CN111759896A

CN111759896A - Pharmaceutical application of total triterpene of pawpaw

Info

Publication number: CN111759896A
Application number: CN202010687895.6A
Authority: CN
Inventors: 贺海波; 石孟琼; 廖勇; 宋利华; 张媛媛; 何毓敏; 卫巧; 程凡; 邹坤; 张继红
Original assignee: China Three Gorges University CTGU
Current assignee: China Three Gorges University CTGU
Priority date: 2020-07-16
Filing date: 2020-07-16
Publication date: 2020-10-13

Abstract

The invention discloses a pharmaceutical application of total triterpene of pawpaw. The application of the total triterpene of the pawpaw in preparing the medicament for promoting the proliferation and cell migration of human skin fibroblast HDF cells damaged by UVB and inhibiting apoptosis is concretely included. And the application of the HUVECs in preparing medicaments for promoting the cell proliferation and cell migration of human umbilical vein endothelial cells damaged by UVB and inhibiting cell apoptosis. And the application in preparing the medicine for healing the postoperative cutting injury wound. And the application in preparing the medicine for treating the regeneration and repair of the damaged skin. Has obvious curative effect.

Description

Pharmaceutical application of total triterpene of pawpaw

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 Nakai, Pyrus callorhinus Linne, Chaenomeles speciosa (Sweet) Nakai, 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 has conducted extensive and intensive research, and finds that the total triterpene of pawpaw extracted from Tujia medicine chaenomeles speciosa can effectively promote the proliferation and migration of human dermal fibroblasts and vascular endothelial cells, has good effects of promoting skin repair and wound healing and reducing scar generation on damaged skin wounds, and has no obvious toxic or side effect. At present, reports related to the preparation method of the total triterpene of the pawpaw prepared by the invention and the promotion of the wound healing of the damaged skin are not found.

The invention aims to provide a pharmaceutical application of total triterpenes in pawpaw. The specific pharmaceutical application is as follows:

the application of the total triterpene of the pawpaw in preparing the medicine for promoting the proliferation and cell migration of human skin fibroblast HDF cells damaged by UVB and inhibiting apoptosis.

Application of total triterpene of fructus Chaenomelis in preparing medicine for promoting cell proliferation and cell migration of HUVECs of human umbilical vein endothelial cell damaged by UVB and inhibiting apoptosis is provided.

Application of total triterpene of fructus Chaenomelis in preparing medicine for healing wound after operation.

Application of total triterpene of fructus Chaenomelis in preparing medicine for treating damaged skin regeneration and repair is provided.

The total triterpenoids of the wrinkled pAN _ SNer majoram prepared by the preparation method have good biocompatibility, nontoxicity, resistibility and good biodegradability, can effectively avoid the first pass effect of a medicine liver, can realize directional administration and have lasting effect, thereby having better application and development prospects.

The invention has the advantages of

The pawpaw total triterpenes prepared by the invention have better effects 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 graph of the effect of total triterpene from papaya on UVB-damaged HDF cell proliferation (MTT assay) (compared to normal group:^# P＜0.05，^## Pless than 0.01; comparison with UVB group:^* P＜0.05，^** P＜0.01；n=4)

FIG. 2 Effect of total triterpene from papaya on proliferation of UVB-damaged HDF cells (plating clone formation experiment) (A: cell clone image, B: number of clone formation; compare with normal group:^# P＜0.05，^## Pless than 0.01; comparison with UVB group:^* P＜0.05，^** P＜0.01；n=4)。

FIG. 3 Effect of Total triterpene from papaya on UVB-damaged HDF apoptosis (A: apoptosis picture, B: number of apoptosis; compare with normal group:^# P＜0.05，^## Pless than 0.01; comparison with UVB group:^* P＜0.05，^** P＜0.01；n=4)。

FIG. 4 Effect of total triterpene from papaya on UVB-damaged HDF cell migration (scratching experiment) (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，^** P＜0.01；n=4)。

figure 5 effect of total triterpene from pawpaw 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，^** P＜0.01；n=4)。

FIG. 6 Effect of Total triterpene from papaya on cell proliferation of UVB-injured HUVECs (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，^** P＜0.01；n=4)。

FIG. 7 Effect of Total triterpene from papaya on UVB-damaged HUVECs apoptosis (A: apoptosis picture, B: number of apoptosis; compare with normal group:^# P＜0.05，^## Pless than 0.01; comparison with UVB group:^* P＜0.05，^** P＜0.01；n=4)。

FIG. 8 Effect of Total triterpene from 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，^** P＜0.01；n=4)。

figure 9 effect of total triterpene from pawpaw on wound healing time in skin injury model rats (compared to model group:^* P＜0.05，^** Pless than 0.01; comparison with scar group:^† P＜0.05，^†† P＜0.01；n=10)。

figure 10 effect of total triterpene from pawpaw on wound healing rate in rat skin injury model (compared to model group:^* P＜0.05，^** Pless than 0.01; comparison with scar group:^† P＜0.05，^†† P＜0.01；n=10)。

FIG. 11 Effect of total triterpene of Chaenomeles speciosa on pathological morphology of skin of rat model of skin injury.

The following specific experimental examples are combined to concretely demonstrate that the total triterpene of the pawpaw 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 1

The following specific experimental examples are combined to specifically illustrate the effects of the total triterpene of pawpaw in promoting cell proliferation, cell migration and cell apoptosis inhibition 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/cm²Irradiation time of 250sThe final total dose of irradiation was 100mJ/cm²(irradiation dose = irradiation intensity × irradiation time.) the UVB-injured HDF cells were randomly divided into UVB group, total triterpene of papaya (2.5, 5, 10, 20, 40, 80 μ g/mL) group and UVB group, total triterpene of papaya (5, 10 and 20 μ g/mL) group, and HDF cells not irradiated with UVB were used as normal group.

(3) MTT (methyl thiazolyl tetrazolium) test effect of total triterpene of pawpaw 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 the papaya total triterpene with the corresponding concentration to each administration, 20. mu.L of MTT solution (5mg/mL) was added to each well after 24 hours and 48 hours of incubation, the culture solution was aspirated after 4 hours of incubation, 150. mu.L of DMSO was added, and the OD value was measured at 490 nm.

(4) Plate clone formation experiment for detecting influence of total triterpene of pawpaw on proliferation of HDF cell damaged by UVB

The experimental groups comprise a normal group, a UVB group, a pawpaw total triterpene group with the concentration of 5 mu g/mL, a pawpaw total triterpene group with the concentration of 10 mu g/mL and a pawpaw total triterpene group with the concentration of 20 mu g/mL, and each group has 3 duplicate wells. 300 single-cell HDF suspensions are inoculated in a 6-well plate, after culturing for 24h, the total triterpene of the pawpaw is added into each dose group of total triterpene of the pawpaw (5, 10 and 20 mug/mL), the culture solution in each well is 3mL, the solution is changed every three days, and the total triterpene of the pawpaw with the corresponding concentration and 5 percent 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 the effect of total triterpene of pawpaw on HDF apoptosis of UVB damage

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,the final total irradiation dose was 100mJ/cm²Adding pawpaw total triterpenes (5, 10 and 20 mu g/mL) with corresponding concentrations 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 total triterpenoids of pawpaw 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 the corresponding concentrations of total triterpene from papaya (5, 10 and 20 μ g/mL) according to (2), and the change in scratch width was observed after 24h of incubation and analyzed using the corresponding image processing software.

(7) 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 Total triterpene of Chaenomeles on UVB-damaged HDF cell proliferation

MTT results show that UVB irradiation has obvious effect of inhibiting proliferation of HDF cells and has significant difference compared with normal group (P<0.01); after being treated with total triterpene of pawpaw (2.5, 5, 10 and 20 mug/mL), the extract has obvious effect of promoting proliferation of HDF cells damaged by UVB, and the total triterpene of pawpaw (40 and 80 mug/mL) inhibits the proliferation of the HDF cells. The low concentration of the total triterpene of the pawpaw can promote the proliferation of HDF cells, and the high concentration of the total triterpene of the pawpaw can inhibit the proliferation activity of the HDF cells. According to the existing research, the proper concentration of the total triterpene of the pawpaw (below 20 mug/mL) is determined) And treatment time (24h) for further in vitro studies (see figure 1).

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 total triterpenoids of pawpaw (5, 10 and 20 mu g/mL), the clone number of HDF cells with UVB damage can be obviously increased, and the clone number has a significant difference compared with UVB group (the difference is that: (the total triterpenoids of pawpaw is not reduced by the total triterpenoids of pawpaw)P<0.05 orP<0.01) (see fig. 2).

2) Effect of Total triterpene of Chaenomeles on apoptosis of HDF injured by UVB

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 typical apoptosis bodies, chromatin condensation, nucleus fragmentation into round bodies with different sizes and the like can be observed to show the phenomenon of 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 total triterpene of Chaenomeles sinensis (5, 10 and 20. mu.g/mL) and with increasing total triterpene of Chaenomeles sinensis dose, normal morphology of cells was increased, the apoptotic character was less pronounced, the number of apoptotic cells was markedly reduced, and there was a significant difference from the UVB group (A)P<0.05 orP<0.01) (see fig. 3).

3) Effect of Total triterpene of Chaenomeles on UVB-damaged HDF 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 total triterpenes of pawpaw (5 and 20 mu g/mL), the pawpaw extract can obviously promote the migration of HDF cells damaged by UVB, obviously reduce the distance from cells at the periphery of a scratch to a central scratch area, obviously improve the healing capacity and have significant difference compared with the UVB group (theP<0.01) (see fig. 4).

Conclusion of the experiment

Experiments on the influence of the total triterpene of the pawpaw on HDF cells damaged by UVB show that the total triterpene of the pawpaw can obviously promote the proliferation of the HDF cells damaged by the UVB, inhibit the apoptosis of the HDF cells and promote the cell migration of the HDF cells damaged by the UVB. Experimental results show that the total triterpene of the pawpaw has a good protective effect on HDF cells damaged by UVB.

Experimental example 2

The following specific experimental examples are combined to specifically demonstrate that the total triterpene in pawpaw can promote the cell proliferation, cell migration and apoptosis inhibition of Human Umbilical Vein Endothelial Cells (HUVECs) damaged by UVB.

Test method

1) Test cell

The cell strain was cultured in DMEM culture medium containing 10% fetal bovine serum at 37 deg.C and 5% CO in the basic medical research institute of Beijing national academy of medical sciences (Beijing) using Human Umbilical Vein Endothelial Cells (HUVECs) in this experiment₂The 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-damaged HUVECs cells were randomly divided into UVB group and wood, respectivelyTotal triterpene of melon (2, 4, 8, 16, 32, 64 μ g/mL) and UVB, total triterpene of papaya (4, 8 and 16 μ g/mL), and HUVECs cells without UVB irradiation were used 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.

(4) Plate clone formation experiment for detecting influence of total triterpene of pawpaw on UVB-damaged HUVECs cell proliferation

The experimental groups comprise a normal group, a UVB group, a pawpaw total triterpene 4 mu g/mL group, a pawpaw total triterpene 8 mu g/mL group and a pawpaw total triterpene 16 mu g/mL group, wherein each group has 3 duplicate wells. Inoculating 300 single cell suspensions of HUVECs into a 6-well plate, culturing for 24h after cell adherence, adding total triterpene of fructus Chaenomelis (4, 8, 16 μ g/mL) into each dosage group of total triterpene of fructus Chaenomelis, changing the culture solution every three days, and adding total triterpene of fructus Chaenomelis 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 total triterpene of pawpaw on apoptosis of HUVECs with UVB injury

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 triterpenes (4, 18 and 16 mu g/mL) with corresponding concentrations 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 total triterpenoids of pawpaw on migration and proliferation of HUVECs

HUVECs in logarithmic growth phase were taken at 15 × 10⁵And (3) inoculating each/mL of the cells into a 6-well plate, vertically scratching the 6-well plate by using a sterile 200-mu L pipette tip, washing the cells by using PBS (phosphate buffer solution), treating the cells by using the total triterpenoids of the pawpaw with corresponding concentrations (4, 8 and 18 mu g/mL) according to the grouping in the step (2), observing the change of the scratch width after culturing for 24 hours, and analyzing the scratch width by using corresponding image processing software.

(7) Statistical analysis

The data of each group are counted as mean ± standard deviation: (

Test results

1) Effect of Total triterpene of Chaenomeles on proliferation of UVB-injured HUVECs

MTT results show that UVB irradiation has obvious effect of inhibiting proliferation of HUVECs cells and has significant difference compared with normal group (P<0.01); after being treated by total triterpenoids of pawpaw (2, 4, 8 and 16 mu g/mL), the extract has obvious effect of promoting the proliferation of HUVECs damaged by UVB, and the total triterpenoids of pawpaw (32 and 64 mu g/mL) inhibit the proliferation of HUVECs. The low-concentration pawpaw total triterpenes can promote the cell proliferation of HUVECs, and the high-concentration pawpaw total triterpenes can inhibit the proliferation activity of the HUVECs. Based on the current study, the appropriate concentration of total triterpene from papaya (below 16 μ g/mL) and treatment time (24h) were determined for further in vitro studies (see FIG. 5).

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 total triterpenoids of pawpaw (4, 8 and 16 mu g/mL), the clone number of HUVECs with UVB damage can be obviously increased, and the clone number has significant difference compared with the UVB group (the)P<0.05 orP<0.01) (see fig. 6).

2) Effect of pawpaw total triterpenes on UVB-damaged HUVECs 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 typical apoptosis bodies, chromatin condensation, nucleus fragmentation into round bodies with different sizes and the like can be observed to show the phenomenon of 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 total triterpene from papaya (4, 8 and 16 μ g/mL) and with increasing dosage of total triterpene from papaya, there was an increase in the number of cells in normal form, less significant apoptotic character, a significant decrease in the number of apoptotic cells, and a significant difference from the UVB group (a)P<0.05 orP<0.01) (see fig. 7).

3) Effect of Total triterpene of Chaenomeles on cell migration of UVB-injured HUVECs

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 total triterpenes of pawpaw (4 and 16 mu g/mL), the extract can obviously promote the migration of UVB-damaged HUVECs cells, obviously reduce the distance from cells at the periphery of a scratch to a central scratch area, obviously improve the healing capacity and have significant difference compared with a UVB group (the extract is a new extract)P<0.01) (see fig. 8).

Conclusion of the experiment

Experiments on the influence of the total triterpene of pawpaw on HUVECs damaged by UVB show that the total triterpene of pawpaw can obviously promote the cell proliferation of HUVECs damaged by UVB, inhibit the apoptosis of the HUVECs damaged by UVB and promote the cell migration of the HUVECs damaged by UVB. Experimental results show that the total triterpene of pawpaw has a good protective effect on HUVECs cells damaged by UVB.

Experimental example 3

The following specific experimental examples are combined to specifically illustrate that the total triterpene from pawpaw obtained in example 1 of the invention has 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 model group, a pawpaw total triterpene group (1.2, 2.4 and 3.6g/kg) and a scar panacea positive drug group (1.0 g/kg). The medicine coating group is respectively coated with corresponding pawpaw total triterpene liniment (dissolving pawpaw total triterpene in physiological saline) and scar panacea at the wound defect; the model groups were then coated with saline. 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 status analysis

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

A group of rats was randomly selected each time on days 3, 14, and 28, 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, Masson 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) Effect of pawpaw total triterpenes on skin injury model rat wound surface

On the 3 rd day after operation, the wound surface exudates of the pawpaw total triterpenoids (1.2, 2.4 and 3.6g/kg) group and the scar-panacea positive medicine group are obviously reduced, the wound surfaces are covered by the blood crust skin, the wound begins to shrink less, the periphery of the wound is dry, the swelling range is small, the wound surface reduction speed of each group is obviously higher than that of the model group along with the time increase, and the effect of promoting the wound healing of the pawpaw total triterpenoids (1.2, 2.4 and 3.6g/kg) group is more obviousObviously, the curative effect is more obvious along with the increase of the dosage of the total triterpene of the pawpaw (1.2 and 2.4 g/kg); however, when the total triterpene dosage of the pawpaw is increased to 3.6g/kg, the curative effect is not obviously increased, and the pawpaw total triterpene dosage has obvious difference compared with a model group (P<0.05 orP<0.01). On day 14 after surgery, the wounds of the group with 2.4g/kg of total triterpene in pawpaw reached the healing standard, and the other groups did 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 triterpenoids (1.2, 2.4 and 3.6g/kg) groups have no obvious difference with 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 have clear wound peripheries, scar tissue formation can be seen, and no obvious hair growth exists, wherein the curative effect of the group with 2.4g/kg of total triterpene of pawpaw is most obvious. The results show that the total triterpene of the pawpaw can obviously shorten the healing time of the wound surface, improve the healing rate of the wound surface, relieve the inflammatory reaction of the wound and promote the generation of granulation tissues.

3) Influence of pawpaw total triterpenes on wound healing time of rat with skin injury model

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 the wound surfaces of the model group to heal is 17.87 +/-1.06 days; the average days for healing of the scar panacea group wound surface is 16.18 +/-1.04; the average days for healing the wound surface of the pawpaw total triterpene (1.2, 2.4 and 3.6g/kg) group are respectively 13.92 +/-1.00, 13.86 +/-0.93 and 15.62 +/-1.15 days, the healing time of the wound surface of the skin injury rat is remarkably shortened along with the increase of the dosage of the pawpaw total triterpene (1.2 and 2.4g/kg), but the healing time of the wound surface of the skin injury rat is not remarkably shortened when the dosage of the pawpaw total triterpene is increased to 3.6g/kg, and the healing time of the wound surface of the skin injury rat is remarkably different from that of a model group and a scar group (the formula: (1.2, 2.4 and 3.6gP<0.05 orP<0.01). The experimental result shows that the total triterpene of the pawpaw can shorten the healing days of the wound (see figure 9).

4) Effect of pawpaw total triterpenes on wound healing rate of rat of skin injury model

Total triterpene of fructus Chaenomelis (1.2, 2.4 and 3.6g/kg) in the group was injured at 3, 7 and 14 days after surgeryThe wound area is reduced in different degrees, the healing rate of the wound of the rat with the skin injury is obviously increased along with the increase of the dosage of the total triterpene of pawpaw (1.2 and 2.4g/kg), but the healing rate of the wound of the rat with the skin injury is not obviously increased when the dosage of the total triterpene of pawpaw is increased to 3.6g/kg, and the healing rate is obviously different from that of a model group and a scar group (the wound area is obviously different from that of the model group and the scar group)P<0.05 orP<0.01). The experimental result shows that the total triterpene of the pawpaw can improve the healing rate of the wound of the rat and shorten the healing time of the wound (see figure 10).

5) Effect of pawpaw total triterpenes on skin pathological morphology of skin injury model rat

Masson staining stained connective tissue and collagen fibers blue, smooth muscle red, and cell nuclei brown. Collagen fibers (blue) were produced in the tissues on days 3, 14, and 28 post-surgery for each group; on days 14 and 28, the number of groups of collagen fibers of the total triterpene group of the pawpaw is less, and the model is formed; on the 28 th day, the total triterpene group of the pawpaw has more collagen fibers than other groups, and the collagen fibers are regularly and orderly arranged and are closer to normal skin. The improvement effect on the skin pathological morphology of the skin injury rats is more obvious with the increase of the dosage of the total triterpene of the pawpaw (1.2 and 2.4g/kg), but the improvement effect on the skin pathological morphology of the skin injury rats is not obvious when the dosage of the total triterpene of the pawpaw is increased to 3.6 g/kg. Experiments suggest that the total triterpenoids in pawpaw can promote the formation and ordered arrangement of collagen fibers at the damaged part of the skin, thereby promoting the healing of the skin wound of a rat (figure 11). Connective tissue and collagen fibers were stained blue, smooth muscle red, and cell nuclei brown.

Conclusion of the experiment

Experiments on the influence of the total triterpenoids of the pawpaw on the skin injury of rats caused by surgical cutting show that the total triterpenoids of the pawpaw 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 the formation of collagen fibers, promote the regeneration and repair of damaged skin and reduce the generation of scars.

Experimental example 4

The following specific experimental examples are combined to specifically show that the total triterpene in pawpaw has obvious therapeutic effect on skin injury of patients with skin trauma.

Test method

1) Clinical data

The 64 patients in the group are from the traditional Chinese medicine clinical medical college of the three gorges university, wherein 32 men and 32 women have the ages of l 6-55 years, and the average age is 38 years; all patients are diagnosed 15 min-2 h after injury. Wound site: 14 upper arm wounds, 18 forearm wounds, 20 calf wounds and 12 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

Dividing 64 patients into a pawpaw total triterpene group and a scar ointment group according to a random number table method; 32 cases of pawpaw total triterpenes and 32 cases of scar ointment 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 fructus Chaenomelis total triterpene and scar eliminating component are applied on the wound surface respectively, and the dressing is changed for 2 times daily until the wound surface is healed. Before dressing change, the original medicine and the liquefied substance are dipped and cleaned and then coated with new medicine, 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 (

Test results

1) Clinical efficacy comparison of two groups of patients

The excellent rate of the curative effect of the wound surface of the patient with the total triterpene group of the pawpaw is 87.50 percent, and no patient with poor curative effect appears; the wound curative rate of the scar group patients is 68.75%, and 3 patients with poor curative effect appear. The excellent rate of the pawpaw total triterpene group is obviously better than 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 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 group is obviously shorter than that of the scar panacea group, and has significant difference compared with the scar panacea 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 group	32	10～14	12.35±1.08^**	0
Scar curing medicine set	32	13～17	15.89±1.48	2

Comparison with scar group:^* P＜0.05，^** P＜0.01.

3) comparison of wound infection of two groups of patients

The scar ling treatment group has 2 patients with different degrees of infection, the wound surface of the patients with the pawpaw total triterpene treatment group has no infection, and the scar ling treatment group has significant difference (P< 0.01) (see Table 2).

Conclusion of the experiment

The pawpaw total triterpenes 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 total triterpenoids of the pawpaw have 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

1. The application of the total triterpene of the pawpaw in preparing the medicine for promoting the proliferation and cell migration of human skin fibroblast HDF cells damaged by UVB and inhibiting apoptosis.

2. Application of total triterpene of fructus Chaenomelis in preparing medicine for promoting cell proliferation and cell migration of HUVECs of human umbilical vein endothelial cell damaged by UVB and inhibiting apoptosis is provided.

3. Application of total triterpene of fructus Chaenomelis in preparing medicine for healing wound after operation.

4. Application of total triterpene of fructus Chaenomelis in preparing medicine for treating damaged skin regeneration and repair is provided.