures afterwards: metal bar (diameter 0.4cm) being ligatured together with pylorus, pays attention to avoiding blood vessel, most
Metal bar is extracted out afterwards.The liquid to exhaust in rat abdominal cavity, closes abdomen after 20,000 IU of saline injection 1ml+ gentamicin, sutures
After give iodophor disinfection wound.Surgical procedure strict aseptic technique.Postoperative fasting for 24 hours, can drink sugar-salt-water.Postoperative 7d starts stomach-filling.
Rats in sham-operated group row sham-operation opens abdomen after anaesthetizing, and stomach and duodenum are proposed, closes abdomen after about 10min, often
Rule disinfection.
2.3 drug therapy
Sham-operation group and model group rats give the physiological saline stomach-filling of 1ml/100g, and esomeprazole group, comparison are real
Group 1, comparative experiments group 2, American cockroach crude extract group, American cockroach extract group are tested, it is corresponding to give 4mg/kg for rat respectively
Drug solution carries out stomach-filling, continuous gavage 14d.
2.4 observation index and measuring method
2.4.1 rat esophagus inflammation index and inhibiting rate
For 24 hours, all 2% yellow Jackets 40mg/kg of rats by intraperitoneal injection are put to death last dose after anesthesia, along cardia to
Upper blunt separation and clip 3~4cm of oesophagus, normal saline flushing is clean, visually observes Esophageal Mucosa lesion degree, referring to " anti-
Fluidity esophagitis diagnosis and treatment guidelines " in classification and scoring (being shown in Table 5), calculate esophagitis index, pass through esophagitis index meter
Damage inhibiting rate is calculated, calculation formula is as follows:
5 esophagitis of table visually observes classification and scoring
2.4.2 inflammatory factor in rat esophagus tissue
The preparation homogenate of feeding tubing, centrifuging and taking supernatant.Enzyme-linked immunization detects interleukin-6 (IL-6), white thin
Born of the same parents' interleukin -8 (IL-8), tumor necrosis factor-alpha (TNF-α), superoxide dismutase (SOD), malonaldehyde (MDA).
3 experimental results
3.1 rat esophagus inflammation indexes and inhibiting rate
It visually observes that rats in sham-operated group Esophageal Mucosa is ruddy, and surface is smooth, has no bleeding and erosion；Model group is all big
There is Esophageal Mucosa erosion in mouse.Compared with model group, esomeprazole group and comparative experiments group 1, comparative experiments group 2, America
Big Lian crude extract group, the coarse-fine object group rat esophagus inflammation index of American cockroach are significantly lower than model group, and inhibiting rate is apparently higher than model
Group (P < 0.5)；The inhibiting rate of inhibition to esophagitis, American cockroach extract group described herein can reach 53.14%,
It is substantially better than the combination of American cockroach crude extract, comparative experiments group 1, comparative experiments group 2 and esomeprazole group.
Table 6 to esophagitis rat esophagus inflammation index and inhibiting rate influence (N=10)
Note: compared with model group, * P < 0.05.
Inflammatory factor in 3.2 rat esophagus tissues
Model group is compared with sham-operation group, and IL-6, IL-8, TNF-α and MDA content obviously rise in model group human esophageal carcinoma
Height, SOD content are substantially reduced (P < 0.05)；Compared with model group, esomeprazole group and comparative experiments group 1, comparative experiments group
2, American cockroach crude extract group, American cockroach extract group can reduce IL-6, IL-8, TNF-α and MDA content, increased SOD and contain
It measures (P < 0.05)；
2 American cockroach extract of experimental example is used for wounds in mice repairing test
1 experimental material
1.1 experimental drugs:
Control group drug: bFGF-ESSEX (Recombinant Bovine Basic Fibroblast Growth Factor for External Use) (hundred million victory bio-pharmaceuticals of Zhuhai
Co., Ltd)
1 drug of comparative experiments group: American-cockroach-extract G is prepared according to 1 the method for comparative example；
2 drug of comparative experiments group: American-cockroach-extract H is prepared according to 2 the method for comparative example；
American cockroach crude extract, American cockroach extract: it is prepared according to method described in embodiment 1；
1.2 experimental animal
6 18~22g of week old Kunming mouse 60 of SPF grade, half male and half female.By Sichuan Provincial Academy of Traditional Chinese Medicine experimental animal
Research department provides, production licence number: SCXK (river) 2015-19.
2 experimental methods
2.1 experimental group
60 healthy mices are randomly divided into model group, control drug group, comparative experiments group 1, comparative experiments group 2, America
Big Lian crude extract group, American cockroach extract group, totally 6 groups, every group each 60.
2.2 modeling method
Test the previous day, back of mice hair is shaved, with 8% Na2The depilation of S solution, warm water cleaning depilation position.Mouse
After fasting can't help drink for 24 hours, after intraperitoneal injection 0.4% yellow Jackets (40mg/kg, 10ml/kg) anesthesia, mouse prostrate is fixed
In operating table, a marking is made with the punch of diameter about 8.2mm, then does full thickness skin excision along the marking with operating scissors.
2.3 drug therapy
Start to be administered on the day of modeling, sprays corresponding medical fluid in the surface of a wound with the medicine bottle with nozzle daily, spray 3 is sprayed (every time every time
Discharge rate is about 0.1ml), 3 times/day, wherein control drug group gives bFGF-ESSEX, and comparative experiments group 1, comparative experiments group 2, America are big
Lian crude extract group, American cockroach extract group, give the medical fluid of corresponding drug respectively, and model group is refused any processing, continuously given
Medicine 11d.
2.4 observation index
The surface of a wound of animal is observed daily, obtains decrustation time, healing time；And it is small in measurement in the 0th, 4,7,11 day
Mouse wound area.
3 experimental results
3.1 decrustation times and healing time
Compared with model group, control drug group and comparative experiments group 1, comparative experiments group 2, American cockroach crude extract, America
The decrustation time of big Lian extract group is obvious or significantly shortens (P < 0.05, P < 0.01), American cockroach crude extract, American cockroach
The healing time of extract group is also obviously shortened (P < 0.05).
7 drug of table to wounds in mice decrustation time and healing time influence (N=10)
Note: compared with model group, * P < 0.05, * * P < 0.01.
3.2 wound area
Compared with model group, control group and comparative experiments group 1, comparative experiments group 2, American cockroach crude extract group, America are big
Lian extract group plays the role of obvious or significant acceleration wound healing (P < 0.05, P < 0.01).
8 drug of table to wounds in mice area influence (N=10)
Note: compared with model group, * P < 0.05**P < 0.01.
Claims (10)
1. a kind of raw material American cockroach extract of Kangfuxin Liquid, which is characterized in that peptides in the American cockroach extract
Component content is 70~90%.
2. a kind of raw material American cockroach extract of Kangfuxin Liquid according to claim 1, which is characterized in that the beauty
The big Lian extract in continent the preparation method comprises the following steps:
1. the fresh worm of American cockroach or dry worm, refluxing extraction, obtain extracting solution A；
2. ethyl alcohol is recovered under reduced pressure in extracting solution A, medicinal extract B is obtained；
3. purified water is added into medicinal extract B, flocculation solution is added, is mixed, is stood, removal precipitating and grease obtain degreaser C；
4. degreaser C collects to obtain ethanol eluate D, through ethyl alcohol is recovered under reduced pressure by nonpolarity and low pole macroporous absorbent resin
And it is concentrated to get medicinal extract；
5. a certain amount of ethyl alcohol is added, stand, filtration, recycling ethyl alcohol simultaneously be concentrated into thick medicinal extract, be dried under reduced pressure, crush America is big
Lian extract.
3. a kind of raw material American cockroach extract of Kangfuxin Liquid according to claim 1, which is characterized in that the beauty
The big Lian extract in continent the preparation method comprises the following steps:
1. the fresh worm of American cockroach or dry worm carry out refluxing extraction using 0~90% ethyl alcohol and water mixed solvent, obtain extracting solution A；
2. ethyl alcohol is recovered under reduced pressure at 50~75 DEG C or less in extracting solution A, medicinal extract B is obtained；
3. the purified water of 6~14 times of medicinal extract weight is added into medicinal extract B, 4~7% gelatin for adding total volume 1%~3% are molten
Liquid mixes, and stands 16~48 hours at 0~10 DEG C, and removal precipitating and grease obtain degreaser C；
4. degreaser C is by nonpolarity and low pole macroporous absorbent resin, first with the pure water rinsing resin of 2~6 times of bed volumes
Column, then with 40%~80% alcohol flushing resin column, collect to obtain ethanol eluate D, ethyl alcohol is recovered under reduced pressure and is concentrated into 60
DEG C detect the medicinal extract that its relative density is 1.10~1.20；
5. a certain amount of ethyl alcohol is added, 16~48 hours are stood, filtration recycles ethyl alcohol and is concentrated into thick medicinal extract, and 55~75 DEG C subtract
Pressure dry, pulverize to obtain American cockroach extract.
4. a kind of raw material American cockroach extract of Kangfuxin Liquid according to claim 1, which is characterized in that the beauty
The big Lian extract in continent the preparation method comprises the following steps:
1. the fresh worm of American cockroach or dry worm carry out refluxing extraction using 0~90% ethyl alcohol and water mixed solvent, obtain extracting solution A；
2. ethyl alcohol is recovered under reduced pressure at 50~75 DEG C in extracting solution A, medicinal extract B is obtained；
3. the purified water of 8~12 times of medicinal extract weight is added into medicinal extract B, 4~7% gelatin for adding total volume 1%~3% are molten
Liquid mixes, and stands 20~40 hours at 3~7 DEG C, and removal precipitating and grease obtain degreaser C；
4. degreaser C is by nonpolarity and low pole macroporous absorbent resin, first with the pure water rinsing resin of 4~6 times of bed volumes
Column, then with 50~75% alcohol flushing resin column, collect to obtain ethanol eluate D, ethyl alcohol is recovered under reduced pressure and is concentrated into 60 DEG C
Detect the medicinal extract that its relative density is 1.10~1.20；
5. a certain amount of ethyl alcohol is added, 16~48 hours are stood, filtration recycles ethyl alcohol and is concentrated into thick medicinal extract, and 60~72 DEG C subtract
Pressure dry, pulverize to obtain American cockroach extract.
5. a kind of raw material American cockroach extract of Kangfuxin Liquid according to claim 1, which is characterized in that the beauty
The big Lian extract in continent the preparation method comprises the following steps:
1. the fresh worm of American cockroach or dry worm carry out refluxing extraction using 70% ethyl alcohol and water mixed solvent, obtain extracting solution A；
2. ethyl alcohol is recovered under reduced pressure at 70 DEG C in extracting solution A, medicinal extract B is obtained；
3. the purified water of 8 times of medicinal extract weight is added into medicinal extract B, mix, stand 24 hours at 5 DEG C, removal precipitating and grease obtain
Degreaser C；
4. degreaser C is by nonpolarity and low pole macroporous absorbent resin, first with the pure water rinsing resin column of 4 times of bed volumes,
Again with 70% alcohol flushing resin column, collect to obtain ethanol eluate D, ethyl alcohol be recovered under reduced pressure and is concentrated into 60 DEG C and detects its phase
The medicinal extract for being 1.10~1.20 to density；
5. a certain amount of ethyl alcohol is added, 16~48 hours are stood, filtration recycles ethyl alcohol and is concentrated into thick medicinal extract, and 70 DEG C of decompressions are dry
It is dry, crush to obtain American cockroach extract, i.e. American cockroach extract.
6. a kind of raw material American cockroach extract of Kangfuxin Liquid according to claims 2 to 5, which is characterized in that described
American cockroach extract preparation method in step 4. described in the high ratio of macroporous absorbent resin its diameter be diameter: it is high=1:
8~12, the macroreticular resin includes any one in D101, AB-8, HPD100, HPD400 and HP20 type；Step is 5.
Described in a certain amount of ethyl alcohol of addition, the amount of the ethyl alcohol are as follows: be added ethyl alcohol to alcohol content reach 80~90%.
7. a kind of raw material American cockroach extract of Kangfuxin Liquid according to claim 1, which is characterized in that the beauty
In the preparation method of the big Lian extract in continent step 4. described in the high ratio of macroporous absorbent resin its diameter be diameter: height=1:10；
Step 5. described in a certain amount of ethyl alcohol of addition, the amount of the ethyl alcohol are as follows: be added ethyl alcohol to alcohol content reach 85%.
8. a kind of raw material American cockroach extract of Kangfuxin Liquid, feature described in any one exist according to claim 1~7
In the American cockroach extract has the application of preparation treatment wound repair drug, health care product, food, daily chemical products.
9. a kind of raw material American cockroach extract of Kangfuxin Liquid, feature described in any one exist according to claim 1~7
In, the American cockroach extract is for oral administration to can be used for preparing treatment stagnation of blood stasis, bleeding of having a stomachache, taste-blindness rate, with
And deficiency of Yin tuberculosis, pulmonary tuberculosis；Diabetic foot ulcer, the alimentary canals surface of a wound such as gastroesophageal reflux, gastritis and ulcerative colitis are repaired
Multiple drug；External application can be used for preparing the surface of a wound drug for the treatment of metal-inflicted wound, wound, ulcer, fistula, burn, scald, bedsore.
10. a kind of raw material American cockroach extract of Kangfuxin Liquid according to claims 1 to 9, which is characterized in that described
American cockroach extract plus pharmaceutically preparation is prepared in common auxiliary material or complementary ingredient.
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| CN111728984B (en) * | 2020-04-08 | 2023-09-12 | 杭州海狮佳科技有限公司 | Preparation method of defatted periplaneta americana extract |
| CN114073720A (en) * | 2020-08-10 | 2022-02-22 | 大理大学 | A compound traditional Chinese medicine for treating damp-heat ulcerative colitis and its preparation method and application |
| CN114073720B (en) * | 2020-08-10 | 2023-03-21 | 大理大学 | Compound traditional Chinese medicine for treating damp-heat type ulcerative colitis and preparation method and application thereof |
| CN114515297A (en) * | 2020-11-20 | 2022-05-20 | 四川好医生攀西药业有限责任公司 | Periplaneta americana extract and amikacin combined compound preparation and preparation method and application thereof |
| CN113274413A (en) * | 2021-07-01 | 2021-08-20 | 大理大学 | Periplaneta americana effective part extract, and extraction method, application and pharmaceutical composition thereof |
| CN114671924A (en) * | 2022-03-24 | 2022-06-28 | 广州市一杰医药科技有限公司 | Periplaneta americana polypeptide extract and application thereof in oral cleaning product |
| CN117562962A (en) * | 2024-01-15 | 2024-02-20 | 四川育强本草生物技术有限公司 | New application of periplaneta americana or extract thereof and veterinary Chinese medicine composition for treating cat stomatitis |
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