CN108785330B - Application of periplaneta americana extract in preparation of medicine for treating wound healing - Google Patents
Application of periplaneta americana extract in preparation of medicine for treating wound healing Download PDFInfo
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Abstract
The invention discloses an application of a periplaneta americana extract in preparing a drug for treating wound healing, and a preparation method of the periplaneta americana extract comprises the following steps: (1) alcohol extraction: pulverizing Periplaneta americana into coarse powder, extracting with ethanol, centrifuging the extractive solution, filtering, and concentrating under reduced pressure; (2) water precipitation: adding purified water into the concentrated solution, stirring, standing for layering, collecting oil layer, concentrating under reduced pressure, and drying to obtain the extract. The invention proves that the extract is an effective part of the periplaneta americana for promoting wound healing through a pharmacodynamic test, the efficacy of the periplaneta americana for promoting wound healing can be effectively improved, and the preparation method of the periplaneta americana extract is scientific and reasonable, simple in process and strong in operability.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of a periplaneta americana extract in preparation of a medicine for treating wound healing.
Background
Periplaneta americana (Periplaneta americana) is a traditional Chinese medicinal material distributed in wide areas of China, is in the class Insecta, subclass pteroidea, order Blattales, family Blattaceae, commonly known as cockroach, is often used as a medicine by dry or fresh adults, has the characteristics of cold nature, salty taste, extremely smelly smell and toxicity, and can strengthen the spleen, eliminate malnutrition, promote blood circulation, promote diuresis, relieve swelling, heal sores and promote granulation. Can be used for treating hypochondriac pain, abdominal mass, malnutritional stagnation, palpitation, asthma and edema. It is used externally to treat scald, various wounds and ulcers.
The main components of Periplaneta americana include proteins and amino acids, peptides, fats and fatty acids, diluted alcohol, diluted acids and hydrocarbons, polyols (such as mannitol, etc.), macrolides, pheromones, saccharides (such as mucopolysaccharide, etc.), enzymes, esterase, coenzyme A and abundant minerals and trace elements. Studies have proved that the periplaneta americana has the effects of protecting the heart, resisting exercise-induced fatigue, promoting tissue repair, resisting bacteria, viruses and tumors, enhancing immunity and protecting the liver. The extraction, separation, identification and gene expression of various chemical components of the American cockroach extract are mainly researched abroad; the domestic main focus is on the clinical application and development of the medicine.
The existing production process of the periplaneta americana preparation is prepared by an alcohol extraction and water precipitation method and after grease removal, wherein whether the grease removal is scientific and reasonable or not still needs to be further researched; it is not clear which type of component in the preparation is more relevant to promoting wound healing.
Disclosure of Invention
Based on the above, the invention aims to overcome the defects of the prior art and provide the application of the periplaneta americana extract in preparing the medicine for treating wound healing.
In order to achieve the purpose, the invention adopts the technical scheme that: the application of the periplaneta americana extract in preparing the medicine for treating wound healing comprises the following steps:
(1) alcohol extraction: pulverizing Periplaneta americana into coarse powder, extracting with ethanol, centrifuging the extractive solution, filtering, and concentrating under reduced pressure;
(2) water precipitation: and (2) adding purified water into the concentrated solution obtained in the step (1), uniformly stirring, standing for layering, collecting an oil layer, concentrating under reduced pressure, and drying to obtain the periplaneta americana extract.
Preferably, the periplaneta americana in the step (1) is crushed into coarse powder, ethanol with volume fraction of 75-90% and weight of 4-8 times of the coarse powder is added, the mixture is extracted for 20-30 hours at room temperature, the extracting solution is centrifuged, filtered, and the filtrate is concentrated under reduced pressure at 40-60 ℃.
More preferably, the periplaneta americana in the step (1) is crushed into coarse powder, ethanol with the volume fraction of 85% and the weight 5 times of the volume of the coarse powder is added, the mixture is extracted for 24 hours at room temperature, the extracting solution is centrifuged, filtered, and the filtrate is concentrated under reduced pressure at 50 ℃.
Preferably, purified water with the volume 3-6 times of the weight of the concentrated solution is added into the concentrated solution in the step (2), the mixture is uniformly stirred, and the mixture is kept stand and layered for 20-30 hours at the temperature of 0-10 ℃.
More preferably, purified water with the volume 5 times of the weight of the concentrated solution is added into the concentrated solution in the step (2), the mixture is stirred uniformly and is kept stand and layered for 24 hours at 4 ℃.
The periplaneta americana extract can effectively treat wound healing, including wound healing of external wounds and internal wounds, wherein the external wounds comprise burns, scalds and the like, and the internal wounds comprise ulcers and the like.
The invention proves that the periplaneta americana extract is an effective part of the periplaneta americana for promoting wound healing through pharmacodynamic test.
The periplaneta americana extract disclosed by the invention can be used for preparing a medicament for treating wound healing.
The invention also provides a medicine for treating wound healing, which comprises the periplaneta americana extract and a pharmaceutically acceptable carrier.
Preferably, the medicine for treating wound healing is an external medicine or an internal medicine.
The medicine containing the periplaneta americana extract can effectively treat the healing of external injury wound surfaces such as burns and scalds and the healing of internal injury wound surfaces such as ulcers.
Preferably, the external medicament form is tincture, cream, powder or ointment.
Preferably, the oral medicament form is tablets, capsules, granules, pills, powder or solution.
Compared with the prior art, the invention has the beneficial effects that: (1) the periplaneta americana extract provided by the invention is an effective part of the periplaneta americana for promoting wound healing, and can remarkably improve the drug effect of the periplaneta americana for promoting wound healing; (2) the preparation method of the periplaneta americana extract is scientific and reasonable, simple in process and high in operability.
Drawings
FIG. 1 is a graph showing wound healing in rats of each group on different days.
FIG. 2 is a graph showing the change in body weight of rats in each group, wherein A: acute toxicity rats; b: rats were tested for drug effect.
Fig. 3 is a graph showing the change of the wound area of rats in each group, wherein a: the upper wound surface; b: and (5) the wound surface is treated.
Fig. 4 is a graph of the percent change in wound healing for rats in each group, wherein a: the upper wound surface; b: and (5) the wound surface is treated.
Fig. 5 is a graph showing the change of the wound healing rate of rats in each group, wherein a: the upper wound surface; b: and (5) the wound surface is treated.
FIG. 6 is a graph showing the results of histopathological examination (HE staining) of rats in each group.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.
Example 1
The embodiment of the preparation method of the periplaneta americana extract comprises the following steps:
(1) alcohol extraction: pulverizing Periplaneta americana into coarse powder, adding 75% ethanol 8 times the coarse powder by volume, extracting at room temperature for 30h, centrifuging the extractive solution, filtering, and concentrating the filtrate at 60 deg.C under reduced pressure to obtain soft extract;
(2) water precipitation: and (2) adding purified water with the volume 3 times of the weight of the concentrated solution into the concentrated solution obtained in the step (1), uniformly stirring, standing at 0 ℃ for layering for 30 hours, collecting an oil layer, concentrating under reduced pressure, and drying to obtain the periplaneta americana extract.
Example 2
The embodiment of the preparation method of the periplaneta americana extract comprises the following steps:
(1) alcohol extraction: pulverizing Periplaneta americana into coarse powder, adding 90% ethanol 4 times the coarse powder by volume, extracting at room temperature for 20 hr, centrifuging the extractive solution, filtering, and concentrating the filtrate at 40 deg.C under reduced pressure to obtain soft extract;
(2) water precipitation: and (2) adding purified water with the volume 6 times of the weight of the concentrated solution into the concentrated solution obtained in the step (1), uniformly stirring, standing at 10 ℃ for layering for 20 hours, collecting an oil layer, concentrating under reduced pressure, and drying to obtain the periplaneta americana extract.
Example 3
The embodiment of the preparation method of the periplaneta americana extract comprises the following steps:
(1) alcohol extraction: pulverizing Periplaneta americana into coarse powder, adding 85% ethanol 5 times the coarse powder by volume, extracting at room temperature for 24 hr, centrifuging the extractive solution, filtering, and concentrating the filtrate at 50 deg.C under reduced pressure to obtain soft extract;
(2) water precipitation: and (2) adding purified water with the volume 5 times of the weight of the concentrated solution into the concentrated solution in the concentrated solution obtained in the step (1), uniformly stirring, standing at 4 ℃ for layering for 24 hours, collecting an oil layer, concentrating under reduced pressure, and drying to obtain the periplaneta americana extract.
Example 4
The periplaneta americana extract prepared in example 3 was taken as an example to study the drug effect of the periplaneta americana extract of the present invention.
(I) Experimental method
1. Grouping animals
The SPF grade SD rats were 64,
respectively, quarantining for 3 days, wherein the weight is 180-220G during the test, and the test is divided into a solvent control group (A group), a rehabilitation new control group (B group), an MDT-1 high dose group (C group), an MDT-1 low dose group (D group), an MDT-2 high dose group (E group), an MDT-2 low dose group (F group), a blank control group (G group) and an EGF control group (H group) according to a weight random block grouping method; the MDT-1 is the American cockroach extract, and the MDT-2 is a water layer obtained after water precipitation in the step (2) of the preparation method of the American cockroach extract. Each group had 8 SD rats, male and female halves, with the same drug in the same rats at high and low doses.
2. Animal model
Before operation, fasting is not forbidden for 16h (17: 30 fasting before operation), on the day of operation, 10% chloral hydrate (300mg/kg, 3mL/kg) is injected into an abdominal cavity to anaesthetize a rat, the back of the rat is shaved into an area of about 7cm multiplied by 4cm, the skin is disinfected by 75% alcohol, a homemade circular mould with the diameter of 2.5cm is attached to the skin by taking the spinal column of the back of the rat as a central line, a graph is drawn by extending a marker pen, an operating knife is used for drawing lines along the edges, tweezers are used for lifting the skin, and ophthalmic scissors are used for cutting off the skin and the superficial fascia along the edges until the superficial surface of the deep fascia, so that the bleeding is completely stopped, the circular wound with the diameter of 2.5cm is formed, and the upper and the lower parts are respectively spaced by 2 cm. And (5) feeding the rats in a single cage after operation.
3. Dosing regimens
The medicine is administered after 0.5h of molding, each group is administered according to the dose, 1mL of medicine is absorbed by a liquid-transferring gun and is beaten on the wound surface for a few times, the wound surface is preferably immersed, and the medicine is put back into the cage after a few minutes. Wherein, the group A is given with a solvent with the same volume, the group B is given with rehabilitation new liquid, the C, D group is respectively given with MDT-1 high-low dose liquid medicine, the E, F group is respectively given with MDT-2 high-low dose liquid medicine, the group G is not given, and the group H is given with EGF with the concentration of 10 mg/mL. The administration site of each group: the control group was given to the lower wound, and not to the upper wound; the upper wound surface of the administration group is given low dose, and the lower wound surface is given high dose. The administration method is detailed in table 1 and table 2. Dressing change was performed 1 time per day for 14 consecutive days.
Table 1 summary of animal numbers for each dose group
Table 2 dosage regimen summary
4. Observation indicator and detection method
(1) Mass of rat body
On days 0, 4, 7, 10, and 14 after the molding was successful, the rats in each group were weighed, and the change in body weight after the administration was examined.
(2) Change of wound surface area
The wound area was measured on days 0, 4, 7, 10, and 14 after the molding was successful. (3) Percent wound healing
And (3) taking a picture of the wound healing condition by using a camera and a fixed phase at the same height under the same condition on 0 th day, 4 th day, 7 th day, 10 th day and 14 th day after the successful molding. The percent of wound healing area was calculated using Image Pro Plus Image analysis software, where the percent of wound healing area is (area of wound before drug administration-area of wound after drug administration)/area of wound before drug administration × 100%.
(4) Rate of wound healing
And calculating the wound healing rate on the 0 th, 4 th, 7 th, 10 th and 14 th days after the molding is successful.
(5) Histopathological examination of wound surface
On day 15 of surgery, the next day of the last dose, a histopathological examination was performed. Chloral hydrate 10% was anesthetized by intraperitoneal injection, the wound surface was carefully removed from the whole skin, and the rats were sacrificed. The excised tissue was fixed in 10% formaldehyde solution and embedded in paraffin. Routine slicing, hematoxylin-eosin (HE) staining, and detecting histopathological changes such as inflammatory cell infiltration, capillary vessel generation, fibroblast hyperplasia, epidermal hyperplasia and collagen formation in wound healing tissue after drug administration.
(6) Data statistics
The measured data is averaged to add or subtract the standard deviation
The mean between groups is compared by One-way analysis of variance (One-WayANOVA), and the mean between groups is compared pairwise by SNK method. Pathological analysis grade data are checked by nonparametric rank sum, the mean number between groups is compared pairwise, a Nemenyi method is adopted, and the pathological analysis grade data are completed by SPSSl3.0 software, wherein alpha is 0.05.
(II) results of experiment
1. General conditions in rats
Two upper and lower skin defect wounds are made on the back of a rat, the rat is raised in a single cage, the drug is administered after 0.5h, 1 time/d and 14 days of drug administration, and the wound healing condition and inflammatory reaction of each rat are observed every day, as shown in detail in figure 1. The results show that on the 4 th day after trauma, due to the rat curling and bowing back, the lower wound surface is pulled, most wound surfaces are enlarged in the 4 th day compared with the 1 st day, and the upper wound surface has rich mucous membrane and blood vessels and better healing capacity. All wounds scabbed and appeared dark red, hair grew around the wound margins, inflammatory exudate appeared on the upper wounds, and a greater number of negative controls appeared. The wound area is smaller and smaller from 7 days to 14 days after the wound, inflammatory exudates are on the upper wound surface, the negative control group has more appearance, and the hair around the wound edge is grown; after 10 days, scabs begin to fall off, the wound surface is cleaner, and less exudate appears.
2. Body weight change in rats
The results of the body weight of the rats in each group are shown in Table 3 and FIG. 2.
TABLE 3 weight changes in rats of each group
The results show that the difference between the body weights of the rats in each group is not statistically significant with the time, which indicates that MDT-1 and MDT-2 have no effect of increasing the body weight of the rats.
3. Wound healing conditions
(1) Change of wound surface area
Because the rat curls, bows and draws the lower wound surface, most wound surfaces are enlarged on the 4 th day than the original ones, and moreover, the mucous membranes and blood vessels of the upper wound surface are abundant and the healing capacity is stronger than that of the lower wound surface, so the results can be only compared in the same direction but not up and down in analysis. The change of the wound area of rats in each group is shown in table 4, table 5 and fig. 3.
TABLE 4 area change of wound surface on rats of each group
Note: p <0.05, p <0.01 compared to blank group.
TABLE 5 area change of lower wound surface of rats in each group
Note: p <0.05, p <0.01 compared to vehicle group.
The results of the change in the area of the upper wound surface (table 4) show that only the MDT-1 low dose group significantly reduced the area of the upper wound surface (p <0.05) at day 10 compared to the blank group; on day 14, the area of the upper wound surface (p <0.01) can be obviously reduced by all the medicines, and the effect of reducing the area of the upper wound surface by the MDT-1 low-dose group is more obvious than that of the EGF group and the MDT-2 low-dose group. The change results of the lower wound surface area (table 5) show that on day 10, although the effect of reducing the lower wound surface area of each group of drugs is not statistically different (p is greater than 0.05) compared with the vehicle group, the effect of reducing the lower wound surface area of the MDT-1 high-dose group is more obvious than that of the convalescent group and the MDT-2 high-dose group; on day 14, the area of the wound surface was significantly reduced (p <0.01) for each group of drugs compared to the vehicle group, and the effect of reducing the area of the wound surface was better for the MDT-1 high dose group than for the convalescent group and the MDT-2 high dose group. The result indicates that MDT-1 (periplaneta americana extract of the invention) has better wound healing capacity, and can play a role in promoting wound healing earlier and more effectively compared with EGF, rehabilitation new and MDT-2.
(2) Percent change in wound healing
The percentage change of wound healing in each group of rats is detailed in tables 6 and 7 and fig. 4.
TABLE 6 percent Change in wound healing in rats of various groups
Note: p <0.05, p <0.01 compared to blank group.
TABLE 7 percentage change in wound healing in rats of each group
Note: p <0.05, p <0.01 compared to vehicle group.
The percentage change in wound healing on the upper surface (Table 6) shows that only the percentage of wound healing in the MDT-1 low dose group was significantly increased (p <0.05) compared to the blank group at day 10; the percent healing of the wound surface on each group of drugs was significantly increased on day 14 (p <0.01), and the percent healing of the wound surface on the MDT-1 low dose group was greater than that of the EGF and MDT-2 low dose groups. The percentage change in wound healing at day 10 (table 7) shows that, although the percentage of wound healing was not statistically different in each group compared to the vehicle group (p >0.05), the percentage of wound healing was higher in the MDT-1 high dose group than in the convalescent group and the MDT-2 high dose group; on day 14, the percent healing of the wound surface was significantly increased in each group compared to the vehicle group (p <0.01), and the percent healing of the wound surface was higher in the MDT-1 high dose group than in the convalescent group and the MDT-2 high dose group. The result indicates that MDT-1 (periplaneta americana extract of the invention) has better wound healing capacity, and can promote wound healing earlier and more effectively compared with EGF, rehabilitation new and MDT-2.
(3) Rate of wound healing change
The change of the wound healing rate of rats in each group is shown in tables 8 and 9 and fig. 5.
TABLE 8 change in wound healing Rate in rats of various groups
Note: p <0.05, p <0.01 compared to blank group.
TABLE 9 change in wound healing Rate in rats of various groups
Note: p <0.05, p <0.01 compared to vehicle group.
The results of the change in the healing rate of the upper wound (Table 8) show that the healing rate of the wound surface of only the MDT-1 low dose group was significantly increased (p <0.05) compared to the blank group at day 10; on day 14, the drug wound healing rate was significantly increased in each group compared to the blank group (p <0.01), and the wound healing rate was higher in the MDT-1 low dose group than in the EGF group. The results of the change in wound healing rate (table 9) show that, on day 10, the wound healing rate was greater in the MDT-1 high dose group than in the convalescent group and the MDT-2 high dose group, although there was no statistical difference (p >0.05) between the drug and vehicle groups; on day 14, the wound healing rate of each group of drugs was significantly increased compared to the vehicle group (p < 0.01). The result indicates that MDT-1 (periplaneta americana extract of the invention) has better wound healing capacity, and can promote wound healing earlier and more effectively compared with EGF, rehabilitation new and MDT-2.
(4) Histopathological detection result of wound surface
The histopathological examination results of the rat wound surface of each group are shown in fig. 6:
negative control-blank: more crusts, moderate capillary hyperplasia, more inflammatory cell infiltration, a small amount of inflammatory exudate, mild epidermal hyperplasia, a large amount of fibroblast and collagen hyperplasia, and mild hemorrhage were observed.
Negative control-vehicle group: small crusts, moderate capillary hyperplasia, large inflammatory cell infiltration, small inflammatory exudate, mild epidermal hyperplasia, large fibroblast and collagen hyperplasia, moderate edema and hemorrhage were seen.
Positive control-EGF group: severe capillary hyperplasia, massive inflammatory cell infiltration, massive inflammatory exudate, severe epidermal hyperplasia, massive fibroblast and collagen hyperplasia, mild hemorrhage and edema can be seen.
Positive control group-new group of convalescence: crusts, moderate capillary hyperplasia, more inflammatory cell infiltration, small inflammatory exudate, mild edema and epidermal hyperplasia, large fibroblast and collagen hyperplasia were seen.
MDT-1 low dose group: small crusts, moderate capillary hyperplasia, large inflammatory cell infiltration, small inflammatory exudate, mild epidermal hyperplasia, large fibroblast and collagen hyperplasia, moderate edema and hyperemia were seen.
MDT-1 high dose group: crusts, moderate capillary hyperplasia, more inflammatory cell infiltration, more inflammatory exudate, mild epidermal hyperplasia, massive fibroblast and collagen hyperplasia, and mild hemorrhage were observed.
MDT-2 low dose group: more crusts, moderate capillary hyperplasia, more inflammatory cell infiltration, large inflammatory exudates, mild epidermal hyperplasia, large fibroblast and collagen hyperplasia, mild edema and hemorrhage were seen.
MDT-2 high dose group: scab skin, extensive capillary hyperplasia, extensive inflammatory cell infiltration, more inflammatory exudate, moderate epidermal hyperplasia, extensive fibroblast and collagen hyperplasia, mild edema and hemorrhage were seen.
The histopathological change grades of the wound surfaces of rats in each group are summarized and the diagnosis score results are shown in tables 10-12:
TABLE 10 histopathological Change staging summary sheet
TABLE 11 histopathological Change staging summary sheet
Table 12 histopathological diagnostic score (
N＝8)
Note: compared with the negative control group, the test results show that, ## P<0.01; compared with the positive control group, the composition has the advantages that, * P<0.05。
as can be seen from the results in table 12, the histopathological diagnostic scores of each group were statistically significant (P <0.01) compared to the negative control group; compared with a positive control group, the histopathological diagnosis score of the MDT-1 (the periplaneta americana extract of the invention) group has statistical significance (P <0.05), and the result shows that the MDT-1 (the periplaneta americana extract of the invention) has the best effect of inducing wound tissue healing.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (7)
1. The application of the periplaneta americana extract in preparing the medicine for treating wound healing is characterized in that the preparation method of the periplaneta americana extract comprises the following steps:
(1) alcohol extraction: pulverizing Periplaneta americana into coarse powder, extracting with ethanol, centrifuging the extractive solution, filtering, and concentrating under reduced pressure;
(2) water precipitation: and (2) adding purified water into the concentrated solution obtained in the step (1), uniformly stirring, standing for layering, collecting an oil layer, concentrating under reduced pressure, and drying to obtain the periplaneta americana extract.
2. The use according to claim 1, wherein the periplaneta americana in the step (1) is crushed into coarse powder, ethanol with a volume fraction of 75-90% and a volume of 4-8 times of the weight of the coarse powder is added, the mixture is extracted at room temperature for 20-30 hours, an extracting solution is centrifuged and filtered, and the filtrate is concentrated under reduced pressure at 40-60 ℃.
3. The use according to claim 2, wherein the periplaneta americana in the step (1) is crushed into coarse powder, ethanol with a volume fraction of 85% and a volume which is 5 times of the weight of the coarse powder is added, the mixture is extracted at room temperature for 24 hours, the extracting solution is centrifuged, filtered, and the filtrate is concentrated under reduced pressure at 50 ℃.
4. The use according to claim 1, wherein purified water with the volume 3-6 times of the weight of the concentrated solution is added into the concentrated solution in the step (2), the mixture is uniformly stirred and is kept stand for layering for 20-30 h at 0-10 ℃.
5. The use according to claim 4, wherein purified water with the volume 5 times of the weight of the concentrated solution is added into the concentrated solution in the step (2), the mixture is stirred uniformly and is kept stand and layered for 24 hours at 4 ℃.
6. A medicine for treating wound healing, which is characterized by comprising the periplaneta americana extract as claimed in any one of claims 1 to 5 and a pharmaceutically acceptable carrier; wherein, the medicine is an external medicine.
7. The medicament for treating wound healing according to claim 6, wherein the external medicament is in the form of tincture, cream, powder or ointment.
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