CN113893265A - Insect extract with fibrinolytic activity, preparation method and application - Google Patents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/63—Arthropods
- A61K35/64—Insects, e.g. bees, wasps or fleas
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
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Abstract
The invention belongs to the technical field of natural extracts, and discloses an insect extract with fibrinolytic activity, a preparation method and application thereof, wherein a mixture of 1-3 parts of periplaneta americana and 1-3 parts of ant lion is collected into a beaker, washed, drained and weighed; adding PBS solution without the mixture, and soaking for 2 hours; pouring into a glass mortar, adding PBS and grinding; pouring the ground homogenate into a beaker, adding PBS with twice volume for soaking for 24 hours, and subpackaging by using a centrifuge tube; centrifuging at 4 deg.C and 10000r/min for 15 min to obtain coarse extract; removing the precipitate in the centrifugal tube, and leaving the supernatant. The insect extract with fibrinolytic activity provided by the invention has stronger fibrinolytic activity. Research shows that with the increase of the concentration of the insect extract, the extract supernatant freeze-dried powder can obviously promote the apoptosis of colon cancer cells of human beings, and the extract supernatant freeze-dried powder has obvious inhibiting effect on the proliferation of the colon cancer cells.
Description
Technical Field
The invention belongs to the technical field of natural extracts, and particularly relates to an insect extracting solution with fibrinolytic activity, a preparation method and application thereof.
Background
Currently, fibrinolytically active protein is a very important alkaline tryptase serine protease, which has good fibrinolytic activity and is widely present in microorganisms, insects and animals. After decades of research, fibrinolytic active protease is separated and purified in a plurality of microorganisms, insects and animals. Some fibrinolysins have been developed as important drugs for clinical treatment of thrombosis, and development of new drugs against tumors by fibrinolysins is also in progress.
Under normal physiological conditions, after a blood vessel is damaged, a thrombus is formed at the damaged part, and when a wound is healed, the formed thrombus is gradually dissolved. Involved in fibrin degradation or thrombolysis is the fibrinolytic system, which antagonizes the coagulation system to prevent thrombus formation and maintain blood flow. The human body relies mainly on plasmin for the cleavage of fibrin. Plasmin cleaves either individual or reticular fibrin. A large amount of active substances with anticoagulation and thrombolysis effects exist in nature. Fibrinolytically active proteases can be broadly divided into two classes: one is plasminogen activator, such as urokinase and glucokinase, and the action principle is that plasminogen activator can activate plasminogen into plasmin, and plasmin with serine protease activity can degrade fibrin forming thrombus skeleton to cause thrombolysis; another class is fibrinolytics, which act to directly degrade fibrin in the blood clot without the need for plasminogen activation. For example, streptokinase, a thrombolytic drug, binds to plasminogen to form an SK-plasminogen complex, which is enzymatically active to convert free plasminogen to plasmin, and finally plasmin lyses fibrin. But lack of specificity can lead to extensive activation of the fibrinolytic system, resulting in lack of antiplasmin, production of free plasmin and even severe bleeding. At present, people are developing novel fibrinolytic proteins which can improve the specificity and the thrombolysis efficiency of fibrin and reduce the risk of intracranial hemorrhage.
Periplaneta americana, the scientific name Periplaneta americana (L.) the common name Blatta Seu Periplaneta, Gejiang, Tenebrio molitor, Veronica officinalis, and Veronica grisea, is of the genus Periplaneta of the family Blatta, the class Insecta, the class Pterialea order Blattaria. Contains various effective components such as protein, amino acids, peptides, saccharides, etc., and has effects of promoting blood circulation for removing blood stasis, removing toxic substance, eliminating malnutrition, inducing diuresis, and relieving swelling. Recorded in Shen nong Ben Cao Jing, it is called salty and cold in flavor. It is used to treat infantile malnutrition, furuncle, carbuncle, tonsillitis, undefined lump, syphilis, snake bite, and centipede bite. The medicinal insect ant lion (Antrion) belongs to the Lepidoptera Ant Ling family, and is also named as Shaniu, Shawangwai, Tuniu, old Fall, etc. The adult is named as Yiling, which belongs to the most widely distributed insects in the order of the vein and wing. The ant lion is regarded as a rare traditional Chinese medicine material. The functions of the medicine mainly comprise removing blood stasis, detumescence, calming the liver, relieving fever, relaxing the bowels, resolving masses and the like, and the medicine is mainly used for treating epilepsy, vasculitis, constipation, calculi and other difficult and complicated diseases, can be used for treating otitis media and the like by external application, and has good curative effect on knife wounds when the ant lion is used as powder. However, no methods for preparing extract solutions from periplaneta americana and lion have been reported in the prior art.
Through the above analysis, the problems and defects of the prior art are as follows: in the prior art, no report is found on a method for preparing an extracting solution from the periplaneta americana and the lion ant together.
The difficulty in solving the above problems and defects is: how to select an effective proportion to obtain the optimal fibrinolytic activity and inhibit the proliferation of human colon cancer cells.
The significance of solving the problems and the defects is as follows: provides theoretical support for jointly preparing the antithrombotic active extract from the periplaneta americana and the ant lion and promotes the practical application of the active extract.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides an insect extracting solution with fibrinolytic activity, a preparation method and application thereof.
The invention is realized in such a way that the preparation method of the insect extract with fibrinolytic activity comprises the following steps:
step one, collecting a mixture of 1-3 parts of periplaneta americana and 1-3 parts of ant lion into a beaker, washing the beaker with distilled water, draining the beaker on a table by using a paper towel, and weighing the beaker after draining.
And step two, adding the PBS solution without the mixture after weighing is finished, and soaking for 2 hours.
And step three, pouring the soaked mixture into a glass mortar, adding 0.5 volume of precooled PBS for grinding, and completely grinding until the solution is completely black paste.
And step four, pouring the ground homogenate into a beaker, adding PBS with the volume twice that of the homogenate for soaking for 24 hours, and subpackaging the homogenate after soaking for 24 hours by using a centrifuge tube.
And fifthly, centrifuging for 15 minutes at 4 ℃ under the condition of 10000r/min to obtain a centrifugal crude extract. Removing the precipitate in the centrifugal tube, and leaving the supernatant. (the fibrinolytic activity of the extract is retained)
The invention also aims to provide the insect extract with fibrinolytic activity, which is prepared by the preparation method of the insect extract with fibrinolytic activity, and the insect extract with fibrinolytic activity consists of 1-3 parts of periplaneta americana and 1-3 parts of lion by mass.
The invention also aims to provide application of the insect extract with fibrinolytic activity in preparing antithrombotic medicaments.
By combining all the technical schemes, the invention has the advantages and positive effects that: the insect extract with fibrinolytic activity provided by the invention has stronger fibrinolytic activity. Research shows that with the increase of the concentration of the insect extract, the extract supernatant freeze-dried powder can obviously promote the apoptosis of colon cancer cells of human beings, and the extract supernatant freeze-dried powder has obvious inhibiting effect on the proliferation of the colon cancer cells.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings used in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a flow chart of a method for preparing an insect extract with fibrinolytic activity according to an embodiment of the present invention.
FIG. 2 is a schematic illustration of the results of the fibrinolysis plate assay of the extract provided in the examples of the present invention;
in fig. 2: 1: urokinase (100 u/ml); 2: precipitating the extract; 3: supernatant of the extract; 4: PBS.
FIG. 3 is a schematic illustration of the effect of temperature on the fibrinolytic activity of an extracted liquid according to an embodiment of the present invention.
FIG. 4 is a graph showing the effect of pH on fibrinolytic activity of an extract according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides an insect extract with fibrinolytic activity, a preparation method and application thereof, and the invention is described in detail with reference to the accompanying drawings.
As shown in fig. 1, the method for preparing the insect extract with fibrinolytic activity provided by the embodiment of the present invention includes the following steps:
s101, collecting a mixture of 1-3 parts of periplaneta americana and 1-3 parts of ant lion into a beaker, washing the beaker with distilled water, draining the beaker on a table by using a paper towel, and weighing the beaker after draining.
And S102, adding a PBS solution without the mixture after weighing, and soaking for 2 hours.
And S103, pouring the soaked mixture into a glass mortar, properly adding a little precooled PBS for grinding, and completely grinding until the solution is completely black paste.
And S104, pouring the ground homogenate into a beaker, adding PBS with the volume twice that of the homogenate for soaking for 24 hours, and subpackaging the homogenate after soaking for 24 hours by using a centrifuge tube.
S105, centrifuging for 15 minutes at 4 ℃ under the condition of 10000r/min to obtain a centrifugal crude extract; removing the precipitate in the centrifugal tube, and leaving the supernatant.
The method for preparing the insect extract with fibrinolytic activity provided by the invention can be implemented by adopting other steps by persons of ordinary skill in the art, and the method for preparing the insect extract with fibrinolytic activity provided by the invention in fig. 1 is only one specific example.
The insect extracting solution with fibrinolytic activity provided by the embodiment of the invention comprises 1-3 parts of periplaneta americana and 1-3 parts of ant lion by mass.
The technical solution of the present invention is further described with reference to the following examples.
The extraction method of the periplaneta americana and the lion ant extract provided by the invention comprises the following steps: 1-3 parts of Periplaneta americana and 1-3 parts of Ant lion are collected into a beaker, washed with distilled water, and then drained on a table top with a paper towel to ensure the influence of water on the quality of the Ant lion, and the mixture is weighed to be 6.55g after being drained. After weighing, the mixture-free PBS solution was added and the mixture was soaked for 2 hours. Pouring the soaked solution into a glass mortar, properly adding a small amount of precooled PBS for grinding, completely grinding until the solution is completely black paste, pouring the ground homogenate into the beaker, adding PBS with twice volume for soaking for 24 hours, subpackaging the homogenate after soaking for 24 hours by using a centrifuge tube, and centrifuging for 15 minutes under the conditions of 4 ℃ and 10000r/min to obtain the centrifugal crude extract. Removing the precipitate in the centrifugal tube, and leaving the supernatant. Or adopts a new technical proposal as follows: cleaning and cleaning mixture of 1-3 parts of periplaneta americana and 1-3 parts of ant lion, filtering to remove residue,
the periplaneta americana and ant lion extracting solution obtained by the method has strong fibrinolytic activity, and is shown in figure 2.
As shown in FIG. 2, urokinase was used as a positive control, and the supernatant of the extract had the strongest activity, while PBS had no activity.
In addition, the invention also detects the inhibition effect of the extract supernatant freeze-dried powder on colon cancer cell proliferation, as shown in table 1.
TABLE 1 results of the Effect of supernatant of extracts on cancer cell growth at different concentration gradients
As can be seen from Table 1, the supernatant lyophilized powder of the extract can significantly promote apoptosis of human colon cancer cells and inhibit growth of the human colon cancer cells with increasing concentration.
The technical effects of the present invention will be described in detail with reference to the following embodiments:
example 1
In the embodiment of the invention, research on the enzymological properties of the insect extract with fibrinolytic activity (1) influence of temperature on enzyme activity is carried out, wherein the extract freeze-dried powder is taken, diluted to a certain concentration by using normal saline, and cultured for 15 minutes in water bath conditions of 4 ℃, 25 ℃, 35 ℃, 37 ℃, 45 ℃, 55 ℃, 65 ℃ and 75 ℃ respectively, and then the activity of the extract freeze-dried powder is detected by using a fiber plate method.
And opening the superclean workbench, putting the required culture dish, the gun head box, the liquid transferring gun, the sterile water, the sterile PBS and the empty beaker into the superclean workbench, and opening the ultraviolet lamp to irradiate and sterilize for about half an hour. Melting sterilized culture medium (heating with microwave oven), placing in 50 deg.C constant temperature water bath kettle, taking out after water bath for 10min, adding prepared bovine fibrinogen 3ml and thrombin 120ul, shaking rapidly and pouring into flat plate. Taking out the pre-prepared crude extract and urokinase, unfreezing in ice water bath, placing on an ice bag after unfreezing, placing on an ultra-clean workbench, and preparing for sample adding. The flat plate is uniformly perforated by a perforator after the local treatment. Sample adding: 20ul of the sample to be tested, the UK standard substance and the PBS reference substance are added into each hole, and marking is done. Culturing: and sealing the plate after the sample is added with a preservative film, and putting the plate into an incubator at 37 ℃ for 24 hours for culture. Taking out the fiber plate which is cultured for 24 hours in a constant temperature incubator at 37 ℃, and adding a pre-prepared Coomassie brilliant blue staining solution for staining; after 15 minutes, the staining solution is disposed of and a destaining solution is added for elution. The decolorizing solution is replaced every 10min for the first 3 times, and the decolorizing solution is replaced every 20min, and after elution for many times, the obvious lysis ring is observed. And observing the size of the sample solution ring and the size of the standard product (UK) solution ring, measuring the diameter of the solution ring, and photographing and recording the experimental result. A small amount of distilled water was added to the surface of the observed plate, and the plate was stored in a refrigerator at 4 ℃.
The results show that: the influence of different temperatures on the enzyme activity is shown in figure 3, the optimal temperature is 45 ℃, the temperature exceeds 65 ℃, the enzyme activity rapidly decreases along with the increase of the temperature, and the temperature which can be tolerated by the plasmin is 65 ℃.
Influence of pH value on enzyme Activity an equal amount of extract solution is dissolved in equal amounts of buffers with different pH values (prepared by phosphate buffer solution with pH 6-8, Tris-HCl buffer solution with pH 8-8.5 and normal saline with pH 9-10.0 adjusted by NaOH), placed at 4 ℃ for half an hour and then subjected to enzyme activity determination by a fibrin plate method.
The results show that: the influence of different pH values on the enzyme activity is shown in FIG. 4, the enzyme activity is increased along with the increase of the pH value, but the enzyme activity is slightly reduced when the pH value is 10, and the optimal pH value of plasmin is 9.0.
The influence of metal ions, denaturant and inhibitor on the activity of the enzyme of the extracting solution is well prepared by 0.1mol/L NaCl and BaCl2、FeCl3EDTA and mercaptoethanol (BME), mixing the same amount of the above reagents (80ul) with the same amount of the extractive solution, standing at 4 deg.C for half an hour, and measuring enzyme activity by fibrin plate method.
The results show that: EDTA solution and Ba2+Has no influence on fibrinolytic activity, and Na+And the denaturant BME (mercaptoethanol) did not completely inhibit its activity. And Fe3+The fibrinolytic activity of the extract was almost completely inhibited (table 2).
TABLE 2 Effect of Metal ions and denaturants on fibrinolytic Activity of the extract
Example 2: research on anti-cancer activity of insect extract with fibrinolytic activity on human colon cancer cells in logarithmic growth phase caco-2, digesting with pancreatin, counting with cell counting plate, and regulating cells with culture solutionTo a concentration of 1.3X 105Taking a 96-well plate, adding 40ul of cell suspension and 200ul of culture solution into each well, taking two experimental groups and a control group, and taking three groups, wherein each group is provided with four parallel wells, and the total number of the wells is 12. Putting the 96-well plate added with the cells into an incubator for culturing for 10-12h, adding 5.5ul of extracting solution and 10ul of PBS solution with each concentration into an experimental group, adding 15.5ul of PBS into a control group, putting the control group into the incubator for continuous culturing for 24h, taking out the experimental group 4h before the culturing is finished, sucking the supernatant, cleaning the experimental group twice by using the PBS, adding 100ul of serum-free culture medium, operating in a dark place, adding 20ul of prepared MTT solution (5mg/ml) into each well, taking out the experimental group after the culturing in the dark place for 4h, carefully sucking the supernatant, adding 150ul of DMSO solution into each well, dissolving purple crystals, and measuring the A value under 490nm by using an enzyme-labeling instrument. According to the formula: the cell inhibition rate is (1-A medicine group/A control group) X100%
The obtained extractive solution has effect in inhibiting proliferation of human colon cancer cells
TABLE 3 gradient setup and sample application table for cancer inhibition experiment
The results show the effect of the extract on the growth of cancer cells. From the table of cellular absorbance values using the formula: viatility (%) ═ atest/Acontrol；
Inhibition(％)＝(1-Atest)/Acontrol
The cell viability and inhibition rates were calculated as shown in Table 4.
TABLE 4 OVCAR-3 absorbance change table after adding extract
As can be seen from the data in Table 5, the crude fibrinolytic protein of the periplaneta americana can obviously promote the apoptosis of human soft ovarian cancer cells and also has a certain inhibition effect on the growth of the human soft ovarian cancer cells along with the increase of the protein concentration.
TABLE 5 cancer cell survival at different concentrations of protein
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any modification, equivalent replacement, and improvement made by those skilled in the art within the technical scope of the present invention disclosed in the present invention should be covered within the scope of the present invention.
Claims (7)
1. A preparation method of insect extract with fibrinolytic activity is characterized by comprising the following steps:
collecting the mixture of the periplaneta americana and the ant lion into a beaker, washing the beaker with distilled water, draining the beaker on a table by using a paper towel, and weighing the beaker after draining;
after weighing, adding a PBS solution without the mixture, and soaking;
pouring the soaked solution into a glass mortar, properly adding precooled PBS (phosphate buffer solution) for grinding, and completely grinding until the solution is completely black paste;
pouring the ground homogenate into a beaker, adding PBS for soaking, and subpackaging the soaked homogenate by a centrifuge tube;
centrifuging to obtain a centrifugal crude extract; the pellet was removed from the centrifuge tube, leaving a supernatant.
2. The method of preparing an insect extract with fibrinolytic activity according to claim 1, wherein 1-3 parts of periplaneta americana and 1-3 parts of ant lion are mixed and collected in a beaker.
3. The method of preparing an insect extract having fibrinolytic activity according to claim 1, wherein after the completion of weighing, a PBS solution without a mixture is added and soaked for 2 hours.
4. The method of claim 1, wherein the ground homogenate is poured into a beaker, twice the volume of PBS is added for 24 hours of soaking, and the homogenate after 24 hours of soaking is dispensed into a centrifuge tube.
5. The method of preparing an insect extract having fibrinolytic activity according to claim 1, wherein the centrifugation is carried out at 4 ℃ and 10000r/min for 15 minutes to obtain a centrifuged crude extract.
6. The insect extract with fibrinolytic activity prepared by the preparation method of the insect extract with fibrinolytic activity according to claim 1 is characterized by comprising 1-3 parts of periplaneta americana and 1-3 parts of ant lion by mass.
7. Use of the insect extract with fibrinolytic activity according to claim 6 in the preparation of an antithrombotic agent.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170000829A1 (en) * | 2015-06-30 | 2017-01-05 | Sichuan Gooddoctor Panxi Pharmaceutical Co.,Ltd. | Periplaneta americana extract or periplaneta americana medicinal powder as well as preparation method thereof and application in preparation for medicine used for preventing and treating radiation-induced damages |
| CN106581634A (en) * | 2017-02-09 | 2017-04-26 | 赵德润 | Periplaneta americana anticancer active peptide and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170000829A1 (en) * | 2015-06-30 | 2017-01-05 | Sichuan Gooddoctor Panxi Pharmaceutical Co.,Ltd. | Periplaneta americana extract or periplaneta americana medicinal powder as well as preparation method thereof and application in preparation for medicine used for preventing and treating radiation-induced damages |
| CN106581634A (en) * | 2017-02-09 | 2017-04-26 | 赵德润 | Periplaneta americana anticancer active peptide and preparation method thereof |
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| 余昕等: ""美洲大蠊不同提取物及不同提取方法对肿瘤细胞的细胞毒性作用"", 《中国实验方剂学杂志》, vol. 22, no. 15, 31 August 2019 (2019-08-31), pages 1 - 2 * |
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