CN117180302B - Application of polysaccharide in repairing oral mucosa and accelerator prepared from polysaccharide - Google Patents
Application of polysaccharide in repairing oral mucosa and accelerator prepared from polysaccharide Download PDFInfo
- Publication number
- CN117180302B CN117180302B CN202311460970.5A CN202311460970A CN117180302B CN 117180302 B CN117180302 B CN 117180302B CN 202311460970 A CN202311460970 A CN 202311460970A CN 117180302 B CN117180302 B CN 117180302B
- Authority
- CN
- China
- Prior art keywords
- polysaccharide
- belladonna
- oral mucosa
- grass
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 150000004676 glycans Chemical class 0.000 title claims abstract description 140
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 140
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 140
- 210000002200 mouth mucosa Anatomy 0.000 title claims abstract description 43
- 241001106067 Atropa Species 0.000 claims abstract description 109
- 244000025254 Cannabis sativa Species 0.000 claims abstract description 42
- 230000008439 repair process Effects 0.000 claims abstract description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 238000001914 filtration Methods 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- 239000012528 membrane Substances 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 239000002244 precipitate Substances 0.000 claims description 6
- 238000004140 cleaning Methods 0.000 claims description 5
- 239000000287 crude extract Substances 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 4
- 238000000034 method Methods 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- 239000012153 distilled water Substances 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 3
- 102000004169 proteins and genes Human genes 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 238000010298 pulverizing process Methods 0.000 claims description 3
- 238000007873 sieving Methods 0.000 claims description 3
- 238000000108 ultra-filtration Methods 0.000 claims description 3
- 210000002919 epithelial cell Anatomy 0.000 abstract description 24
- 241000222122 Candida albicans Species 0.000 abstract description 13
- 229940095731 candida albicans Drugs 0.000 abstract description 13
- 230000001737 promoting effect Effects 0.000 abstract description 12
- 230000005012 migration Effects 0.000 abstract description 11
- 238000013508 migration Methods 0.000 abstract description 11
- 230000006378 damage Effects 0.000 abstract description 9
- 230000035755 proliferation Effects 0.000 abstract description 9
- 230000000694 effects Effects 0.000 abstract description 7
- 208000025865 Ulcer Diseases 0.000 abstract description 5
- 231100000397 ulcer Toxicity 0.000 abstract description 5
- 208000027418 Wounds and injury Diseases 0.000 abstract description 3
- 208000014674 injury Diseases 0.000 abstract description 2
- 244000052616 bacterial pathogen Species 0.000 abstract 1
- 238000011160 research Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 31
- 238000011282 treatment Methods 0.000 description 31
- 239000000243 solution Substances 0.000 description 22
- 239000001963 growth medium Substances 0.000 description 20
- 239000002609 medium Substances 0.000 description 15
- 238000005374 membrane filtration Methods 0.000 description 8
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 239000002994 raw material Substances 0.000 description 6
- 239000006285 cell suspension Substances 0.000 description 5
- 210000000981 epithelium Anatomy 0.000 description 5
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- 208000002399 aphthous stomatitis Diseases 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 210000004379 membrane Anatomy 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 3
- 230000001464 adherent effect Effects 0.000 description 3
- 208000020670 canker sore Diseases 0.000 description 3
- 230000004663 cell proliferation Effects 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 229920006395 saturated elastomer Polymers 0.000 description 3
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 108010087230 Sincalide Proteins 0.000 description 2
- 229920002385 Sodium hyaluronate Polymers 0.000 description 2
- 229930003268 Vitamin C Natural products 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 238000010609 cell counting kit-8 assay Methods 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 239000001525 mentha piperita l. herb oil Substances 0.000 description 2
- 210000000214 mouth Anatomy 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 235000019477 peppermint oil Nutrition 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 2
- 229940010747 sodium hyaluronate Drugs 0.000 description 2
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 235000019154 vitamin C Nutrition 0.000 description 2
- 239000011718 vitamin C Substances 0.000 description 2
- 101000577180 Aspergillus oryzae (strain ATCC 42149 / RIB 40) Neutral protease 2 Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 208000019505 Deglutition disease Diseases 0.000 description 1
- 206010013887 Dysarthria Diseases 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- 208000007117 Oral Ulcer Diseases 0.000 description 1
- 208000025157 Oral disease Diseases 0.000 description 1
- 241000209051 Saccharum Species 0.000 description 1
- 206010044546 Traumatic ulcer Diseases 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 230000036528 appetite Effects 0.000 description 1
- 235000019789 appetite Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000005178 buccal mucosa Anatomy 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000001055 chewing effect Effects 0.000 description 1
- 235000015218 chewing gum Nutrition 0.000 description 1
- 229940112822 chewing gum Drugs 0.000 description 1
- 210000004913 chyme Anatomy 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000000431 effect on proliferation Effects 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 238000000643 oven drying Methods 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000000306 recurrent effect Effects 0.000 description 1
- 239000012487 rinsing solution Substances 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 208000026473 slurred speech Diseases 0.000 description 1
- 235000021259 spicy food Nutrition 0.000 description 1
- 210000004876 tela submucosa Anatomy 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Abstract
The invention provides an application of polysaccharide in repairing oral mucosa and a prepared accelerator thereof, belonging to the technical field of stomatology. The polysaccharide provided by the invention is extracted and separated from belladonna grass with the molecular weight of 10-30kDa, and experimental researches show that the belladonna grass polysaccharide has the effect of promoting proliferation and migration of human oral mucosa epithelial cells and can remarkably inhibit adhesion of oral pathogenic bacteria candida albicans. Meanwhile, the polysaccharide is used for preparing the oral mucosa repair promoter, so that the oral mucosa repair promoter can be used for effectively promoting the repair of oral mucosa and treating various oral mucosa injuries such as dental ulcer.
Description
Technical Field
The invention belongs to the technical field of stomatology, and particularly relates to application of polysaccharide in repairing oral mucosa and a prepared accelerator thereof.
Background
Oral ulcers are a very common disease of the oral mucosa, which is mainly manifested by limited localized pain or ulcerated lesions, i.e. ulcers, on the oral mucosa. Canker sores can occur anywhere in the mouth, such as the buccal mucosa, tongue, gingival sulcus, and the like, and can also be typed according to different etiologies, such as traumatic ulcers, recurrent aphtha ulcers, and the like.
The canker sore can cause obvious pain or burning to the patient, is usually more obvious when eating and speaking, and seriously affects the appetite and the life quality of the patient. Part of ulcers have longer disease course and repeated attacks, and the pain of patients is increased. Furthermore, canker sores also increase the likelihood of the patient chewing dysphagia, and slurred speech. Complications such as bleeding, infection and the like can also be caused in severe cases.
The occurrence of canker sores is closely related to the destruction of the integrity of oral mucosal tissue. When the oral mucosal epithelium and basal layer are damaged by various factors, it may result in loss of normal integrity and protection of the mucosa, followed by a destructive ulcer injury. Factors that lead to the destruction of the oral mucosa are very diverse. Among them, the most common causes are mechanical wounds of the oral cavity, such as residual chyme, foreign matter scraping, improper tooth cleaning, etc., causing damage to the mucosal surface. Second, chemical irritation such as tobacco, alcohol, chewing gum, spicy foods, etc. can also cause damage to the oral mucosa.
Therefore, although the mechanism of occurrence of canker sore is complex and various, it is mostly related to the damage of the integrity of the oral mucosa tissue, so how to effectively improve the restoration ability of the oral mucosa is important for the cure and prognosis of canker sore.
Disclosure of Invention
The invention aims to provide an application of polysaccharide in repairing oral mucosa and an accelerator prepared by the polysaccharide, so as to promote rapid repairing of the oral mucosa.
In order to achieve the above purpose, the present invention provides the following technical solutions:
firstly, the invention provides application of polysaccharide in preparing an oral mucosa repair promoter, wherein the polysaccharide is belladonna grass polysaccharide.
Preferably, the preparation method of the belladonna grass polysaccharide comprises the following steps:
(1) Cleaning belladonna grass, and drying in a drying oven until the weight is unchanged;
(2) Pulverizing dried belladonna grass, and sieving to obtain belladonna grass powder;
(3) Extracting belladonna grass powder with 15 times of distilled water at 80deg.C for 2 hr to obtain extractive solution;
(4) Taking out the extracting solution, filtering with gauze to remove insoluble substances, centrifuging in a centrifuge, and collecting supernatant to obtain polysaccharide crude extracting solution;
(5) Concentrating the polysaccharide crude extract to 1/5 of the original volume by using a rotary evaporator under the water bath condition of 60 ℃ to obtain crude polysaccharide liquid;
(6) Adding 4 times of absolute ethyl alcohol, standing for 24 hours to separate out a precipitate;
(7) Centrifuging in a centrifuge, and collecting precipitate to obtain coarse belladonna polysaccharide;
(8) Dissolving coarse belladonna polysaccharide, and removing protein by Sevage method to obtain belladonna polysaccharide solution;
(9) Filtering the belladonna polysaccharide solution with ultrafiltration membrane of 30kDa and 10kDa to obtain belladonna polysaccharide component of 10-30 kDa;
(10) Freeze drying belladonna polysaccharide component to obtain belladonna polysaccharide.
Preferably, the oral mucosa repair promoter exerts an oral mucosa repair promoting effect by promoting proliferation and migration ability of an oral mucosa epithelium.
Preferably, the oral mucosa promoter exerts an oral mucosa repair promoting effect by reducing oral lesions by reducing the ability of candida albicans to adhere to oral mucosa epithelial cells.
Preferably, the concentration of belladonna polysaccharide in the accelerator is greater than 0.1mg/ml.
The invention further provides application of the polysaccharide in preparing a promoter for promoting proliferation of oral mucosa epithelial cells, wherein the polysaccharide is belladonna grass polysaccharide, and the belladonna grass polysaccharide is prepared by the belladonna grass polysaccharide preparation method.
The invention further provides application of the polysaccharide in preparing the accelerator for promoting the migration capacity of the oral mucosa epithelial cells, wherein the polysaccharide is belladonna grass polysaccharide, and the belladonna grass polysaccharide is prepared by the belladonna grass polysaccharide preparation method.
The invention further provides application of the polysaccharide in preparing an inhibitor for inhibiting the adhesion capacity of candida albicans, wherein the polysaccharide is belladonna grass polysaccharide, and the belladonna grass polysaccharide is prepared by the belladonna grass polysaccharide preparation method.
Secondly, the present invention provides an accelerator for accelerating oral mucosa repair by improving proliferation and migration ability of oral mucosa epithelial cells, the preparation method of the accelerator comprising the steps of:
(1) The raw materials are prepared according to the following formula: 2% of vitamin C,10% of sodium hyaluronate, 5% of glycerol, 10% of belladonna polysaccharide, 2% of peppermint oil and 71% of sterile water;
(2) And (3) after the raw materials are uniformly stirred, sterilizing and subpackaging the raw materials into spray bottles to obtain the oral mucosa repair promoter.
Preferably, the belladonna grass polysaccharide is prepared by the belladonna grass polysaccharide preparation method.
The beneficial effects of the invention are as follows:
the invention extracts and separates belladonna polysaccharide with molecular weight between 10-30kDa from belladonna grass, and experimental results show that belladonna polysaccharide can promote proliferation of oral epithelial cells in a dose-dependent manner within a certain concentration range; transwell migration experiments also prove that belladonna polysaccharide can promote the migration capacity of oral epithelial cells. In addition, it has been found that belladonna polysaccharide inhibits the adhesion of pathogenic candida albicans to oromucosal cells.
In view of the above results, we have found that the specific polysaccharide component in belladonna grass extract has a promoting effect on repairing damage to oral mucosa.
Drawings
FIG. 1 is a graph showing the results of migration cell numbers of human oral mucosal epithelial cells after treatment with different belladonna polysaccharide;
FIG. 2 is a statistical plot of the number of migrating cells of human oral mucosal epithelial cells after treatment with different belladonna polysaccharide;
in fig. 2, P < 0.001 is shown.
Detailed Description
Example 1: 1. the oral mucosa obtained from volunteers was repeatedly rinsed 5 times with physiological saline in an ultra clean bench;
2. washing for 5 times by using a rinsing solution containing double antibodies to remove residual blood stains on the surface;
3. shearing thicker submucosa tissues by using ophthalmic scissors, adding 0.25% neutral protease II, and separating surface epithelium from lower layer by using ophthalmic forceps after digestion for 16-18h at 4 ℃;
4. cutting surface epithelium into 1mm×1mm small pieces, adding 0.25% trypsin, digesting at 37deg.C for 10min, adding foetal calf serum to stop digestion, and blowing into single cell suspension;
5. collecting cells into a centrifuge tube, placing the centrifuge tube into a centrifuge for centrifugation at 1000rpm for 5min, removing the supernatant, adding Defined Keratinocyte-SFM culture medium and blowing into single cell suspension;
6. after counting the cells, the cells were counted according to 1X 10 4 The cells were inoculated into a culture dish at 37℃and 5% CO at a density of/ml 2 Culturing in a saturated humidity cell incubator to obtain human oral mucosa epithelial cells;
7. the first change of liquid was performed on the third day of culture, followed by 1 change of liquid every 2 days and used for the subsequent experiments.
Example 2:1. cleaning belladonna grass, and oven drying at 60deg.C until the weight is unchanged;
2. pulverizing dried belladonna grass, and sieving with 40 mesh sieve to obtain belladonna grass powder;
3. taking 100g of belladonna grass powder, adding 1500ml of distilled water, placing into an electric water bath kettle, and leaching at 80 ℃ for 2h;
4. taking out the extract, filtering with 4 layers of gauze to remove insoluble substances, centrifuging at 8000rpm in a centrifuge for 20min, and collecting supernatant to obtain polysaccharide crude extract;
5. concentrating the polysaccharide crude extract to 300ml by using a rotary evaporator under the water bath condition of 60 ℃ to obtain crude polysaccharide liquid;
6. adding 1200ml of absolute ethyl alcohol, standing for 24 hours to separate out a precipitate;
7. centrifuging at 8000rpm in a centrifuge for 20min, collecting precipitate to obtain coarse belladonna polysaccharide;
8. dissolving coarse belladonna polysaccharide, repeating the process with Sevage method for 7 times to remove protein to obtain belladonna polysaccharide solution;
9. sequentially filtering belladonna polysaccharide solution with ultrafiltration membranes of 50kDa,30kDa and 10kDa to obtain belladonna polysaccharide component 1 smaller than 10kDa, belladonna polysaccharide component 2 of 10-30kDa and belladonna polysaccharide component 3 of 30-50 kDa;
10. freeze drying belladonna polysaccharide components 1,2 and 3 to obtain belladonna polysaccharide components 1,2 and 3.
Example 3:1. human oral mucosal epithelial cells in growth phase were seeded in 96 plates, 100 μl per well;
2. after overnight culture of cells to complete adherence, cells were treated according to the following groupings:
control group: an added Defined Keratinocyte-SFM medium;
belladonna grass polysaccharide 1 treatment group (hereinafter referred to as polysaccharide 1 treatment group): 1mg/ml of a culture medium solution of belladonna polysaccharide 1 prepared using Defined Keratinocyte-SFM medium and sterilized by filtration using a 0.22 μm membrane was added;
belladonna grass polysaccharide 2 treatment group (hereinafter referred to as polysaccharide 2 treatment group): 1mg/ml of a culture medium solution of belladonna polysaccharide 2 prepared using Defined Keratinocyte-SFM medium and sterilized by filtration using a 0.22 μm membrane was added;
belladonna grass polysaccharide 3 treatment group (hereinafter referred to as polysaccharide 3 treatment group): 1mg/ml of a culture medium solution of belladonna polysaccharide 3 prepared using Defined Keratinocyte-SFM medium and sterilized by filtration using a 0.22 μm membrane was added;
3. the treated cells were subjected to 5% CO at 37 ℃ 2 Culturing in a saturated humidity cell incubator;
4. after 5 days of culture, taking out the 96-well plate, adding 10 mu L of CCK-8 solution into each well, and then placing the 96-well plate in a cell culture box again for incubation for 3 hours;
5. the OD450nm values of each group and the OD450nm values of the pure medium were measured using an enzyme-labeled instrument, and a table was drawn.
TABLE 1 OD values of human oral mucosal epithelial cells after treatment with different belladonna polysaccharide
From table 1, we can see that the three belladonna polysaccharide prepared by the invention has certain measured promotion effect on proliferation of human mouth mucosa epithelium, wherein compared with the control group, the cell proliferation rate of the belladonna polysaccharide 1 treatment group is improved to 0.707+/-0.051, the proliferation rate is improved by 22.5%, and the difference is statistically significant in the two groups compared with p= 0.0437;
the highest cell proliferation rate of the belladonna grass polysaccharide 2 treatment group reaches 0.971+/-0.058, which is 68.3 percent higher than that of the control group, and the difference is very obvious when the two groups are compared with p=0.0012.
The cell proliferation rate of belladonna grass polysaccharide 3 treatment group is 0.792+/-0.052, which is improved by 37.3% compared with control group, and the difference is statistically significant in the two groups compared with p=0.0089.
In combination, the promotion effect of the belladonna grass polysaccharide 2 is very remarkable compared with that of the belladonna grass polysaccharide 1 and the belladonna grass polysaccharide 3, which is probably caused by the different structures of the polysaccharides with different molecular weights. The embodiment preliminarily verifies that belladonna polysaccharide has promoting effect on proliferation of oral mucosa, and can be used for promoting oral mucosa.
Example 4: 1. human oral mucosal epithelial cells in growth phase were seeded in 96 plates, 100 μl per well;
2. after overnight culture of cells to complete adherence, cells were treated according to the following groupings:
control group: an added Defined Keratinocyte-SFM medium;
0.1mg/ml polysaccharide 2 treatment group: adding a medium solution of 0.1mg/ml belladonna polysaccharide 2 prepared using Defined Keratinocyte-SFM medium and sterilized using 0.22um membrane filtration;
0.5 mg/ml polysaccharide 2 treatment group: adding a medium solution of 0.5 mg/ml belladonna polysaccharide 2 prepared using Defined Keratinocyte-SFM medium and sterilized using 0.22um membrane filtration;
1mg/ml polysaccharide 2 treatment group: 1mg/ml of a culture medium solution of belladonna polysaccharide 2 prepared using Defined Keratinocyte-SFM medium and sterilized using 0.22um membrane filtration was added;
5mg/ml polysaccharide 2 treatment group: adding 5mg/ml belladonna polysaccharide 2 culture medium solution prepared with Defined Keratinocyte-SFM culture medium and sterilized with 0.22um membrane filtration;
10 mg/ml polysaccharide 2 treatment group: adding 5mg/ml belladonna polysaccharide 2 culture medium solution prepared with Defined Keratinocyte-SFM culture medium and sterilized with 0.22um membrane filtration;
3. the treated cells were subjected to 5% CO at 37 ℃ 2 Culturing in a saturated humidity cell incubator;
4. after 5 days of culture, taking out the 96-well plate, adding 10 mu L of CCK-8 solution into each well, and then placing the 96-well plate in a cell culture box again for incubation for 3 hours;
5. the OD450nm values of each group and the OD450nm values of the pure medium were measured using an enzyme-labeled instrument, and a table was drawn.
TABLE 2 OD values of human oral mucosal epithelial cells after treatment with different concentrations of belladonna polysaccharide 2
As can be seen from Table 2, the belladonna polysaccharide 2 concentration of 0.1mg/ml can promote the oral mucosa epithelial cells of human, which means that the minimum effective concentration of belladonna polysaccharide 2 is 0.1mg/ml, and the OD value is highest when the belladonna polysaccharide 2 concentration reaches 5mg/ml, and the concentration promotion effect is not significantly increased when the belladonna polysaccharide 2 concentration is continued, which means that 5mg/ml is the optimal concentration of belladonna polysaccharide 2.
Example 5:1. human oral mucosal epithelial cells were treated with the aid of 6-well culture plates, after cell attachment, according to the following group:
control group: an added Defined Keratinocyte-SFM medium;
belladonna grass polysaccharide 1 treatment group (hereinafter referred to as polysaccharide 1 treatment group): adding 5mg/ml of a culture medium solution of belladonna polysaccharide 1 prepared using Defined Keratinocyte-SFM culture medium and sterilized using 0.22um membrane filtration;
belladonna grass polysaccharide 2 treatment group (hereinafter referred to as polysaccharide 2 treatment group): adding 5mg/ml belladonna polysaccharide 2 culture medium solution prepared with Defined Keratinocyte-SFM culture medium and sterilized with 0.22um membrane filtration;
belladonna grass polysaccharide 3 treatment group (hereinafter referred to as polysaccharide 3 treatment group): adding 5mg/ml belladonna polysaccharide 3 culture medium solution prepared with Defined Keratinocyte-SFM culture medium and sterilized with 0.22um membrane filtration;
2. after 24h of culture, the cells of the different treatment groups were digested and resuspended using Defined Keratinocyte-SFM medium without serum to prepare a cell suspension;
3. the Transwell chamber was placed in a 24-well plate, and 500. Mu.L of Defined Keratinocyte-SFM medium containing 10% fetal bovine serum was added to the 24-well plate;
4. cells of different treatment groups were seeded into the upper chamber of the transwell chamber at 20000 cells per well, 24-well plates were placed in 37℃with 5% CO 2 A cell incubator incubated for 24 hours to allow migration of cells from the upper chamber to the lower chamber;
5. after the incubation, carefully taking out the Transwell chamber by using sterile forceps, adding PBS into the upper chamber and the lower chamber respectively, lightly washing for 2 times, then adding enough 4% paraformaldehyde fixing solution into the upper chamber, and fixing for 30 minutes at room temperature to fix the cells migrating to the lower layer;
6. the PBS is used for cleaning the upper chamber and the lower chamber for 2 times to remove paraformaldehyde, and then a sterile cotton swab is used for carefully wiping off redundant non-migrated cells in the upper chamber;
7. adding a proper amount of crystal violet working solution into the lower chamber for dyeing for 20 minutes, and then washing with water to remove unbound dye;
8. the transwell chamber was photographed under a microscope and the number of migrated cells was counted by selecting a plurality of fields.
As can be seen from the figures 1 and 2, compared with the control group, the average migration cell number of the 3 polysaccharide treatment groups is increased to different degrees, which indicates that the belladonna polysaccharide 1,2 and 3 prepared by the invention have obvious promotion on the migration capacity of human oral mucosa epithelial cells, and the belladonna polysaccharide 2 has the best promotion effect similar to the proliferation promotion effect.
Meanwhile, compared with the effect of promoting proliferation of human oral epithelial cells, the belladonna polysaccharide has more remarkable migration promoting effect.
Example 6:1. inoculating candida albicans standard strain ATCC 10231 into a Saccharum liquid culture medium, and culturing at 37 ℃ and 120rpm for 24 hours;
2. collecting Candida albicans, and adjusting the concentration of the bacterial liquid to 1×10 6 CFU/mL；
3. Preparing human oral mucosa epithelial cells into cell suspension, and adjusting concentration to 1×10 5 Individual/ml;
4. taking a pre-cleaned slide, adding 100 mu L of mucosal cell suspension, and standing at room temperature for 30 minutes to attach cells to the slide;
5. carefully wash 2 times to remove non-adherent cells;
6. the control group is added with 400 mu L Defined Keratinocyte-SFM culture medium, the polysaccharide 1 treatment group is added with 400 mu L of 5mg/ml belladonna polysaccharide 1 culture medium, the polysaccharide 2 treatment group is added with 400 mu L of 5mg/ml belladonna polysaccharide 2 culture medium, and the polysaccharide 3 treatment group is added with 400 mu L of 5mg/ml belladonna polysaccharide 3 culture medium;
7. adding 400 mu L of candida albicans bacterial liquid into each group, placing the groups at 37 ℃ for incubation for 2 hours, and washing the groups for 2 times by PBS to remove non-adhered candida albicans;
8. after fixation with ethanol, gram staining was performed to count the number of adherent candida in the field of view, adhesion rate = number of cells of adherent candida/number of cells of the field of view observed.
TABLE 3 Effect of different belladonna polysaccharides on Candida albicans on human oral mucosal epithelial cell adhesion
As can be seen from Table 3, candida albicans has strong adhesion to human oral mucosa epithelial cells, and the adhesion rate can reach 55.49%. After three different belladonna polysaccharide treatments, the adhesion rate of candida to epithelial cells is reduced to different degrees, but the difference of the influence of belladonna polysaccharide 1 on the adhesion rate of candida albicans to oral epithelial cells has no statistical significance;
the belladonna polysaccharide 2 and the belladonna polysaccharide 3 can obviously inhibit the adhesion of candida albicans to oral cells. Wherein, the belladonna polysaccharide 2 has optimal adhesion inhibiting effect. The result shows that the belladonna polysaccharide 2 prepared by the invention can obviously inhibit the adhesion of candida albicans to oral epithelial cells and reduce the damage to oral mucosa, so that the belladonna polysaccharide 2 can be used for developing an accelerator for repairing the oral mucosa.
Example 7: 1. the raw materials are prepared according to the following formula: 2% of vitamin C,10% of sodium hyaluronate, 5% of glycerol, 10% of belladonna polysaccharide 2,2% of peppermint oil and 71% of sterile water;
2. and (3) after the raw materials are stirred and evenly stirred, sterilizing and subpackaging the raw materials into spray bottles to obtain the oral mucosa repair promoter.
Claims (1)
1. The application of polysaccharide in preparing an oral mucosa repair promoter is characterized in that the polysaccharide is belladonna grass polysaccharide, and the preparation method of the belladonna grass polysaccharide comprises the following steps:
(1) Cleaning belladonna grass, and drying in a drying oven until the weight is unchanged;
(2) Pulverizing dried belladonna grass, and sieving to obtain belladonna grass powder;
(3) Extracting belladonna grass powder with 15 times of distilled water at 80deg.C for 2 hr to obtain extractive solution;
(4) Taking out the extracting solution, filtering with gauze to remove insoluble substances, centrifuging in a centrifuge, and collecting supernatant to obtain polysaccharide crude extracting solution;
(5) Concentrating the polysaccharide crude extract to 1/5 of the original volume by using a rotary evaporator under the water bath condition of 60 ℃ to obtain crude polysaccharide liquid;
(6) Adding 4 times of absolute ethyl alcohol, standing for 24 hours to separate out a precipitate;
(7) Centrifuging in a centrifuge, and collecting precipitate to obtain coarse belladonna polysaccharide;
(8) Dissolving coarse belladonna polysaccharide, and removing protein by Sevage method to obtain belladonna polysaccharide solution;
(9) Filtering the belladonna polysaccharide solution with ultrafiltration membrane of 30kDa and 10kDa to obtain belladonna polysaccharide component of 10-30 kDa;
(10) Freeze drying belladonna polysaccharide component to obtain belladonna polysaccharide.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311460970.5A CN117180302B (en) | 2023-11-06 | 2023-11-06 | Application of polysaccharide in repairing oral mucosa and accelerator prepared from polysaccharide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311460970.5A CN117180302B (en) | 2023-11-06 | 2023-11-06 | Application of polysaccharide in repairing oral mucosa and accelerator prepared from polysaccharide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117180302A CN117180302A (en) | 2023-12-08 |
| CN117180302B true CN117180302B (en) | 2024-01-12 |
Family
ID=88994586
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311460970.5A Active CN117180302B (en) | 2023-11-06 | 2023-11-06 | Application of polysaccharide in repairing oral mucosa and accelerator prepared from polysaccharide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117180302B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6277370B1 (en) * | 1998-04-30 | 2001-08-21 | Renata Maria Anna Cavaliere Ved. Vesely | Pharmaceutical compositions containing lactobacilli for treatment of vaginal infections and related method |
| CN102697960A (en) * | 2012-05-21 | 2012-10-03 | 马艺 | Medicament for treating recurrent oral ulcer and preparation method thereof |
| WO2016106313A1 (en) * | 2014-12-23 | 2016-06-30 | Kiromic, Llc | Oral care compositions |
| CN111683653A (en) * | 2017-10-11 | 2020-09-18 | 伊拉斯吹斯制药有限公司 | Methods and compositions for topical delivery |
| CN111956711A (en) * | 2020-10-09 | 2020-11-20 | 河北金兴制药有限公司 | Preparation method of medicine for treating peptic ulcer and chronic gastritis |
| CN113274436A (en) * | 2021-04-06 | 2021-08-20 | 福建力传生物有限公司 | Belladonna extract preparation method |
-
2023
- 2023-11-06 CN CN202311460970.5A patent/CN117180302B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6277370B1 (en) * | 1998-04-30 | 2001-08-21 | Renata Maria Anna Cavaliere Ved. Vesely | Pharmaceutical compositions containing lactobacilli for treatment of vaginal infections and related method |
| CN102697960A (en) * | 2012-05-21 | 2012-10-03 | 马艺 | Medicament for treating recurrent oral ulcer and preparation method thereof |
| WO2016106313A1 (en) * | 2014-12-23 | 2016-06-30 | Kiromic, Llc | Oral care compositions |
| CN111683653A (en) * | 2017-10-11 | 2020-09-18 | 伊拉斯吹斯制药有限公司 | Methods and compositions for topical delivery |
| CN111956711A (en) * | 2020-10-09 | 2020-11-20 | 河北金兴制药有限公司 | Preparation method of medicine for treating peptic ulcer and chronic gastritis |
| CN113274436A (en) * | 2021-04-06 | 2021-08-20 | 福建力传生物有限公司 | Belladonna extract preparation method |
Non-Patent Citations (3)
| Title |
|---|
| 引起口腔疾病的药物知多少;陈育民;;健康天地(第10期);全文 * |
| 湖南产颠茄草不同生长期8个成分的动态变化及不同部位分布特征;张文婷;张春阳;黄伟;程维明;黄琴伟;岳超;;中草药(第22期);全文 * |
| 茄科药用植物中多糖成分提取及药理作用的研究进展;黄越燕;潘琳琳;周吉芳;;中国药房(第28期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117180302A (en) | 2023-12-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20220096352A1 (en) | Human placental collagen compositions and methods of making and using the same | |
| KR20050002906A (en) | Collagen biofabric and methods of preparation and use therefor | |
| CN113509590B (en) | Wound dressing with exosome combined with hyaluronic acid and preparation method and application thereof | |
| CN113527467B (en) | Sturgeon skin collagen polypeptide extraction method, application, cosmetic raw material and preparation method thereof | |
| CN1311072C (en) | Preparation method of dried active amnion | |
| CN110585062A (en) | Hyaluronic acid oral care composition and preparation method and application thereof | |
| CN110938587A (en) | Preparation method and application of epidermal stem cell suspension | |
| US5770205A (en) | Tissue fractions of sea cucumber for the treatment of inflammation | |
| US5876762A (en) | Process for obtaining medically active fractions from sea cucumbers | |
| CN117180302B (en) | Application of polysaccharide in repairing oral mucosa and accelerator prepared from polysaccharide | |
| KR20220085035A (en) | Composition for improving oral health comprising extracts of Stewartia koreana Nakai and the product comprising the same | |
| KR100785441B1 (en) | Physiological saline solution with antibacterial activity comprising minerals of deep sea water | |
| CN110882236A (en) | Anti-tumor composition, pharmaceutical preparation for treating cancer and application of pharmaceutical preparation | |
| CN114591396A (en) | Preparation method of phenol amide peptide and anti-inflammatory and bacteriostatic traditional Chinese medicine composition formed by phenol amide peptide | |
| CN113876657B (en) | Application of cercis chinensis flower extract in oral care products | |
| CN113897321B (en) | Marseillella and preparation method and application thereof | |
| CN114904045B (en) | Hydrogel medical dressing containing traditional Chinese medicine extract and preparation method thereof | |
| CN112245488A (en) | Bone marrow mesenchymal stem cell composition for treating oral ulcer and preparation method thereof | |
| CN112891295A (en) | Exosome mouthwash as well as preparation method and application thereof | |
| CN117427202A (en) | Exosome and collagen sponge biological scaffold and preparation method and application thereof | |
| KR20220131021A (en) | Composition for treating conjunctivitis containing membrane free stem cell extract and a eye drop thereby | |
| EP4106827A1 (en) | Porcine scaffolds and methods of preparation | |
| CN111643712A (en) | Wound dressing and preparation method thereof | |
| CN117599239A (en) | Dual-slow-release wound healing-promoting and anti-scar dressing and preparation method thereof | |
| CN1970094A (en) | Process for preparing hyaluronic acid-chitosan biomembrane |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |