CN112843094A - Extraction method of periplaneta americana extract - Google Patents
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- CN112843094A CN112843094A CN202110137690.5A CN202110137690A CN112843094A CN 112843094 A CN112843094 A CN 112843094A CN 202110137690 A CN202110137690 A CN 202110137690A CN 112843094 A CN112843094 A CN 112843094A
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Abstract
The invention discloses an extraction method of a periplaneta americana extract, which comprises the following steps: (1) inactivating and crushing; (2) degreasing treatment; (3) extracting by percolation; (4) concentrating under reduced pressure; (5) degreasing and filtering; (6) macroporous adsorption resin column chromatography; (7) concentrating under reduced pressure; (8) and (5) vacuum freeze drying. The percolation method is adopted to extract the periplaneta americana, so that the effective components can be completely leached, the extraction efficiency is high, the reproducibility is good, a heating process is avoided, the effective components are prevented from being heated and decomposed, and the biological activity of each component is ensured; through macroporous adsorption resin column chromatography, the polypeptide substances can be separated and purified, the obtained polypeptide content is high, and the obtained periplaneta americana extract is more suitable for ulcer and promotion of wound repair.
Description
Technical Field
The invention relates to the technical field of medicines, and particularly relates to an extraction method of a periplaneta americana extract.
Background
Periplaneta americana belongs to the genus Periplaneta of the family Blattaria, and the main common cockroach is Periplaneta americana. Periplaneta americana is the largest indoor cockroach, generally 27-40mm in body length, but also smaller or larger individuals. Female adults and male adults are about the same size, but female adults are relatively obese. The body is reddish brown. The anterior chest back plate is provided with a large black brown butterfly-shaped spot, the midline of the spot extends backwards to form a small tail, and a T-shaped yellow stripe is arranged in front of the midline; the rear edge is gray yellow, and the color spots are wider.
Modern researches find that the periplaneta americana epidermis contains sclerite and chitin, and elements such as bromine, zinc, nickel, manganese, potassium, calcium, titanium, chlorine, sulfur, silicon, aluminum, magnesium and the like. Muscle hydrolyzes 13 amino acids. In addition, the body stores vitamins B1 and B2, nicotinic acid and ascorbic acid, etc., and lymph contains trehalose, trehalase, glycoprotein, inositol, protocatechuic acid glucoside, etc. The whole body contains ergothioneine, lobster creatine, trigonelline, glycine, betaine, trimethylamine, adenine, etc.
The periplaneta americana extract comprises protein, fatty acid, nucleic acid, mucopolysaccharide, pheromone, insect neuropeptide, isoflavone and the like, has the effects of promoting tissue repair, resisting tumors and improving immunity, also has the effects of bacteriostasis and antioxidation, improves the functions of organisms and the activity of cells, and has wide prospect and application value in the aspect of drug development.
Polypeptide molecules, polyols, mucoglycine and the like in the periplaneta americana extract have the effects of resisting bacteria, repairing wounds and accelerating wound repair, and can effectively promote wound healing of ulcers, scalds, burns and the like and shorten ulcer repair time. However, the traditional Chinese medicine extracting solution contains a large amount of ineffective components and substances, such as oil molecules, which have no effect of repairing wounds but aggravate the damage degree of wound surfaces, and in order to enrich effective components as much as possible, increase the curative effect of the medicine and reduce the dosage, the traditional Chinese medicine extracting solution needs to be purified to a certain extent, so that the extraction method of the periplaneta americana extract which has the highest content of polypeptide components, fatty acids and amino acids and no fishy smell is developed, and the extraction method has potential application value.
Disclosure of Invention
The invention provides an extraction method of a periplaneta americana extract, which has high leaching efficiency, and the extract obtained by the method has high contents of polypeptide and amino acid, can effectively promote tissue repair and shorten the time of wound repair, and has the functions of bacteriostasis and antioxidation.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for extracting a periplaneta americana extract comprises the following steps:
(1) inactivation and crushing: inactivating fresh American cockroach, drying, crushing and sieving to obtain American cockroach coarse powder;
(2) degreasing treatment: soaking and degreasing the periplaneta americana coarse powder obtained in the step (1) for 2 times by using petroleum ether to obtain degreased periplaneta americana powder;
(3) extracting by a percolation method: adding ethanol into the degreased periplaneta americana powder obtained in the step (2), uniformly stirring, sealing, placing, filling into a percolation cylinder after the powder is fully expanded, adding 5-10 times of ethanol for soaking, percolating at a certain speed, and collecting filtrate;
(4) and (3) concentrating under reduced pressure: concentrating the filtrate obtained in the step (3) under reduced pressure to obtain a concentrated solution;
(5) degreasing and filtering: adding water into the concentrated solution obtained in the step (4), separating the grease layer, and filtering to obtain filtrate;
(6) macroporous adsorption resin column chromatography: pretreating macroporous adsorption resin, soaking the macroporous adsorption resin in ethanol to fully swell the macroporous adsorption resin, filling the macroporous adsorption resin into a column by a wet method, washing the macroporous adsorption resin with the ethanol until the effluent ethanol is mixed with distilled water and is not turbid, finally, fully washing the effluent ethanol with the distilled water until no alcohol smell exists, loading the filtrate obtained in the step (5) at a certain speed, flowing the filtrate through the macroporous adsorption resin column, eluting the filtrate with the ethanol, and collecting eluent;
(7) and (3) concentrating under reduced pressure: concentrating the eluent obtained in the step (6) under reduced pressure to obtain an extract;
(8) vacuum freeze drying: and (3) putting the extract into a freeze drying tray, transferring the extract into a vacuum freeze dryer, and dehydrating the material to obtain the periplaneta americana extract.
As a preferable scheme, the mesh number sieved in the step (1) is 50-80 meshes.
As a preferable scheme, the mass of the petroleum ether added in the step (2) is 3 times of that of the periplaneta americana coarse powder, and the soaking time of each degreasing treatment is 7-9 h.
As a preferable scheme, the mass percentage of the ethanol in the step (3) is 90%, the ethanol soaking time is 40-50h, and the percolation speed is 1-3 mL/min/kg.
Preferably, the vacuum degree of the reduced pressure concentration in the step (4) is-0.03 to-0.06 Mpa, the concentration temperature is 45 to 58 ℃, and the concentration is stopped when the concentration is carried out until the relative density is 1.08 to 1.15.
As a preferable scheme, the amount of the water used in the step (5) is 5 to 8 times of the weight of the concentrated solution.
As a preferable scheme, in the step (6), the type of the macroporous adsorption resin is HP20, the sample loading flow rate of the macroporous adsorption resin is 1.5 BV/h-2.5 BV/h, the sample loading solution concentration is 0.1-0.3 g/mL, and the mass concentration of the ethanol for elution is 50-80%.
Preferably, in the step (6), the elution speed is 1-3 BV/h, and the amount of ethanol used for elution is 1.5-3 BV.
Preferably, in the step (7), the temperature of the reduced pressure concentration is 45-58 ℃, and the reduced pressure concentration is carried out until the extract with the relative density of 1.25-1.35 is obtained.
As a preferable scheme, in the step (8), the conditions of freeze-drying are as follows: the temperature is-15 to-40 ℃, the pressure is 1.0 to 10Pa, and the time is 25 to 45 hours.
Has the advantages that:
(1) the granularity of the powder screened by the American cockroach smashing treatment is 50-80 meshes, so that the subsequent degreasing treatment only needs to be carried out twice, and the time of the third degreasing treatment is saved;
(2) the percolation method is adopted for extraction, so that the effective components can be completely leached, the extraction efficiency is high, the reproducibility is good, a heating process is avoided, the effective components are prevented from being heated and decomposed, and the biological activity of each component is ensured; the percolation speed is controlled to be 1-3 mL/min/kg, so that ethanol can permeate into histiocytes of periplaneta americana with a thicker wall layer;
(3) the HP20 macroporous resin is adopted to separate and purify the periplaneta Americana extracting solution, polypeptide components can be separated out in a concentrated mode, Van der Waals force between hydrophobic groups of compounds in the extracting solution and a nonpolar adsorbent is used for adsorption, the polarity of the eluent is reduced, and the elution capacity is improved.
Detailed Description
The invention will be further understood by reference to the following detailed description of preferred embodiments of the invention and the examples included therein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. To the extent that a definition of a particular term disclosed in the prior art is inconsistent with any definition provided in the present disclosure, the definition of the term provided in the present disclosure controls.
As used herein, a feature that does not define a singular or plural form is also intended to include a plural form of the feature unless the context clearly indicates otherwise. It will be further understood that the term "prepared from …," as used herein, is synonymous with "comprising," including, "comprising," "having," "including," and/or "containing," when used in this specification means that the recited composition, step, method, article, or device is present, but does not preclude the presence or addition of one or more other compositions, steps, methods, articles, or devices. Furthermore, the use of "preferred," "preferably," "more preferred," etc., when describing embodiments of the present invention, is meant to refer to embodiments of the invention that may provide certain benefits, under certain circumstances. However, other embodiments may be preferred, under the same or other circumstances. In addition, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.
In order to realize the purpose, the invention provides an extraction method of a periplaneta americana extract, which comprises the following steps:
(1) inactivation and crushing: inactivating fresh American cockroach, drying, crushing and sieving to obtain American cockroach coarse powder;
(2) degreasing treatment: soaking and degreasing the periplaneta americana coarse powder obtained in the step (1) for 2 times by using petroleum ether to obtain degreased periplaneta americana powder;
(3) extracting by a percolation method: adding ethanol into the degreased periplaneta americana powder obtained in the step (2), uniformly stirring, sealing, placing, filling into a percolation cylinder after the powder is fully expanded, adding 5-10 times of ethanol for soaking, percolating at a certain speed, and collecting filtrate;
(4) and (3) concentrating under reduced pressure: concentrating the filtrate obtained in the step (3) under reduced pressure to obtain a concentrated solution;
(5) degreasing and filtering: adding water into the concentrated solution obtained in the step (4), separating and filtering the grease layer to obtain filtrate;
(6) macroporous adsorption resin column chromatography: pretreating macroporous adsorption resin, soaking the macroporous adsorption resin in ethanol to fully swell the macroporous adsorption resin, filling the macroporous adsorption resin into a column by a wet method, washing the macroporous adsorption resin with the ethanol until the effluent ethanol is mixed with distilled water and is not turbid, finally, fully washing the effluent ethanol with the distilled water until no alcohol smell exists, loading the filtrate obtained in the step (5) at a certain speed, flowing the filtrate through the macroporous adsorption resin column, eluting the filtrate with the ethanol, and collecting eluent;
(7) and (3) concentrating under reduced pressure: concentrating the eluent obtained in the step (6) under reduced pressure to obtain an extract;
(8) vacuum freeze drying: and (3) putting the extract into a freeze drying tray, transferring the extract into a vacuum freeze dryer, and dehydrating the material to obtain the periplaneta americana extract.
In some preferred embodiments, the mesh number sieved in the step (1) is 50-80 meshes, the mesh number is finer compared with that of the conventional crushing treatment, the specific surface area is enlarged, the uniform powder granularity is ensured, only two times of degreasing are needed in the subsequent degreasing treatment, the time of the third degreasing treatment is saved, and the influence of the grease on the quality of the extract and the influence of the grease on the percolation extraction can be effectively removed.
In some preferred embodiments, the mass of the petroleum ether added in the step (2) is 3 times of that of the periplaneta americana coarse powder, and the soaking time of each degreasing treatment is 7-9 h.
The percolation method is a method in which a moderately pulverized medicinal material is placed in a percolation cylinder, a solvent is continuously added from the upper part, and the solvent is percolated through a medicinal material layer to leach out medicinal material components in the downward flowing process. The percolation belongs to a dynamic leaching method, the utilization rate of a solvent is high, the effective components are completely leached, the leachate can be directly collected, a heating process is not needed, the effective components are prevented from being heated and decomposed, the periplaneta americana belongs to insects, the cell wall of the periplaneta americana is thick, the adoption of slow percolation is favorable for promoting ethanol to permeate into tissue cells, and the periplaneta americana is extracted by a percolation process and has good reproducibility and reliability.
In some preferred embodiments, the ethanol in step (3) is 90% by mass, the ethanol soaking time is 40-50h, and the percolation speed is 1-3 mL/min/kg.
The effective components of the periplaneta americana are polypeptide and amino acid substances, the peptide bond of the protein is broken due to overhigh temperature, the structure is changed, the activity is lost, the decomposition of heat-sensitive substances can be prevented or reduced by adopting reduced pressure concentration, the concentration time can be shortened, in some preferred embodiments, the vacuum degree of the reduced pressure concentration in the step (4) is-0.03 to-0.06 Mpa, the concentration temperature is 45 to 58 ℃, and the concentration is stopped when the concentration is carried out until the relative density is 1.08 to 1.15.
Relative density refers to the density relative to water, equal to the ratio of the mass of the solution to the same volume of water.
Method for measuring relative density:
(1) taking a clean, dry and precisely weighed pycnometer, and filling the pycnometer with a test solution; (2) installing a thermometer (no bubbles exist in the bottle), and placing the bottle in a water bath at 20 ℃ for 10-20 minutes to enable the temperature of the object to be measured in the bottle to reach 20 ℃;
(3) removing the solution overflowing from the side tube by using filter paper, and immediately covering the cover;
(4) taking out the pycnometer from the water bath, wiping the outer surface of the pycnometer with filter paper, precisely weighing, and subtracting the weight of the pycnometer to obtain the weight of the test sample;
(5) the test sample is poured off, the pycnometer is cleaned, and the bottle is filled with fresh boiled cold water.
(6) And (4) measuring the weight of the water at the same temperature by the method, and calculating according to the following formula to obtain the water-saving agent.
Relative density of test solution ═ weight of test solution/weight of water
In some preferred embodiments, the amount of water used in step (5) is 5 to 8 times the weight of the concentrate.
Generally, the extract of traditional Chinese medicine contains a large amount of ineffective components and substances, in order to enrich the effective components as much as possible and increase the curative effect of the medicine, the extract needs to be purified to a certain extent, the main effective component of the periplaneta americana for treating ulcer or promoting wound repair is a polypeptide substance, the polypeptide in the periplaneta americana extract is purified by a macroporous adsorption resin separation and purification technology, and the extract of the periplaneta americana can be lightened in color and effectively deodorized by the purification method.
The macroporous adsorption resin is a high molecular polymer which is insoluble in acid, alkali and organic solvent and is polymerized by taking methyl methacrylate, styrene and the like as raw materials and adding a certain amount of pore-foaming agent (divinylbenzene), and is divided into a plurality of types of non-polarity, weak polarity and polarity according to the difference of polarity. The macroporous adsorption resin has certain polar groups and larger total surface area of particles, so that the macroporous adsorption resin has larger adsorption capacity, and has a reticular hole structure with different pore sizes, so that the macroporous adsorption resin has certain selectivity according to different molecular weights of compounds, and the adsorption can be increased or desorbed by adjusting the hydrophilic and hydrophobic balance of the system and the properties of the compounds.
In some preferred embodiments, in the step (6), the macroporous adsorption resin is HP20 type macroporous adsorption resin, the sample loading flow rate of the macroporous adsorption resin is 1.5 BV/h-2.5 BV/h, the sample loading solution concentration is 0.1-0.3 g/mL, and the mass concentration of the ethanol for elution is 50% -80%.
HP20 macroporous adsorbent resin is nonpolar, and mainly adsorbs hydrophobic groups of compounds with van der waals force between nonpolar adsorbents, so as to reduce the polarity of the eluent and increase the elution capacity.
In some preferred embodiments, in the step (6), the elution speed is 1-3 BV/h, and the amount of ethanol used for elution is 1.5-3 BV.
When the dosage of the eluent is fixed, the smaller the flow rate of the sample loading is, the longer the contact time of the eluent and the resin is, the more sufficient the desorption is, the better the elution effect is, but the time and the labor are wasted when the flow rate is too small.
In some preferred embodiments, in the step (7), the temperature of the reduced pressure concentration is 45-58 ℃, and the reduced pressure concentration is carried out until the extract with the relative density of 1.25-1.35 is obtained.
In some preferred embodiments, in the step (8), the conditions of freeze-drying are: the temperature is-15 to-40 ℃, the pressure is 1.0 to 10Pa, and the time is 25 to 45 hours.
Examples
Example 1
The embodiment provides an extraction method of a periplaneta americana extract, which comprises the following steps:
(1) inactivation and crushing: freezing fresh Periplaneta americana at-25 ℃, repeatedly cleaning with deionized water for 3 times, draining, transferring to-80 ℃ for freezing and inactivating for 48 hours, drying, crushing and sieving to obtain Periplaneta americana coarse powder with the particle size of 50-80 meshes;
(2) degreasing 100g of periplaneta americana coarse powder obtained in the step (1), wherein the degreasing process comprises the following specific steps: the first degreasing treatment is to add petroleum ether into the periplaneta americana powder, the mass of the added petroleum ether is 3 times of that of the powder, then soak the powder for 8 hours at room temperature, and filter the powder to obtain a first petroleum ether mixed solution and first degreased periplaneta americana powder; and then carrying out secondary degreasing treatment on the American cockroach powder subjected to primary degreasing, wherein the process is the same as that of the primary degreasing treatment, and thus obtaining the degreased American cockroach powder.
(3) Extracting by a percolation method: adding 90% ethanol by mass concentration into the degreased periplaneta americana powder obtained in the step (2), uniformly stirring, sealing, placing, filling into a percolation cylinder after the powder is fully expanded, adding 8 times of 90% ethanol by weight, soaking for 45 hours, percolating at the speed of 3mL/min/kg, and collecting filtrate;
(4) and (3) concentrating under reduced pressure: concentrating the filtrate obtained in the step (3) under reduced pressure under the conditions that the vacuum degree is-0.05 MPa and the temperature is 52 ℃, and stopping concentrating when the relative density is 1.10 to obtain concentrated solution;
(5) degreasing and filtering: adding deionized water 7 times the weight of the concentrated solution obtained in the step (4), heating to 55 ℃, stirring for 20min, then preserving heat and standing for 10h, cooling to 15 ℃, continuing to stand for 20h, separating and filtering the solution after standing, wherein the filtering aperture is 5 mu m, so as to obtain a filtrate;
(6) macroporous adsorption resin column chromatography: soaking a proper amount of HP20 macroporous adsorption resin in 95% ethanol with mass concentration of 3 times of the weight of the HP20 macroporous adsorption resin for 24 hours to fully swell the HP20 macroporous adsorption resin, filling the HP20 macroporous adsorption resin into a column by a wet method, washing the column by the 95% ethanol with mass concentration at the flow rate of 3BV/h until the effluent ethanol and distilled water are not mixed and turbid, and fully washing the column by the distilled water at the flow rate of 3BV/h until no alcohol smell exists; enabling the filtrate obtained in the step (5) to flow through a macroporous adsorption resin column by sampling at 2BV/h, standing for 1h, eluting with 75% ethanol by mass concentration at the elution speed of 2BV/h and the use amount of an eluent of 2BV, and collecting the eluent;
(7) and (3) concentrating under reduced pressure: concentrating the eluate obtained in step (6) at 55 deg.C under vacuum degree of-0.05 MPa until the relative density is 1.26, and stopping concentration to obtain extract;
(8) vacuum freeze drying: and (3) putting the extract into a freeze drying tray, transferring the extract into a vacuum freeze dryer, dehydrating the material at the temperature of-30 ℃ and under the pressure of 5Pa, and freeze-drying for 36 hours to obtain the periplaneta americana extract.
Example 2
The embodiment provides an extraction method of a periplaneta americana extract, which comprises the following steps:
(1) inactivation and crushing: freezing fresh Periplaneta americana at-25 ℃, repeatedly cleaning with deionized water for 3 times, draining, transferring to-80 ℃ for freezing and inactivating for 48 hours, drying, crushing and sieving to obtain Periplaneta americana coarse powder with the particle size of 50-80 meshes;
(2) degreasing 100g of periplaneta americana coarse powder obtained in the step (1), wherein the degreasing process comprises the following specific steps: the first degreasing treatment is to add petroleum ether into the periplaneta americana powder, the mass of the added petroleum ether is 3 times of that of the powder, then soak the powder for 9 hours at room temperature, and filter the powder to obtain a first petroleum ether mixed solution and first degreased periplaneta americana powder; and then carrying out secondary degreasing treatment on the American cockroach powder subjected to primary degreasing, wherein the process is the same as that of the primary degreasing treatment, and thus obtaining the degreased American cockroach powder.
(3) Extracting by a percolation method: adding 90% ethanol by mass concentration into the defatted periplaneta americana powder obtained in the step (2), uniformly stirring, sealing, placing, filling into a percolation cylinder after the powder is fully expanded, adding 10 times of 90% ethanol by weight, soaking for 42 hours, percolating at the speed of 1mL/min/kg, and collecting filtrate;
(4) and (3) concentrating under reduced pressure: concentrating the filtrate obtained in the step (3) under reduced pressure under the conditions that the vacuum degree is-0.04 MPa and the temperature is 52 ℃, and stopping concentrating when the relative density is 1.09 to obtain a concentrated solution;
(5) degreasing and filtering: adding deionized water 8 times the weight of the concentrated solution obtained in the step (4), heating to 55 ℃, stirring for 30min, then preserving heat and standing for 12h, cooling to 10 ℃, continuing to stand for 24h, separating and filtering the solution after standing, wherein the filtering aperture is 5 mu m, so as to obtain a filtrate;
(6) macroporous adsorption resin column chromatography: soaking a proper amount of HP20 macroporous adsorption resin in 95% ethanol with mass concentration of 3 times of the weight of the HP20 macroporous adsorption resin for 24 hours to fully swell the HP20 macroporous adsorption resin, filling the HP20 macroporous adsorption resin into a column by a wet method, washing the column by 90% ethanol with mass concentration at the flow rate of 2BV/h until the effluent ethanol and distilled water are not turbid when mixed, and then fully washing the column by distilled water at the flow rate of 2BV/h until no alcohol smell exists; enabling the filtrate obtained in the step (5) to flow through a macroporous adsorption resin column by sampling at 2BV/h, standing for 1h, eluting with 75% ethanol by mass concentration at the elution speed of 2BV/h and the use amount of an eluent of 3BV, and collecting the eluent;
(7) and (3) concentrating under reduced pressure: concentrating the eluate obtained in step (6) at 52 deg.C under vacuum degree of-0.04 MPa until the relative density is 1.30, and stopping concentration to obtain extract;
(8) vacuum freeze drying: and (3) putting the extract into a freeze drying tray, transferring the extract into a vacuum freeze dryer, dehydrating the material at the temperature of-20 ℃ and under the pressure of 8Pa, and freeze-drying for 44 hours to obtain the periplaneta americana extract.
Example 3
The embodiment provides an extraction method of a periplaneta americana extract, which comprises the following steps:
(1) inactivation and crushing: freezing fresh Periplaneta americana at-25 ℃, repeatedly cleaning with deionized water for 3 times, draining, transferring to-80 ℃ for freezing and inactivating for 48 hours, drying, crushing and sieving to obtain Periplaneta americana coarse powder with the particle size of 50-80 meshes;
(2) degreasing 100g of periplaneta americana coarse powder obtained in the step (1), wherein the degreasing process comprises the following specific steps: the first degreasing treatment is to add petroleum ether into the periplaneta americana powder, the mass of the added petroleum ether is 3 times of that of the powder, then soak the powder for 8 hours at room temperature, and filter the powder to obtain a first petroleum ether mixed solution and first degreased periplaneta americana powder; and then carrying out secondary degreasing treatment on the American cockroach powder subjected to primary degreasing, wherein the process is the same as that of the primary degreasing treatment, and thus obtaining the degreased American cockroach powder.
(3) Extracting by a percolation method: adding 90% ethanol by mass concentration into the degreased periplaneta americana powder obtained in the step (2), uniformly stirring, sealing, placing, filling into a percolation cylinder after the powder is fully expanded, adding 10 times of 90% ethanol by weight, soaking for 50 hours, percolating at the speed of 2mL/min/kg, and collecting filtrate;
(4) and (3) concentrating under reduced pressure: concentrating the filtrate obtained in the step (3) under reduced pressure under the conditions that the vacuum degree is-0.06 MPa and the temperature is 56 ℃, and stopping concentrating when the relative density is 1.13 to obtain concentrated solution;
(5) degreasing and filtering: adding deionized water 6 times the weight of the concentrated solution obtained in the step (4), heating to 50 ℃, stirring for 25min, then preserving heat and standing for 12h, cooling to 12 ℃, continuing to stand for 22h, separating and filtering the solution after standing, wherein the filtering aperture is 5 mu m, so as to obtain a filtrate;
(6) macroporous adsorption resin column chromatography: soaking a proper amount of HP20 macroporous adsorption resin in 90% ethanol with mass concentration of 3 times of the weight of the HP20 macroporous adsorption resin for 30 hours to fully swell the HP20 macroporous adsorption resin, filling the HP20 macroporous adsorption resin into a column by a wet method, washing the column by 90% ethanol with mass concentration at a flow rate of 2BV/h until the effluent ethanol and distilled water are not turbid when mixed, and then fully washing the column by distilled water at a flow rate of 2BV/h until no alcohol smell exists; enabling the filtrate obtained in the step (5) to flow through a macroporous adsorption resin column at a sample loading rate of 2.5BV/h, standing for 1h, eluting with 75% ethanol by mass concentration at an elution speed of 2BV/h and an eluent dosage of 2.5BV, and collecting the eluent;
(7) and (3) concentrating under reduced pressure: concentrating the eluate obtained in step (6) at 48 deg.C under-0.06 MPa until the relative density is 1.28, and stopping concentration to obtain extract;
(8) vacuum freeze drying: and (3) putting the extract into a freeze drying tray, transferring the extract into a vacuum freeze dryer, dehydrating the material at the temperature of minus 35 ℃ and under the pressure of 2Pa, and freeze-drying for 30 hours to obtain the periplaneta americana extract.
Comparative example 1
The comparative example provides an extraction method of the periplaneta americana extract, which is different from the extraction method of example 1 in that the ethanol reflux method is adopted in the step (3), and the specific operations are as follows: adding 90% of Periplaneta americana powder obtained in the step (2) by weight in an amount which is 10 times that of the Periplaneta americana powder, soaking for 50 hours, refluxing for 1 hour, filtering, adding 90% of ethanol by weight in an amount which is 8 times that of the Periplaneta americana powder, refluxing for 2 times, 1 hour each time, filtering, and combining the filtrates for 3 times.
Comparative example 2
The comparative example provides an extraction method of the periplaneta americana extract, and the difference from the example 1 is that the model of the macroporous adsorption resin used in the step (6) is AB-8.
Determination of polypeptide content
The periplaneta americana extracts obtained in examples 1-3 and comparative examples 1-2 were subjected to polypeptide content measurement, and the results are shown in table 1.
Referring to the general rules of the four pharmacopoeias of China (2015 edition), 0731 protein assay: the detection by the Folin phenol method (Lowry method) is as follows:
reagent: preparing an alkaline copper test solution;
liquid A: mixing and dissolving 10g of sodium hydroxide, 50g of sodium carbonate and 400mL of purified water;
b, liquid B: dissolving 0.25g of copper sulfate and 30mL of purified water, dissolving 0.5g of potassium tartrate and 50mL of purified water, and combining the two solutions;
mixing the first solution and the second solution before use, and adding purified water to 500mL to prepare the alkaline copper test solution.
(1) Preparation of control solutions: precisely weighing 22.4mg bovine serum albumin reference substance (BSA) in a 50ml volumetric flask, adding purified water to dissolve to a scale, shaking up, and preparing into 0.448mg/ml bovine serum albumin reference substance solution.
(2) Linear range investigation: precisely absorbing 0ml, 0.2 ml, 0.4 ml, 0.6 ml, 0.8 ml and 1.0ml of BSA reference substance solution into a 10ml glass test tube with a plug, adding purified water to 1.0ml, adding 5.00ml of alkaline copper test solution, uniformly mixing, standing for 10min, then quickly adding 4.0ml of Folin phenol reagent, quickly shaking uniformly, standing for 30min at room temperature, measuring the absorbance of the solution at the wavelength of 500nm by using an ultraviolet spectrophotometry, and preparing a BSA standard curve by using a polypeptide concentration X as a horizontal coordinate and an absorbance Y as a vertical coordinate.
(3) Weighing a proper amount of periplaneta americana concentrated solution into a 10ml glass test tube with a plug, adding purified water to 1.0ml, adding 5.00ml of alkaline copper test solution, uniformly mixing, standing for 10min, then quickly adding 4.0ml of Folin phenol reagent, quickly shaking uniformly, standing for 30min at room temperature, measuring the absorbance of the periplaneta americana concentrated solution at the wavelength of 500nm by using an ultraviolet spectrophotometry, and calculating the content of the sample according to the absorbance.
TABLE 1 determination of the polypeptide content
As can be seen from Table 1, the Periplaneta americana extracted by the percolation method can completely leach main components, has higher extraction rate, can effectively separate out polypeptide by adopting HP20 macroporous adsorption resin, removes other ineffective components, and can be better suitable for repairing ulcers and wound surfaces.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and variations can be made in the present invention without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. The extraction method of the periplaneta americana extract is characterized by comprising the following steps of:
(1) inactivation and crushing: inactivating fresh American cockroach, drying, crushing and sieving to obtain American cockroach coarse powder;
(2) degreasing treatment: soaking and degreasing the periplaneta americana coarse powder obtained in the step (1) for 2 times by using petroleum ether to obtain degreased periplaneta americana powder;
(3) extracting by a percolation method: adding ethanol into the degreased periplaneta americana powder obtained in the step (2), uniformly stirring, sealing, placing, filling into a percolation cylinder after the powder is fully expanded, adding 5-10 times of ethanol for soaking, percolating at a certain speed, and collecting filtrate;
(4) and (3) concentrating under reduced pressure: concentrating the filtrate obtained in the step (3) under reduced pressure to obtain a concentrated solution;
(5) degreasing and filtering: adding water into the concentrated solution obtained in the step (4), separating and filtering the grease layer to obtain filtrate;
(6) macroporous adsorption resin column chromatography: pretreating macroporous adsorption resin, soaking the macroporous adsorption resin in ethanol to fully swell the macroporous adsorption resin, filling the macroporous adsorption resin into a column by a wet method, washing the macroporous adsorption resin with the ethanol until the effluent ethanol is mixed with distilled water and is not turbid, finally, fully washing the effluent ethanol with the distilled water until no alcohol smell exists, loading the filtrate obtained in the step (5) at a certain speed, flowing the filtrate through the macroporous adsorption resin column, eluting the filtrate with the ethanol, and collecting eluent;
(7) and (3) concentrating under reduced pressure: concentrating the eluent obtained in the step (6) under reduced pressure to obtain an extract;
(8) vacuum freeze drying: and (3) putting the extract into a freeze drying tray, transferring the extract into a vacuum freeze dryer, and dehydrating the material to obtain the periplaneta americana extract.
2. The extraction method of the periplaneta americana extract according to claim 1, wherein the mesh number sieved in the step (1) is 50-80 meshes.
3. The extraction method of the periplaneta americana extract according to claim 1, wherein the mass of the petroleum ether added in the step (2) is 3 times of that of the periplaneta americana coarse powder, and the soaking time of the defatting treatment is 7-9h each time.
4. The extraction method of the periplaneta americana extract according to claim 1, wherein the ethanol in the step (3) accounts for 90% by mass, the ethanol soaking time is 40-50h, and the percolation speed is 1-3 mL/min/kg.
5. The extraction method of the periplaneta americana extract according to claim 1, wherein the vacuum degree of the vacuum concentration in the step (4) is-0.03 to-0.06 Mpa, the concentration temperature is 45 to 58 ℃, and the concentration is stopped when the concentration is carried out until the relative density is 1.08 to 1.15.
6. The extraction method of periplaneta americana extract according to claim 1, wherein the amount of water used in step (5) is 5-8 times the weight of the concentrate.
7. The extraction method of the periplaneta americana extract according to claim 1, wherein in the step (6), the type of the macroporous adsorption resin is HP20 macroporous adsorption resin, the sampling flow rate of the macroporous adsorption resin is 1.5 BV/h-2.5 BV/h, the concentration of the sampling solution is 0.1-0.3 g/mL, and the mass concentration of the ethanol for elution is 50-80%.
8. The extraction method of the periplaneta americana extract according to claim 1, wherein in the step (6), the elution speed is 1-3 BV/h, and the amount of ethanol used for elution is 1.5-3 BV.
9. The extraction method of the periplaneta americana extract according to claim 1, wherein in the step (7), the temperature of the reduced pressure concentration is 45-58 ℃, preferably 50-55 ℃, and the concentration is carried out until the extract with the relative density of 1.25-1.35 is obtained.
10. The extraction method of the periplaneta americana extract according to claim 1, wherein the freeze-drying condition in the step (8) is: the temperature is-15 to-40 ℃, the pressure is 1.0 to 10Pa, and the time is 25 to 45 hours.
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