CN111773277A - Preparation method and application of pawpaw total triterpene hydrogel - Google Patents
Preparation method and application of pawpaw total triterpene hydrogel Download PDFInfo
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Abstract
The invention discloses a preparation method and application of pawpaw total triterpene hydrogel. The pawpaw total triterpene hydrogel is prepared from pawpaw total triterpene, monomethoxy polyethylene glycol, caprolactone and alpha-cyclodextrin, and the prepared hydrogel is applied to the application of promoting the healing of skin wound. Firstly, taking mPEG and-CL as substrates, and carrying out ring-opening polymerization reaction on the-CL and the mPEG under the catalysis of ethyl ether hydrochloride to synthesize mPEG-polycaprolactone with a hydrophilic chain segment and a hydrophobic chain segment; then, preparing the pawpaw total triterpene drug-loaded micelle by a thin film hydration method by taking mPEG-PCL as a material; and uniformly mixing the pawpaw total triterpene drug-loaded micelle and the alpha-CD on a vortex mixer to obtain the pawpaw total triterpene hydrogel. The method has the advantages of simple process and easily obtained raw materials; the prepared pawpaw total triterpene hydrogel can effectively promote the healing of skin wound surfaces, shorten the healing time of wounds, promote the regeneration and repair of damaged skin and reduce the generation of scars.
Description
Technical Field
The invention belongs to the technical field of skin repair, and particularly relates to a preparation process and application of pawpaw total triterpene hydrogel.
Background
The skin is the largest organ of the human body, has a plurality of important functions of metabolism, absorption, protection, thermoregulation, secretion, sensation and the like, and is easy to cause damage when being stimulated by external chemical, physical and other factors. The healing process of skin after acute trauma is very easy to cause scar and wound contraction, and normal skin function, beauty and psychology are affected, so that the regeneration and function repair of the skin after injury become focus of attention of clinicians. Particularly, a patient with full-thickness skin defect can heal the wound mainly through the migration of the skin epithelial cells and the regeneration of the stem cells of the residual skin appendages, if the skin defect area of the patient is too large or the full-thickness skin is damaged/defected, the skin cannot be completely regenerated, the wound surface forms scars, the healing is delayed or not healed, the patient suffers from serious dysfunction, and the patient suffers great pain. Therefore, it is important to promote the healing of the skin wound and reduce the scar formation.
At present, methods for healing skin wounds mainly include gene engineering application, bioengineering material application, nanotechnology application, nutrition adjuvant therapy and the like, but have the defects of large individual difference of curative effect, high price, poor patient compliance, easy scar generation after healing and the like, the traditional Chinese medicine has a long history of medical record for treating skin, is rich in experience, creates a plurality of different dosage forms, and has good curative effect in treating skin diseases by extracting traditional Chinese medicines along with scientific development in recent years.
Fructus Chaenomelis is of Rosaceae plant Chaenomelis speciosa (sweet) nakaiChaenomeles speciosa(Sweet) dried nearly mature fruit of Nakai, also known as chaenomeles speciosa, barberry, chaenomeles speciosa, sour chaenomeles, chaenomeles speciosa, etc. Pawpaw is called 'Baiyi fruit' and is a very important ornamental and edible plant, and is listed as one of the first medical and edible foods identified by the ministry of health in 2003. It is used as an edible fruit and is also used in the field of cosmetic and health care products. The total triterpene of fructus Chaenomelis is an important active ingredient in Chaenomeles speciosa nakai, and its main ingredients include oleanolic acid, ursolic acid, betulinic acid, 3-O-acetyl ursolic acid, 3-O-acetyl palygosamine acid, maslinic acid, tormentic acid and specificosaperoxide. The content of the total triterpene of the pawpaw in the fresh fruit is more than 0.32 percent, and the total content of the enriched total triterpene of the pawpaw is 90.02 percent. Specific methods are described in our issued patents: a Chaenomeles speciosa extract, its extraction method and its application (patent No. ZL201510660595.8) are described in detail.
Disclosure of Invention
The inventor of the invention prepares the pawpaw total triterpene hydrogel from the pawpaw total triterpene extracted from the Tujia medicine wrinkled pAN _ SNer pawpaw through extensive and intensive research, and finds that the pawpaw total triterpene can effectively promote the proliferation and migration of human dermal fibroblasts and vascular endothelial cells, has better promotion of skin repair and wound healing on damaged skin wound surfaces and reduces the generation of scars through research, and has no obvious toxic or side effect. At present, reports related to the preparation method of the pawpaw total triterpene hydrogel prepared by the invention and the promotion of wound healing of damaged skin are not found.
The invention aims to provide a preparation method for preparing pawpaw total triterpene into pawpaw total triterpene hydrogel. The method comprises the steps of synthesizing monomethoxy polyethylene glycol-polycaprolactone (mPEG-PCL), preparing the pawpaw total triterpene micelle and preparing pawpaw total triterpene hydrogel.
The invention is realized by the following technical scheme:
a) heating and azeotropy mPEG and toluene in a three-neck flask, and recovering the toluene to obtain anhydrous mPEG;
b) dissolving anhydrous mPEG obtained in the step a) in dichloromethane, sequentially adding caprolactone (-CL) and 2.0mol/L ethyl hydrochloride, reacting at 25 ℃, and then recovering dichloromethane and ethyl ether to obtain a crude product of mPEG-PCL;
c) precipitating the mPEG-PCL crude product obtained in the step b) in ether, and filtering to obtain purified white to off-white star-shaped mPEG-PCL crystals.
d) Uniformly mixing mPEG-PCL obtained in the step c) and the total triterpene of the pawpaw, adding dichloromethane, stirring to a transparent solution, and recovering the dichloromethane in the solution to obtain the light brown crude micelle of the total triterpene of the pawpaw.
e) Adding distilled water into the light brown crude pawpaw total triterpene micelle obtained in the step d), heating, uniformly mixing, filtering by using a filter membrane, removing unencapsulated precipitates such as pawpaw total triterpene and the like to obtain a pawpaw total triterpene micelle solution, or freeze-drying to obtain the pawpaw total triterpene micelle.
f) Dissolving the pawpaw total triterpene micelle solution or the pawpaw total triterpene micelle obtained in the step e) by using distilled water, and then adding alpha-cyclodextrin (alpha-CD) with a certain concentration for uniformly mixing to obtain the pawpaw total triterpene hydrogel.
In a specific embodiment of the invention in the synthesis of mPEG-PCL: firstly, during the process of removing water by using mPEG, dissolving the mPEG and toluene in a three-neck flask according to the mass-volume ratio of 1: 2-10 (g: mL); heating and azeotroping for 1-5 hours at the temperature of 100-160 ℃, and then recovering toluene; repeating the operation once more, and recovering the toluene until no toluene smell exists to obtain the anhydrous mPEG. Dissolving anhydrous mPEG and dichloromethane in a rotary evaporation bottle according to the mass-volume ratio of 1: 2-10 (m: V), and adding-CL in the mass ratio of 1: 1-5 (m: m) of mPEG to uniformly mix; then adding 2.0mol/L ethyl ether hydrochloride with the volume ratio of 1: 0.1-0.5 (v: v), uniformly mixing, stirring at 25 ℃ for 12-36 hours, and then recovering dichloromethane and ethyl ether to obtain a crude product of mPEG-PCL. And thirdly, adding diethyl ether into the mPEG-PCL crude product according to the mass-volume ratio of 1: 2-10 (m: V) for washing and precipitation, and then filtering to obtain the purified white to off-white star-shaped mPEG-PCL crystal.
The synthesis process is simple, feasible and quick; the yield and the purity of mPEG-PCL obtained by the process are both higher (the purity reaches 99.5%); the recovered product and the catalyst can be recycled, so that the production cost is reduced, and the pollution to the environment is reduced; in a word, the synthesis process is very suitable for large-scale synthesis of mPEG-PCL by pharmaceutical enterprises.
In the specific embodiment of the pawpaw total triterpene micelle, the invention comprises the following two types: uniformly mixing mPEG-PCL and total triterpene of pawpaw according to the mass ratio (m: m) of 1: 0.05-0.5, adding dichloromethane (mixture: dichloromethane mass volume ratio 1: 25-100 (m: V)) and continuously stirring until a transparent solution is obtained, and recovering dichloromethane in the solution by a rotary evaporation method to obtain the crude micelle of the light brown total triterpene of pawpaw. Adding distilled water into the obtained light brown crude papaya triterpene micelles according to the mass volume ratio (m: V) of 1: 25-100, heating to 60 ℃, uniformly mixing by using a vortex instrument to obtain a papaya triterpene micelle solution, filtering by using a filter membrane of at least 0.22 mu m, removing unencapsulated precipitates such as papaya total triterpene and the like to obtain a papaya total triterpene micelle solution, or freeze-drying to obtain the papaya total triterpene micelle.
In specific embodiments of the papaya total triterpene hydrogels of the present invention: adding the pawpaw total triterpene micelle solution into an alpha-cyclodextrin (alpha-CD) solution with the concentration of 15-25% according to the volume ratio of 1: 0.25-1 (v/v), and uniformly mixing by using a vortex instrument at 25 ℃ to obtain the wrinkled pawpaw total triterpene hydrogel; or adding distilled water into the obtained pawpaw total triterpene micelle according to the mass volume ratio (m: V) of 1: 25-100, heating to 60 ℃, uniformly mixing, cooling to 25 ℃, adding an alpha-CD solution with the concentration of 15-25% according to the volume ratio of 1: 0.1-0.5 (V: V), and uniformly mixing by using a vortex instrument to obtain the pawpaw total triterpene hydrogel.
The total triterpene hydrogel of the wrinkled pAN _ SNer majoram prepared by the preparation method has good biocompatibility, nontoxicity, resistibility and good biodegradability, can effectively avoid the first pass effect of a medicine liver, can realize directional administration and has lasting effect, thereby having better application and development prospects.
The invention has the advantages of
The pawpaw total triterpene hydrogel prepared by the invention has better functions of promoting proliferation, inhibiting apoptosis and promoting cell migration in human dermal fibroblasts and vascular endothelial cell injury models; the Chinese medicinal composition has good treatment effect on rat skin injury models and skin trauma patients caused by surgical cutting, and can remarkably promote the healing of skin wound surfaces, shorten the healing time of wounds, promote the regeneration and repair of damaged skin and reduce the generation of scars.
Drawings
Fig. 1 is a preparation process and application technology route chart of pawpaw total triterpene hydrogel.
Figure 2 effect of total triterpene hydrogels of pawpaw on UVB damaged HDF cell proliferation (MTT assay) (compare to normal group:# P<0.05,## Pless than 0.01; comparison with UVB group:* P<0.05,** Pless than 0.01; comparison with blank hydrogel group:† P<0.05,†† P<0.01;n=4)。
FIG. 3 Effect of Total triterpene hydrogels of papaya on UVB-damaged HDF cell proliferation (plating clone formation experiment) (A: cell clone panel, B: number of clone formation; compare with normal group:# P<0.05,## Pless than 0.01; comparison with UVB group:* P<0.05,** Pless than 0.01; comparison with blank hydrogel group:† P<0.05,†† P<0.01;n=4)。
FIG. 4 HDF cell withering of Total triterpene hydrogels of papaya against UVB injuryEffects of apoptosis (A: apoptosis picture, B: number of apoptosis; compared to normal group:# P<0.05,## Pless than 0.01; comparison with UVB group:* P<0.05,** Pless than 0.01; comparison with blank hydrogel group:† P<0.05,†† P<0.01;n=4)。
FIG. 5 Effect of Total triterpene hydrogels of papaya on UVB-damaged HDF cell migration (scratch test) (A: cell migration picture, B: migration area; compare with normal group:# P<0.05,## Pless than 0.01; comparison with UVB group:* P<0.05,** Pless than 0.01; comparison with blank hydrogel group:† P<0.05,†† P<0.01;n=4)。
FIG. 6 Effect of Total triterpene hydrogels of papaya on UVB-damaged HDF cell migration (Transwell experiment) (A: image of cell migration, B: number of migrating cells; compare with normal group:# P<0.05,## Pless than 0.01; comparison with UVB group:* P<0.05,** Pless than 0.01; comparison with blank hydrogel group:† P<0.05,†† P<0.01;n=4)。
figure 7 effect of total triterpene hydrogels of papaya on UVB-damaged HUVECs cell proliferation (MTT assay) (compare to normal group:# P<0.05,## Pless than 0.01; comparison with UVB group:* P<0.05,** Pless than 0.01; comparison with blank hydrogel group:† P<0.05,†† P<0.01;n=4)。
FIG. 8 Effect of pawpaw total triterpene hydrogels on UVB-damaged HUVECs cell proliferation (plating clone formation experiment) (A: cell clone picture, B: number of clone formation; compare with normal group:# P<0.05,## Pless than 0.01; comparison with UVB group:* P<0.05,** Pless than 0.01; comparison with blank hydrogel group:† P<0.05,†† P<0.01;n=4)。
FIG. 9 Effect of pawpaw total triterpene hydrogels on UVB-damaged HUVECs apoptosis (A: apoptosis picture, B: apoptosis number; compared to normal group:# P<0.05,## Pless than 0.01; comparison with UVB group:* P<0.05,** Pless than 0.01; comparison with blank hydrogel group:† P<0.05,†† P<0.01;n=4)。
FIG. 10 Effect of Total triterpene hydrogels of papaya on cell migration of UVB-injured HUVECs (scratch test) (A: cell migration picture, B: migration area; compare with normal group:# P<0.05,## Pless than 0.01; comparison with UVB group:* P<0.05,** Pless than 0.01; comparison with blank hydrogel group:† P<0.05,†† P<0.01;n=4)。
FIG. 11 Effect of Total triterpene hydrogels of papaya on cell migration of UVB-injured HUVECs (Transwell experiment) (A: cell migration picture, B: number of migrated cells; compared with normal group:# P<0.05,## Pless than 0.01; comparison with UVB group:* P<0.05,** Pless than 0.01; comparison with blank hydrogel group:† P<0.05,†† P<0.01;n=4)。
figure 12 effect of total triterpene hydrogel from papaya on wound surface of rat model of skin injury.
Figure 13 effect of total triterpene hydrogel of papaya on wound healing time in rat skin injury model (compared to blank hydrogel group:* P<0.05,** Pless than 0.01; comparison with scar group:† P<0.05,†† P<0.01;n=10)。
figure 14 effect of total triterpene hydrogel of papaya on wound healing rate of rat wound in skin injury model (compared to blank hydrogel group:* P<0.05,** Pless than 0.01; comparison with scar group:† P<0.05,†† P<0.01;n=10)。
FIG. 15 Effect of pawpaw total triterpene hydrogels on pathological morphology of skin of rats in skin injury model.
Detailed Description
The present invention will be further described with reference to the following examples, which are intended to be illustrative only and not to be limiting of the invention in any way, and any person skilled in the art can modify the present invention by applying the teachings disclosed above and applying them to equivalent embodiments with equivalent modifications. Any simple modification or equivalent changes made to the above embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.
Example 1 preparation of pawpaw total triterpene hydrogels
(1) Removing water by monomethoxy polyethylene glycol (mPEG)
Weighing 10.0g of monomethoxypolyethylene glycol (mPEG) and placing the obtained product in a three-neck flask, adding 40mL of toluene according to the mass-volume ratio of 1:4(g: mL), heating for azeotropic distillation for 2 hours at 120 ℃, and then recovering the toluene; the above operation was repeated once more, and toluene was recovered until no toluene smell was observed to obtain a white anhydrous mPEG powder, which was weighed to obtain 8.25 g.
(2) Synthesis of monomethoxypolyethylene glycol-polycaprolactone (mPEG-PCL)
The white anhydrous mPEG powder obtained in the above (1) is put into a rotary evaporation bottle, 41.25mL of dichloromethane is added according to the mass-volume ratio of 1:5(g: mL) to dissolve the white anhydrous mPEG powder, 16.50g of caprolactone (-CL) with the mass ratio of 1:2(g: g) is added to mix evenly, then 12.5mL of 2.0mol/L ethyl ether hydrochloride with the volume ratio of 1:0.25(v: v) is added to stir at 25 ℃ for 24 hours, and then dichloromethane and ethyl ether are recovered to obtain 24.62g of crude mPEG-PCL.
(3) Refinement of mPEG-PCL
The crude mPEG-PCL obtained in the step (2) is added with 123.75mL of diethyl ether according to the mass-to-volume ratio of 1:5(g: mL), washed, precipitated and filtered to obtain purified white to off-white star-shaped mPEG-PCL crystals, and the crystals are weighed to obtain 18.47g (see figure 1).
(4) Preparation of pawpaw total triterpene micelle
And (3) uniformly mixing 18.47g of mPEG-PCL crystals obtained in the step (3) and 4.62g of total triterpene of pawpaw in a rotary evaporation bottle according to the mass ratio of 1:0.25, adding dichloromethane with the mass volume ratio of 1:50 (g: mL) to continuously stir until a transparent solution is obtained, and recovering dichloromethane in the solution by a rotary evaporation method to obtain 23.05g of crude micelle of the light brown total triterpene of pawpaw.
(5) Refinement of pawpaw total triterpene micelle
Adding distilled water into 23.05g of the light brown pawpaw total triterpene crude micelle obtained in the step (4) according to the mass volume ratio (g/mL) of 1:50, heating to 60 ℃, uniformly mixing by using a vortex instrument to obtain a chaenomeles speciosa total triterpene micelle solution, filtering by using a 0.22 mu m filter membrane, removing precipitates such as the chaenomeles speciosa total triterpene and the like to obtain 1105mL of the chaenomeles speciosa total triterpene micelle solution, or freeze-drying to obtain 22.10g of the chaenomeles speciosa total triterpene micelle (shown in figure 1).
(6) Preparation of pawpaw total triterpene hydrogel
Adding 221mL of alpha-cyclodextrin (alpha-CD) into the pawpaw total triterpene micelle solution obtained in the step (5) according to the volume ratio (v/v) of 1:0.2, and uniformly mixing by using a vortex apparatus to obtain the chaenomeles speciosa total triterpene hydrogel; or adding 1105mL of distilled water into the obtained pawpaw total triterpene micelle according to the mass volume ratio (g/mL) of 1:50, heating to 60 ℃, uniformly mixing, cooling, adding 221mL of alpha-cyclodextrin (alpha-CD) according to the volume ratio (v/v) of 1:0.2, and uniformly mixing by using a vortex instrument to obtain the wrinkled pawpaw total triterpene hydrogel (shown in figure 1).
The following specific experimental examples are combined to specifically illustrate that the pawpaw total triterpene hydrogel obtained in the embodiment 1 of the invention has better effects of promoting proliferation, inhibiting apoptosis and promoting cell migration on skin cell injury; can remarkably promote the healing of skin wound, shorten the healing time of the wound, promote the regeneration and repair of damaged skin and reduce the generation of scars for an animal skin injury model and a patient with skin trauma.
Experimental example 2
The following will specifically illustrate the effects of the pawpaw total triterpene hydrogel obtained in example 1 of the invention in promoting cell proliferation, cell migration and inhibiting apoptosis of UVB-damaged human skin fibroblasts (HDF).
Test method
1) Test cell
Cell lines in this experiment human skin fibroblasts (HDF) were purchased from Saimer Feishell science and technology (China) Co., Ltd and cultured in DMEM culture medium containing 10% fetal bovine serum at 37 ℃ with 5% CO2The cells are grown in an incubator, and cells in a logarithmic growth phase are taken during experiments.
2) Method of producing a composite material
(1) Cell culture
Culturing normal HDF cells in DMEM medium containing 10% imported fetal calf serum at 37 deg.C under 5% CO2Relative saturation humidity condition, sterile culture. The growth condition of the cells is observed every day, and the passage is carried out when the adherent of the monolayer cells reaches about 80 percent. And (3) discarding the supernatant during passage, washing the cells for 2-3 times by PBS, discarding the PBS, adding 2mL of 0.25% trypsin for digestion for 3min, adding a serum-containing culture solution to stop digestion, gently blowing down adherent cells to form a cell suspension, centrifuging at 1400rpm for 4min, discarding the supernatant, adding a fresh culture solution to blow and beat the adherent cells to form a single cell suspension, wherein the passage ratio is 1:3, and transferring the single cell suspension into a culture bottle for continuous culture. Cells in logarithmic growth phase were taken at the time of the experiment.
(2) Establishment and grouping of HDF cellular models of medium-wave Ultraviolet (UVB) damage
HDF cells were cultured in DMEM medium containing 10% imported fetal bovine serum, and the concentration of the cells was 1 × 10 when the cells grew to the logarithmic growth phase5Inoculating single cell suspension/mL into 96-well plate, culturing for 24 hr, and irradiating with ultraviolet ray at intensity of 0.4mW/cm2The irradiation time was 250s, and the final total irradiation dose was 100mJ/cm2(irradiation dose = irradiation intensity × irradiation time.) UVB-damaged HDF cells were randomly divided into UVB group, blank hydrogel group, pawpaw total triterpene hydrogel [2.5, 5, 10, 20, 40, 80 μ g (containing pawpaw total triterpene)/mL, respectively]Group and UVB group, blank hydrogel group, pawpaw total triterpene hydrogel [5, 10 and 20 μ g (containing pawpaw total triterpene)/mL]Group, HDF cells not irradiated with UVB were additionally set as normal group. Before the experiment, the total triterpene hydrogel of the pawpaw is prepared into a solution of 100mg/mL by dimethyl sulfoxide (DMSO), and the solution is filtered by a filter membrane of 0.22 mu m and stored at 4 ℃ for later use.
(3) MTT (methyl thiazolyl tetrazolium) detection effect of pawpaw total triterpene hydrogel on UVB (ultraviolet B) damaged HDF (high density fibroblast proliferation) cell proliferation
The HDF cell culture method and group are shown in (2). After adding pawpaw total triterpene hydrogel with corresponding concentration into each administration, respectively culturing for 24h and 48h, adding 20 mu L of MTT solution (5mg/mL) into each well, completely sucking out the culture solution after culturing for 4h, adding 150 mu LDMSO, and measuring OD value at 490nm wavelength.
(4) Plate clone formation experiment for detecting influence of pawpaw total triterpene hydrogel on UVB damaged HDF cell proliferation
The experimental groups were normal, UVB, blank hydrogel, pawpaw total triterpene hydrogel 5 μ g (containing pawpaw total triterpene)/mL, pawpaw total triterpene hydrogel 10 μ g (containing pawpaw total triterpene)/mL, pawpaw total triterpene hydrogel 20 μ g (containing pawpaw total triterpene)/mL. 300 single-cell HDF suspensions are inoculated in a 6-well plate per well, after culturing for 24h, the pawpaw total triterpene hydrogel is added into each dosage group of the pawpaw total triterpene hydrogel (5, 10 and 20 mug/mL), the culture solution in each well is 3mL, the solution is changed every three days, and pawpaw total triterpene with corresponding concentration and 5% CO are added while the solution is changed2Culturing at 37 deg.C under relative saturated humidity for about 2 weeks, and irradiating with ultraviolet ray at intensity of 0.4mW/cm except for normal group2The irradiation time was 250s, and the final total irradiation dose was 100mJ/cm2And continuously culturing for 24h, then carrying out crystal violet staining, and calculating the number of formed clones.
(5) Hoechst 33258 staining detection of influence of pawpaw total triterpene hydrogel on UVB-damaged HDF apoptosis
5×1051mL of HDF single cell suspension per mL is inoculated in a six-well plate, and after the cells adhere to the wall, the drug is added for treatment, and the cells are grouped in (2). The groups were irradiated with ultraviolet rays at an intensity of 0.4mW/cm except for the normal group2The irradiation time was 250s, and the final total irradiation dose was 100mJ/cm2Adding pawpaw total triterpene hydrogel (5, 10 and 20 mug/mL) with corresponding concentration into each administration, culturing for 24h, discarding culture solution in the hole, adding 0.5mL of 4% paraformaldehyde fixing solution, and fixing for 10 min; washing with PBS for 2 times, adding 1mL of Hoechst 33258 staining solution, and lightly shaking the mixture by using a shaking table to stain for 5 minutes; washing with PBS 2 timesAnd the liquid is sucked up. And (5) detecting and photographing under a fluorescence microscope.
(6) Scratch test for detecting influence of pawpaw total triterpene hydrogel on migration and proliferation of HDF (high density lipoprotein)
HDF cells in logarithmic growth phase were taken at 15 × 105each/mL was inoculated into a 6-well plate, 3 scratches were vertically scribed in the 6-well plate using a sterile 200 μ L tip, then washed with PBS, cells were treated with pawpaw total triterpene hydrogels (5, 10 and 20 μ g/mL) of corresponding concentrations according to (2), and the change in scratch width was observed after incubation for 24h and analyzed using corresponding image processing software.
(7) Transwell experiment detects influence of pawpaw total triterpene hydrogel on migration and proliferation of HDF
When the cells grow in the logarithmic phase, the cells are subjected to conventional digestion and centrifugation, and the cells are resuspended in serum-free DMEM medium to prepare 5 × 105one/mL cell suspension, 100. mu.L of cell suspension was added to each upper chamber; adding a culture medium containing 10% FBS into a lower chamber of a normal group, adding a serum-free culture medium into a model group, and adding a serum-free culture medium containing pawpaw total triterpene hydrogel (5, 10 and 20 mu g/mL) with corresponding concentration into an administration group; incubating for 24 hours in an incubator at 37 ℃; taking out the upper chamber, washing the upper chamber for 2 times by PBS, wiping off cells on the surface of the bottom membrane of the upper chamber by a cotton ball, adding crystal violet (0.1%) for dyeing for 10min, washing off redundant dye by PBS, observing under a microscope, recording by photography, and counting the number of the cells by software.
(8) Statistical analysis
The data of each group are counted as mean ± standard deviation: (S) is shown. Data analysis was performed on the SPSS21.0 statistical software package, comparisons of differences between groups were performed with one-way ANOVA, differences in mean between groups were compared with Dunnett-t test,P<0.05 indicated that the difference was statistically significant. Each experiment was repeated 4 times.
Test results
1) Effect of pawpaw total triterpene hydrogel on UVB-damaged HDF cell proliferation
MTT results show, UVB illuminationHas obvious effect of inhibiting the proliferation of HDF cells and has significant difference compared with a normal group (P<0.01); after being treated by pawpaw total triterpene hydrogel (2.5, 5, 10 and 20 mu g/mL), the gel has obvious effect of promoting proliferation of HDF cells damaged by UVB, and 40 and 80 mu g/mL pawpaw total triterpene hydrogel inhibit the proliferation of the HDF cells. The low-concentration pawpaw total triterpene hydrogel can promote HDF cell proliferation, and the high-concentration pawpaw total triterpene hydrogel can inhibit the proliferation activity. Based on the current study, the appropriate concentration of pawpaw total triterpene hydrogels (below 20 μ g/mL) and treatment time (24h) were determined for further in vitro studies (see figure 2).
The experimental result of plate clone formation shows that the UVB irradiation can obviously inhibit the clone number of HDF cells and has significant difference compared with a normal group (P<0.01); after being treated by pawpaw total triterpene hydrogel (5, 10 and 20 mu g/mL), the clone number of HDF cells damaged by UVB can be obviously increased, and the obvious difference is shown in comparison with UVB group and blank hydrogel group (theP<0.05 orP<0.01); the blank hydrogel group had no significant effect on the number of UVB-damaged HDF cell clones (see fig. 3).
2) Effect of pawpaw total triterpene hydrogel on UVB-damaged HDF apoptosis
The results of Hoechst 33258 staining show that the cells of the normal control group show weak blue fluorescence, the shape is uniform and complete, and the chromatin is distributed uniformly. More strong blue fluorescent staining cells can be seen in the UVB damage model group and the blank hydrogel group, and typical apoptosis bodies, chromatin condensation, nucleus fragmentation into round bodies with different sizes and the like can be observed to show the phenomenon of cell apoptosis; the apoptosis number of the cells is obviously increased, and the significant difference is shown in (A) and (B) compared with a normal groupP<0.01); the phenomenon is weakened after the pawpaw total triterpene hydrogel (5, 10 mu and 20 mu g/mL) is treated, and with the increase of the dosage of the pawpaw total triterpene hydrogel, cells with normal forms are increased, the apoptosis characteristics are less obvious, the apoptosis number is obviously reduced, and the pawpaw total triterpene hydrogel has significant difference compared with a UVB group and a blank hydrogel group (theP<0.05 orP<0.01); the blank hydrogel group showed no significant improvement in the number of UVB-damaged HDF apoptotic cells (see fig. 4).
3) Effect of pawpaw total triterpene hydrogel on migration of UVB-damaged HDF cells
The scratch test result shows that: UVB irradiation obviously inhibits the migration capability of cells, the healing capability of the cells is obviously reduced, and the obvious difference is shown compared with a normal group (P<0.01); after being treated by pawpaw total triterpene hydrogel (5 and 20 mu g/mL), the pawpaw total triterpene hydrogel can obviously promote the migration of HDF cells damaged by UVB, obviously reduce the distance from the cells at the periphery of a scratch to a central scratch area, obviously improve the healing capability and have significant difference compared with the UVB group and a blank hydrogel group (theP<0.01); no HDF cell migration promoting UVB damage was seen in the blank hydrogel group (see fig. 5).
The results of the Transwell experiment show that: the ability of the UVB-irradiated damaged HDF cells to penetrate the upper chamber membrane was significantly reduced, resulting in a significant reduction in the number of membrane-penetrating cells, with significant differences compared to the normal group (a)P<0.01); the membrane penetrating capacity of the HDF cells damaged by UVB can be obviously promoted after the pawpaw total triterpene hydrogel (5 and 20 mu g/mL) is treated, so that the number of the membrane penetrating cells is obviously increased, and the obvious difference is shown in comparison with the UVB group and the blank hydrogel group (theP<0.01); the blank hydrogel group did not see any enhancement of the membrane penetration of UVB-damaged HDF cells (see fig. 6).
Conclusion of the experiment
Experiments on the influence of the pawpaw total triterpene hydrogel on UVB-damaged HDF cells show that the pawpaw total triterpene hydrogel can remarkably promote the proliferation of the UVB-damaged HDF cells, inhibit the apoptosis of the UVB-damaged HDF cells and promote the cell migration of the UVB-damaged HDF cells. Experimental results show that the pawpaw total triterpene hydrogel has a good protection effect on HDF cells damaged by UVB.
Experimental example 3
The following will specifically illustrate the effects of the pawpaw total triterpene hydrogel obtained in example 1 of the invention in promoting cell proliferation, cell migration and inhibiting apoptosis of Human Umbilical Vein Endothelial Cells (HUVECs) damaged by UVB by combining with specific experimental examples.
Test method
1) Test cell
Cell lines in this experiment Human Umbilical Vein Endothelial Cells (HUVECs) were purchased from Beijing ChinaThe institute of basic medicine of medical academy of sciences, culturing in DMEM medium containing 10% fetal calf serum at 37 deg.C and 5% CO2The cells are grown in an incubator, and cells in a logarithmic growth phase are taken during experiments.
2) Method of producing a composite material
(1) Cell culture
Culturing normal HUVECs (bovine serum albumin) cells in DMEM (DMEM) culture solution containing 10% of imported fetal calf serum at constant temperature of 37 ℃ and 5% of CO2Relative saturation humidity condition, sterile culture. The growth condition of the cells is observed every day, and the passage is carried out when the adherent of the monolayer cells reaches about 80 percent. And (3) discarding the supernatant during passage, washing the cells for 2-3 times by PBS, discarding the PBS, adding 2mL of 0.25% trypsin for digestion for 3min, adding a serum-containing culture solution to stop digestion, gently blowing down adherent cells to form a cell suspension, centrifuging at 1400rpm for 4min, discarding the supernatant, adding a fresh culture solution to blow and beat the adherent cells to form a single cell suspension, wherein the passage ratio is 1:3, and transferring the single cell suspension into a culture bottle for continuous culture. Cells in logarithmic growth phase were taken at the time of the experiment.
(2) Establishment and grouping of HUVECs cell model damaged by medium-wave Ultraviolet (UVB)
HUVECs cells were cultured in DMEM medium containing 10% imported fetal bovine serum, and the cell concentration was 1 × 10 when the cells grew to logarithmic growth phase5Inoculating single cell suspension/mL into 96-well plate, culturing for 24 hr, and irradiating with ultraviolet ray at intensity of 0.4mW/cm2The irradiation time was 250s, and the final total irradiation dose was 100mJ/cm2(irradiation dose = irradiation intensity × irradiation time.) UVB-injured HUVECs cells were randomly divided into UVB group, blank hydrogel group, pawpaw total triterpene hydrogel [2, 4, 8, 16, 32, 64 μ g (containing pawpaw total triterpene)/mL, respectively]Group and UVB group, blank hydrogel group, pawpaw total triterpene hydrogel [4, 8 and 16 μ g (containing pawpaw total triterpene)/mL]In the group, HUVECs cells that were not irradiated with UVB were used as the normal group. Before the experiment, the total triterpene hydrogel of the pawpaw is prepared into a solution of 100mg/mL by dimethyl sulfoxide (DMSO), and the solution is filtered by a filter membrane of 0.22 mu m and stored at 4 ℃ for later use.
(3) MTT (methyl thiazolyl tetrazolium) detection method for influence of pawpaw total triterpene hydrogel on UVB-damaged HUVECs (human umbilical cord blood cells) cell proliferation
The culture method and the grouping of HUVECs are shown in (2). After adding pawpaw total triterpene hydrogel with corresponding concentration into each administration, respectively culturing for 24h and 48h, adding 20 mu L of MTT solution (5mg/mL) into each well, completely sucking out the culture solution after culturing for 4h, adding 150 mu LDMSO, and measuring OD value at 490nm wavelength.
(4) Plate clone formation experiment for detecting influence of pawpaw total triterpene hydrogel on UVB damaged HUVECs cell proliferation
The experimental groups were normal, UVB, blank hydrogel, pawpaw total triterpene hydrogel 4 μ g (containing pawpaw total triterpene)/mL, pawpaw total triterpene hydrogel 8 μ g (containing pawpaw total triterpene)/mL, pawpaw total triterpene hydrogel 16 μ g (containing pawpaw total triterpene)/mL, each group had 3 multiple wells. Inoculating 300 single cell suspensions of HUVECs into a 6-well plate, culturing for 24h, adding pawpaw total triterpene hydrogel (4, 8 and 16 mu g/mL) into each dosage group of pawpaw total triterpene hydrogel, changing the culture solution every three days, and adding pawpaw total triterpene hydrogel with corresponding concentration and 5% CO while changing the solution2Culturing at 37 deg.C under relative saturated humidity for about 2 weeks, and irradiating with ultraviolet ray at intensity of 0.4mW/cm except for normal group2The irradiation time was 250s, and the final total irradiation dose was 100mJ/cm2And continuously culturing for 24h, then carrying out crystal violet staining, and calculating the number of formed clones.
(5) Hoechst 33258 staining detection of influence of pawpaw total triterpene hydrogel on apoptosis of UVB-damaged HUVECs
5×1051mL of HUVECs single cell suspension per mL is inoculated in a six-well plate, and after the cells are attached to the wall, the drug is added for treatment, and the cells are grouped in (2). The groups were irradiated with ultraviolet rays at an intensity of 0.4mW/cm except for the normal group2The irradiation time was 250s, and the final total irradiation dose was 100mJ/cm2Adding pawpaw total triterpene hydrogel (4, 8 and 16 mug/mL) with corresponding concentration into each administration, culturing for 24h, discarding culture solution in the hole, adding 0.5mL of 4% paraformaldehyde fixing solution, and fixing for 10 min; washing with PBS for 2 times, adding 1mL of Hoechst 33258 staining solution, and lightly shaking the mixture by using a shaking table to stain for 5 minutes; wash 2 times with PBS and aspirate off the liquid. And (5) detecting and photographing under a fluorescence microscope.
(6) Scratch test for detecting influence of pawpaw total triterpene hydrogel on HUVECs migration and proliferation
HUVECs in logarithmic growth phase were taken at 15 × 105each/mL was inoculated into a 6-well plate, 3 scratches were vertically scribed in the 6-well plate using a sterile 200 μ L tip, then washed with PBS, cells were treated with pawpaw total triterpene hydrogels (4, 8 and 16 μ g/mL) at corresponding concentrations according to the panel division in (2), and the change in scratch width was observed after incubation for 24h and analyzed using corresponding image processing software.
(7) Transwell experiment detection of influence of pawpaw total triterpene hydrogel on HUVECs migration and proliferation
When the cells grow in the logarithmic phase, the cells are subjected to conventional digestion and centrifugation, and the cells are resuspended in serum-free DMEM medium to prepare 5 × 105one/mL cell suspension, 100. mu.L of cell suspension was added to each upper chamber; adding a culture medium containing 10% FBS into a lower chamber of a normal group, adding a serum-free culture medium into a model group, and adding a serum-free culture medium containing pawpaw total triterpene hydrogel (4, 8 and 16 mu g/mL) with corresponding concentration into an administration group; incubating for 24 hours in an incubator at 37 ℃; taking out the upper chamber, washing the upper chamber for 2 times by PBS, wiping off cells on the surface of the bottom membrane of the upper chamber by a cotton ball, adding crystal violet (0.1%) for dyeing for 10min, washing off redundant dye by PBS, observing under a microscope, recording by photography, and counting the number of the cells by software.
(8) Statistical analysis
The data of each group are counted as mean ± standard deviation: (S) is shown. Data analysis was performed on the SPSS21.0 statistical software package, comparisons of differences between groups were performed with one-way ANOVA, differences in mean between groups were compared with Dunnett-t test,P<0.05 indicated that the difference was statistically significant. Each experiment was repeated 4 times.
Test results
1) Effect of pawpaw total triterpene hydrogel on UVB-damaged HUVECs cell proliferation
MTT results show that UVB irradiation has obvious effect of inhibiting proliferation of HUVECs cells and has the effect of inhibiting proliferation compared with normal groupsSignificant difference (P<0.01); after being treated by pawpaw total triterpene hydrogel (2, 4, 8 and 16 mu g/mL), the gel has obvious effect of promoting proliferation of HUVECs cells damaged by UVB, and 32 and 64 mu g/mL pawpaw total triterpene hydrogel inhibit the proliferation of the HUVECs cells. The low-concentration pawpaw total triterpene hydrogel can promote the cell proliferation of HUVECs, and the high-concentration pawpaw total triterpene hydrogel can inhibit the proliferation activity of the HUVECs. Based on the current study, the appropriate concentration of pawpaw total triterpene hydrogels (below 16 μ g/mL) and treatment time (24h) were determined for further in vitro studies (see figure 7).
The plate clone formation experiment result shows that UVB irradiation can obviously inhibit the clone number of HUVECs and has significant difference compared with a normal group (P<0.01); after being treated by pawpaw total triterpene hydrogel (4 and 16 mu g/mL), the cell clone number of HUVECs damaged by UVB can be obviously increased, and the cell clone number has significant difference compared with UVB group and blank hydrogel group (the method is that the cell clone number of the HUVECs damaged by UVB is obviously increased by adding the pawpaw total triterpene hydrogel to the pawpaw total triterpene, wherein the pawpaw total triterpene is shown in theP<0.05 orP<0.01); the blank hydrogel group had no significant effect on UVB-injured HUVECs cell clone numbers (see figure 8).
2) Effect of pawpaw total triterpene hydrogel on UVB-damaged HUVECs cell apoptosis
The results of Hoechst 33258 staining show that the cells of the normal control group show weak blue fluorescence, the shape is uniform and complete, and the chromatin is distributed uniformly. More strong blue fluorescent staining cells can be seen in the UVB damage model group and the blank hydrogel group, and typical apoptosis bodies, chromatin condensation, nucleus fragmentation into round bodies with different sizes and the like can be observed to show the phenomenon of cell apoptosis; the apoptosis number of the cells is obviously increased, and the significant difference is shown in (A) and (B) compared with a normal groupP<0.01); this phenomenon was reduced after treatment with pawpaw total triterpene hydrogel (4 and 16 μ g/mL) and with increasing pawpaw total triterpene hydrogel dose, there was an increase in normal morphology cells, less significant apoptotic character, significantly reduced apoptotic numbers, and significant differences in comparison to UVB and blank hydrogel groups (a)P<0.05 orP<0.01); the blank hydrogel group showed no significant improvement in the apoptotic numbers of UVB-damaged HUVECs (see fig. 9).
3) Effect of pawpaw total triterpene hydrogel on UVB-damaged HUVECs cell migration
The scratch test result shows that: UVB irradiation obviously inhibits the migration capability of cells, the healing capability of the cells is obviously reduced, and the obvious difference is shown compared with a normal group (P<0.01); after being treated by pawpaw total triterpene hydrogel (4 and 16 mu g/mL), the pawpaw total triterpene hydrogel can obviously promote the cell migration of HUVECs damaged by UVB, obviously reduce the distance from cells at the periphery of scratches to a central scratch area, obviously improve the healing capability, has obvious difference compared with a UVB group and a blank hydrogel group, and shows obvious aging and dose-effect relationship (2)P<0.05 orP<0.01); the blank hydrogel group did not show cell migration of HUVECs that promoted UVB damage (see fig. 10).
The results of the Transwell experiment show that: the ability of the HUVECs damaged by UVB irradiation to penetrate the upper ventricular membrane is obviously reduced, so that the number of the cells penetrating the membrane is obviously reduced, and the obvious difference is shown in comparison with the normal group (P<0.01); the membrane penetrating capacity of HUVECs damaged by UVB can be obviously promoted after the pawpaw total triterpene hydrogel (4 and 16 mu g/mL) is treated, so that the number of the membrane penetrating cells is obviously increased, and the obvious difference is shown in comparison with the UVB group and the blank hydrogel group (theP<0.05 orP<0.01); the blank hydrogel group did not see any enhancement of the membrane penetration ability of UVB-damaged HUVECs cells (see fig. 11).
Conclusion of the experiment
Experiments on the influence of the pawpaw total triterpene hydrogel on UVB-damaged HUVECs show that the pawpaw total triterpene hydrogel can remarkably promote the proliferation of UVB-damaged HUVECs, inhibit the apoptosis of the UVB-damaged HUVECs and promote the cell migration of the UVB-damaged HUVECs. Experimental results show that the pawpaw total triterpene hydrogel has a good protective effect on HUVECs cells damaged by UVB.
Experimental example 4
The following specific experimental examples are combined to specifically illustrate that the pawpaw total triterpene hydrogel obtained in the example 1 of the invention has an obvious therapeutic effect on rat skin injury caused by surgical incision.
Test method
1) Laboratory animal
Animals and feeds were purchased from the experimental animals center of the university of three gorges, 50 SPF grade Sprague Dawley (SD) rats weighing 180-220 g, half male and female, quality certification numbers of experimental animals: SCXK (jaw) 2017-0061. Animals were housed in a normal-grade animal room dry, ventilated, quiet environment of 5 animals per cage. Animal experiments followed the guidelines of the ethical committee on experimental animals of the university of three gorges and experimental studies were developed under its supervision.
2) Method of producing a composite material
(1) Preparation of skin injury rat model
The mold was made using surgical cutting methods. Two days before molding, the two sides of the dorsal spine of the rat are dehaired by 8% sodium sulfide and prepared into skin. Before operation, 20% ethyl carbamate (1.5g/kg) is injected into abdominal cavity for anesthesia, a rat prone position is taken, the back is exposed, a sterile towel is laid, after conventional iodophor disinfection in an operation area, cornea trephines with the diameter of 1.2cm are used for making a full-layer skin defect wound surface at positions 4.0cm away from the back of ears and 1.0cm beside two sides of the middle of a spine, the left side and the right side of the wound surface are respectively provided with 1, and the wound is stopped and washed by normal saline. In the process of making a wound surface, the punching position, the size and the depth are consistent as much as possible, only the whole layer of skin is taken as much as possible, the subcutaneous fascia is damaged less, deep blood vessels and nerves are not damaged as much as possible, and the individual difference of model making is reduced.
(2) Grouping and administration of skin injury model rats
The rats successfully made into the skin injury model are randomly divided into a skin injury blank hydrogel group, a pawpaw total triterpene hydrogel group (1.2, 2.4 and 3.6g/kg) and a scar panacea positive drug group (1.0 g/kg). After the medicine coating group coats corresponding pawpaw total triterpene hydrogel and scar panacea on the wound defect part, the periphery of the wound is sewed, wrapped and coated with vaseline to ensure that the sterile gauze completely covers the hydrogel and the wound, and the hydrogel is prevented from falling off and the wound is prevented from being infected; the blank hydrogel group was coated with the blank hydrogel, and the other treatments were the same as the drug treatment group. The day of molding was taken as day 0. The preparation is administered 1 time per day in the morning and evening for 4 weeks.
(3) General situation observation
The rats were observed for food intake, mental status, wound infection, etc. during the experiment.
(4) Wound healing image collection
The wound was photographed with a camera every 3 days, starting on day 0 of molding. The rats are anesthetized before each photographing, then the wound surfaces and hairs growing on the periphery of the wound surfaces are trimmed, straight rulers are placed at positions with equal distances from the wound surfaces, the photographing is carried out by using the same focal distance and distance, the left and right wound surfaces corresponding to the serial numbers of the rats are recorded, and the wound surface healing condition is observed every day. Calculating the wound area by using Image J software Image analysis software, wherein the wound healing rate = (the wound area after modeling-the current residual wound area)/the wound area after modeling is multiplied by 100 percent, and the wound healing rate of more than 90 percent is taken as the healing standard.
(5) Morphological analysis of skin tissue
One group of rats was randomly selected each time on days 3, 11, and 21, respectively, after anesthetizing, hairs were removed from the periphery of the wound, a specimen containing the entire skin and subcutaneous tissue was taken 1cm from the periphery of the wound, and the animals were sacrificed. Fixing tissue specimen in 4% paraformaldehyde, gradient dehydrating with ethanol, embedding in paraffin, slicing, HE staining, and observing under light microscope.
(6) Statistical analysis
The experimental results are given as mean. + -. standard deviation (S) data analysis was performed on SPSS21.0 statistical software package, comparison of differences between groups was performed by one-way analysis of variance, Dunnett-tChecking and comparing the difference between the two groups;P<0.05 was considered statistically different.
Test results
1) General conditions in rats
The experimental rat is slightly declined after operation and slowly increased after operation, the rat moves normally during feeding, and no obvious infection and suppuration of the wound after operation occur.
2) Influence of pawpaw total triterpene hydrogel on skin injury model rat wound surface
On the 3 rd day after operation, the wound surface exudates of the pawpaw total triterpene hydrogel group and the scar panacea positive drug group are obviously reduced, the wound surface is covered by the scab skin, the wound begins to shrink and becomes smaller, the periphery of the wound is dry, the swelling range is small, and the wound surface reduction speed of each group of wounds is obviously faster than that of the blank water along with the prolonging of timeThe gel group has more obvious wound healing promoting effect, and the curative effect is more obvious along with the increase of the dosage of the total triterpene hydrogel of the pawpaw (1.2, 2.4 and 3.6 g/kg); however, when the total triterpene hydrogel dosage of the pawpaw is increased to 3.6g/kg, the curative effect is not obviously increased, and the pawpaw hydrogel dosage has obvious difference compared with a blank hydrogel group and a scar group (1)P<0.05 orP<0.01). On the 9 th day after the operation, the wounds of the group with 2.4g/kg of pawpaw total triterpene hydrogel reach the healing standard, and the other groups do not reach the healing standard. On the 28 th day after the operation, the wound surfaces of all groups are healed, the wound surfaces of the pawpaw total triterpene hydrogel (1.2, 2.4 and 3.6g/kg) groups are not obviously different from the peripheral skin, no obvious scar bulge exists, and a large amount of new hairs grow out in the wound healing area; the other groups had clear wound peripheries, visible scar tissue formation and no significant hair growth, with the group having 2.4g/kg total triterpene hydrogel of papaya (see fig. 12) having the most significant therapeutic effect. The results show that the pawpaw total triterpene hydrogel can moisturize the wound surface, form a protective layer on the wound surface, prevent large-area contact between the skin defect part and the outside, reduce the probability of wound infection, create better conditions for wound healing to promote skin wound healing, shorten the wound healing time, promote the regeneration and repair of damaged skin and reduce scar generation.
3) Influence of pawpaw total triterpene hydrogel on healing time of skin injury model rat wound surface
Each group was observed daily for wound healing and the wounds were photographed every third day. After photographing, Image J software Image analysis software is used for calculating the area of the residual wound, and the area of the healed wound surface is more than 90 percent as the healing standard. The results show that the average days for wound healing of the blank hydrogel group is 17.94 +/-0.90 days; the average days for healing of the wound surfaces of the scar panacea group is 15.76 +/-0.84; the average days for healing the wound surfaces of the group of the pawpaw total triterpene hydrogel (1.2, 2.4 and 3.6g/kg) are respectively 16.56 +/-0.77, 8.91 +/-0.61 and 8.98 +/-0.94 days, the healing time of the wound surfaces of the skin injury rats is remarkably shortened along with the increase of the dosage of the pawpaw total triterpene hydrogel (1.2 and 2.4g/kg), but the healing time of the wound surfaces of the skin injury rats is not remarkably shortened and is combined with blank water when the dosage of the pawpaw total triterpene hydrogel is increased to 3.6g/kgThe gel group and the scar group have significant differenceP<0.05 orP<0.01). The experimental result shows that the pawpaw total triterpene hydrogel can shorten the healing days of the wound (see figure 13).
4) Influence of pawpaw total triterpene hydrogel on wound healing rate of skin injury model rat
The wound surface areas of the pawpaw total triterpene hydrogel (1.2, 2.4 and 3.6g/kg) groups are reduced to different degrees at 3, 7 and 11 days after the operation, the wound surface healing rate of the skin injury rat is obviously increased along with the increase of the dosage of the pawpaw total triterpene hydrogel (1.2 and 2.4g/kg), but when the dosage of the pawpaw total triterpene hydrogel is increased to 3.6g/kg, the wound surface healing rate of the skin injury rat is not obviously increased, and the pawpaw total triterpene hydrogel has significant difference compared with a blank hydrogel group and a scar panacea group (the formula is shown in the specification and has the following characteristics that (the formula is shown in the specification and has the characteristicsP<0.05 orP<0.01). The experimental result shows that the pawpaw total triterpene hydrogel can improve the healing rate of the rat wound and shorten the healing time of the wound (see figure 14).
5) Effect of pawpaw total triterpene hydrogel on skin pathological morphology of rat model with skin injury
Post-operative day 3 HE staining analysis: a large amount of granulation tissues are formed on each group of wound surfaces and grow into the wound, a large amount of capillary vessels are generated in the wound surface area, a large amount of macrophages and neutrophils infiltrate and collagen fibers are generated, but the wound surfaces are not covered by complete epidermis, the inflammatory reaction of the pawpaw total triterpene hydrogel group is light relative to other groups, and the generation of the capillary vessels and the collagen fibers is more than that of a blank hydrogel group and a scar panacea group. The improvement effect on the skin pathological morphology of the rats with the skin injury is more obvious with the increase of the dosage of the pawpaw total triterpene hydrogel (1.2 and 2.4g/kg), but the improvement effect on the skin pathological morphology of the rats with the skin injury is not obvious when the dosage of the pawpaw total triterpene hydrogel is increased to 3.6 g/kg.
Post-operative day 11 HE staining analysis: each group of granulation tissues is more compact than the 3 rd day after operation, the wound surface of the blank hydrogel group still can be infiltrated by inflammatory cells, and the arrangement of collagen fibers is disordered; the scar panacea group has reduced inflammatory cell infiltration on the wound surface compared with other groups, collagen fibers are relatively disordered, and granulation tissues are converted into scar tissues; the wound surface of the pawpaw total triterpene hydrogel group has complete epidermis covering the wound surface, a small amount of inflammatory cells are arranged on the wound surface, and collagen fibers are arranged more orderly and regularly. The improvement effect on the skin pathological morphology of the rats with the skin injury is more obvious with the increase of the dosage of the pawpaw total triterpene hydrogel (1.2 and 2.4g/kg), but the improvement effect on the skin pathological morphology of the rats with the skin injury is not obvious when the dosage of the pawpaw total triterpene hydrogel is increased to 3.6 g/kg.
Post-operative day 21 HE staining analysis: each group of wound surfaces has complete epidermis to cover the wound surfaces, thickened regenerated epidermis and dermis have obvious boundaries, collagen fibers are arranged orderly compared with the previous group, inflammatory cells are not obvious, and no obvious new sebaceous gland is formed; however, the regenerated epidermis of the pawpaw total triterpene hydrogel group wound surface is closer to the normal epidermis, and has the formation of new hair follicles, and the quantity and the density of the new hair follicles are lower than those of the normal skin. Therefore, the pawpaw total triterpene hydrogel can relieve inflammatory reaction of wounds, promote generation of granulation tissues and capillary vessels, promote regeneration and repair of damaged skin and reduce scar generation. The improvement effect on the skin pathological morphology of the skin injury rats is more obvious with the increase of the dosage of the pawpaw total triterpene hydrogel (1.2 and 2.4g/kg), but the improvement effect on the skin pathological morphology of the skin injury rats is not more obvious when the dosage of the pawpaw total triterpene hydrogel is increased to 3.6g/kg (see figure 15).
Conclusion of the experiment
The experiment that the pawpaw total triterpene hydrogel influences the skin injury of a rat caused by surgical cutting shows that the pawpaw total triterpene hydrogel can obviously shorten the healing time of a wound surface, improve the healing rate of the wound surface, relieve the inflammatory reaction of the wound, promote the generation of granulation tissues and capillary vessels, promote the regeneration and repair of the damaged skin and reduce the generation of scars.
Experimental example 5
The following specific experimental examples are combined to specifically illustrate that the pawpaw total triterpene hydrogel obtained in the example 1 of the invention has obvious therapeutic effect on skin injury of patients with skin trauma.
Test method
1) Clinical data
The 82 patients in the group are from the traditional Chinese medicine clinical medical college of the three gorges university, wherein 40 men and 40 women have the ages of l 6-57 years, and the average age is 36 years; all patients are diagnosed 15 min-2 h after injury. Wound site: 13 upper arm wounds, 26 forearm wounds, 28 shank wounds and 14 thigh wounds are small-area superficial skin tissue defects above subcutaneous tissues; the area of the wound surface is 1.5cm multiplied by 5.0cm to 5.0cm multiplied by 13.0 cm.
2) Method of producing a composite material
(1) Grouping method
82 patients were divided into a pawpaw total triterpene hydrogel group and a scar ointment group according to a random number table method; 41 cases of pawpaw total triterpene hydrogel groups and 41 cases of scar ointment groups are treated by corresponding therapies respectively.
(2) Method of treatment
After the patient is admitted, all patients are subjected to primary debridement to remove necrotic and contaminated tissues of the wound surface, then the wound surface is washed by normal saline, and then the sterile dry gauze is used for dipping dry water, and the wound surface depth and the damage condition of surrounding tissues are explored. After debridement, different methods are adopted for treatment according to the condition of the wound surface. The pawpaw total triterpene hydrogel group and the scar panacea group are respectively coated with the pawpaw total triterpene hydrogel and the scar panacea on the surface of a wound, the thickness is about 1.0mm, the surface of the wound is exposed, and the dressing is changed for 2 times every day until the surface of the wound is healed. Before dressing change, the original medicine and the liquefied substance are dipped and cleaned and then coated with new ointment, and the dressing change follows the three principles of no pain on wound surface, no bleeding and no damage to normal tissues.
(3) Evaluation of therapeutic Effect
Observation indexes are as follows: the wound infection and the wound healing conditions of two groups of patients are observed, and the scar hyperplasia condition is observed after the follow-up visit is 8-21 months.
The therapeutic effect judgment standard is as follows: and (3) excellent: the wound surface is completely healed, and clinical symptoms completely disappear; good: the wound surface healing area is more than 50%, and clinical symptoms completely disappear; can be as follows: the wound healing area is 20-50%, the exudate is reduced, and the clinical symptoms are improved; difference: the wound surface is unchanged or enlarged, and the clinical symptoms are not improved.
(6) Statistical analysis
The experimental results are given as mean. + -. standard deviation (S) data analysis was performed on SPSS21.0 statistical software package, comparison of differences between groups was performed by one-way analysis of variance, Dunnett-tChecking and comparing the difference between the two groups;P<0.05 was considered statistically different.
Test results
1) Clinical efficacy comparison of two groups of patients
The excellent rate of the wound curative effect of the patients with the pawpaw total triterpene hydrogel group is 92.68%, and no patients with poor curative effect appear; the wound curative rate of the scar group patients is 70.73%, and 3 patients with poor curative effect appear. The excellent rate of the pawpaw total triterpene hydrogel group is obviously superior to that of the scar group, and has significant difference compared with the scar group (P< 0.01) (see Table 1).
TABLE 1 comparison of clinical efficacy of two groups of patients
Comparison with scar group:* P<0.05,** P<0.01.
2) comparison of wound healing time of two groups of patients
The average healing time of the wound surface of the patients with the pawpaw total triterpene hydrogel group is 11.10 +/-1.13 days, and the average healing time of the wound surface of the patients with the scar panacea group is 15.62 +/-1.34 days. The average healing time of the pawpaw total triterpene hydrogel group is obviously shorter than that of the scar group, and the pawpaw total triterpene hydrogel group has significant difference compared with the scar group (P< 0.01) (see Table 2).
TABLE 2 comparison of clinical efficacy of two groups of patients
Group of | Number of cases (n) | Healing time (Tian) | Average healing time (day) | Number of infection cases (n) |
Pawpaw total triterpene hydrogel group | 41 | 9~13 | 11.10±1.13** | 0 |
Scar curing medicine set | 41 | 13~17 | 15.62±1.34 | 3 |
Comparison with scar group:* P<0.05,** P<0.01.
3) comparison of wound infection of two groups of patients
The scar panacea treatment group has 3 patients with different degrees of infection, the wound surface of the pawpaw total triterpene hydrogel treatment group has no infection, and the scar panacea treatment group has significant difference (the scar panacea treatment group has the following characteristics ofP< 0.01) (see Table 2).
Conclusion of the experiment
The pawpaw total triterpene hydrogel can effectively promote the healing speed of the wound surface epidermis, increase the elasticity of skin tissues, reduce the generation of scars and improve the healing quality of the wound surface.
In conclusion, the pawpaw total triterpene hydrogel has better effects of promoting proliferation, inhibiting apoptosis and promoting cell migration on skin cell injury; can obviously promote the healing of skin wound, shorten the healing time of the wound, promote the regeneration and repair of damaged skin and reduce the generation of scars for an animal skin injury model and a patient with skin trauma. Compared with the current clinically used medicines for treating skin injury, the traditional Chinese medicine composition has the advantages of good effect, small toxic and side effects, nature and no stimulation.
The above examples and experimental examples are illustrative of the present invention. Those skilled in the art can, using the teachings disclosed above, modify many equivalent embodiments without departing from the spirit or scope of the invention. Any simple modification or equivalent changes made to the above embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.
Claims (10)
1. The preparation method of the pawpaw total triterpene hydrogel is characterized by adding a pawpaw total triterpene micelle solution into an alpha-cyclodextrin solution, and uniformly mixing at normal temperature to obtain the wrinkled pawpaw total triterpene hydrogel, wherein the mass concentration of the alpha-cyclodextrin solution is 15-25%; the volume ratio of the pawpaw total triterpene micelle solution to the alpha-cyclodextrin (alpha-CD) solution with the mass concentration of 15-25% is 1: 0.25-1.
2. The method for preparing the pawpaw total triterpene hydrogel according to claim 1, which is characterized in that the method for preparing the pawpaw total triterpene micelle comprises the following steps:
(a) uniformly mixing monomethoxy polyethylene glycol-polycaprolactone and the total triterpene of the pawpaw, adding dichloromethane, stirring to obtain a transparent solution, and evaporating to recover the dichloromethane to obtain the light brown total triterpene crude micelle of the pawpaw;
(b) adding distilled water into the light brown crude papaya triterpene micelle obtained in the step (a), heating to 50-60 ℃, uniformly mixing to obtain a papaya total triterpene micelle solution, filtering by a filter membrane to obtain the papaya total triterpene micelle solution, or freeze-drying to obtain the papaya total triterpene micelle.
3. The preparation method of the pawpaw total triterpene hydrogel according to claim 2, wherein the mass ratio of the monomethoxypolyethylene glycol-polycaprolactone to the pawpaw total triterpene in the step (a) is 1: 0.05-0.5; the filter membrane used in the filtration in step (b) is at least 0.22 μm.
4. The preparation method of the pawpaw total triterpene hydrogel according to claim 2, wherein the synthesis method of the monomethoxy polyethylene glycol-polycaprolactone comprises the following steps:
(1) heating the mPEG and toluene to azeotropy, and recovering the toluene to obtain anhydrous mPEG;
(2) adding dichloromethane into the anhydrous mPEG obtained in the step (1) to dissolve, sequentially adding caprolactone and ethyl ether hydrochloride, reacting at normal temperature, and recovering dichloromethane and ethyl ether to obtain a crude product of mPEG-PCL;
(3) and (3) precipitating the mPEG-PCL crude product obtained in the step (2) in ethyl ether, and filtering to obtain white to off-white star-shaped mPEG-PCL crystals, namely the monomethoxy polyethylene glycol-polycaprolactone.
5. The preparation method of the pawpaw total triterpene hydrogel according to claim 2, wherein mPEG and toluene are dissolved according to a mass-volume ratio of 1: 2-10 (m: V) in the step (1); and then heating and azeotropy for 1-5 hours at the temperature of 100-160 ℃, and recovering toluene to obtain anhydrous mPEG.
6. The preparation method of the pawpaw total triterpene hydrogel according to claim 2, which is characterized in that after the anhydrous mPEG and the dichloromethane are dissolved according to the mass-volume ratio of 1: 2-10 (m: V) in the step (2), the caprolactone is added and uniformly mixed according to the mass ratio of mPEG: caprolactone to 1: 1-5; the concentration of the ethyl ether hydrochloride is 1.0-2.5 mol/L; the volume ratio of the addition amount of 1.0-2.5mol/L ethyl ether hydrochloride to the volume of the mixed liquid of anhydrous mPEG, dichloromethane and caprolactone is 0.1-0.5: 1.
7. Use of the pawpaw total triterpene hydrogel prepared according to any one of claims 1 to 6 in preparation of medicaments for promoting proliferation and cell migration and inhibiting apoptosis of human skin fibroblast HDF cells damaged by UVB.
8. The application of the pawpaw total triterpene hydrogel prepared according to any one of claims 1 to 6 in preparing medicaments for promoting cell proliferation, cell migration and apoptosis of human umbilical vein endothelial cells HUVECs damaged by UVB.
9. Use of the pawpaw total triterpene hydrogel prepared according to any one of claims 1 to 6 in preparation of a medicament for healing wounds caused by postoperative cutting injury.
10. Use of the pawpaw total triterpene hydrogel prepared according to any one of claims 1 to 6 in preparation of a medicament for treating and repairing damaged skin regeneration.
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