CN110075146B - Oral ulcer spray and preparation method thereof - Google Patents
Oral ulcer spray and preparation method thereof Download PDFInfo
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- CN110075146B CN110075146B CN201910513108.3A CN201910513108A CN110075146B CN 110075146 B CN110075146 B CN 110075146B CN 201910513108 A CN201910513108 A CN 201910513108A CN 110075146 B CN110075146 B CN 110075146B
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
- A61K31/525—Isoalloxazines, e.g. riboflavins, vitamin B2
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
- A61K9/006—Oral mucosa, e.g. mucoadhesive forms, sublingual droplets; Buccal patches or films; Buccal sprays
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
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- A61K9/10—Dispersions; Emulsions
- A61K9/12—Aerosols; Foams
Abstract
The invention provides a spray for treating oral ulcer and a preparation method thereof, belonging to the technical field of treating oral ulcer and comprising the following components in parts by volume: 75-85 parts of alcohol extract of the Japanese basil, 5-15 parts of dry cell extract, 3-8 parts of deionized water, 2-6 parts of preservative and vitamin B20.1-0.5 part; the concentration of exosome protein in the stem cell extract is 25-35 mg/mL; the pH value of the oral ulcer spray is 6.6-7.11. The oral ulcer spray provided by the invention has good treatment effect, is mild and comfortable to use, has no scar after healing, and has a total effective rate of 97.22% for oral ulcer patients.
Description
Technical Field
The invention relates to the technical field of treating oral ulcer, and particularly relates to an oral ulcer spray and a preparation method thereof.
Background
The ulcerated areas are usually found on the tongue surface, lips, gums, etc. The oral ulcer has severe pain and obvious local burning pain during the attack, serious patients can influence diet and speaking, and can also have halitosis, chronic pharyngitis and the like, and if the oral ulcer is not treated in time, the daily life is greatly inconvenient. Its induction factors are many, such as local trauma, irritants, bacterial infections, endocrine imbalances, depression, mental stress, etc., all cause ulcers.
Recurrent oral ulceration is roughly divided into three types: minor aphthae (minor aphthous ulcers), major aphthae (major aphthous ulcers) and herpetiform ulcers (andeerpetiform ulcers), which are most common, have a high recurrence rate. The clinical treatment of aphthous canker sores can be divided into: (1) symptomatic support therapy; (2) special treatment; (3) and (4) preventive treatment. Standard topical treatment regimens typically use a number of drugs for symptomatic relief of pain, including analgesics, antimicrobials, anti-inflammatory agents, steroids, vitamin B2And the like. At present, the recurrent oral ulcer is not completely cured clinically,in general, after healing, a large and deep canker sore (major aphthous ulcers) will form a scar locally, and the scar formed after healing of the ulcer will gradually become soft, absorbed and small with time, but the local mucous membrane is difficult to recover as before.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides the oral ulcer spray and the preparation method thereof.
The invention provides an oral ulcer spray which comprises the following components in parts by volume:
75-85 parts of alcohol extract of the Japanese basil, 5-15 parts of dry cell extract, 3-8 parts of deionized water, 2-6 parts of preservative and vitamin B20.1-0.5 part;
the concentration of exosome protein in the stem cell extract is 25-35 mg/mL;
the pH value of the oral ulcer spray is 6.6-7.11.
Preferably, the composition comprises the following components in parts by volume:
80 parts of alcohol extract of the Japanese creeper, 10 parts of dry cell extract, 5 parts of deionized water, 4 parts of preservative and vitamin B20.3 part;
the concentration of exosome protein in the stem cell extract was 30 mg/mL.
Preferably, the preparation method of the alcohol extract of the guava comprises the following steps:
1) mixing flos basjoliae with a first ethanol solution, extracting for 1-2 h, and filtering to obtain a first filtrate and a first filter residue; the first ethanol solution is 60-70% in volume percentage;
2) mixing the first filter residue obtained in the step 1) with a second ethanol solution, extracting for 1-2 h, and filtering to obtain a second filtrate and a second filter residue; the volume percentage content of the second ethanol solution is 55-65%;
3) mixing the second filter residue obtained in the step 2) with a third ethanol solution, extracting for 1-2 h, and filtering to obtain a third filtrate and a third filter residue; the volume percentage content of the third ethanol solution is 55-65%;
4) mixing the first filtrate obtained in the step 1), the second filtrate obtained in the step 2) and the third filtrate obtained in the step 3) to obtain a filtrate, and concentrating the filtrate under reduced pressure to obtain an alcohol extract of the basjoo flower; the volume of the alcohol extract of the guava and the weight ratio of the alcohol extract of the guava to the guava are (1-5): 1.
Preferably, the mass ratio of the bava flower in the step 1) to the first ethanol solution is 1 (15-20).
Preferably, the mass ratio of the first filter residue to the second ethanol solution in the step 2) is 1 (8-12).
Preferably, the mass ratio of the second filter residue to the third ethanol solution in the step 3) is 1 (6-8).
Preferably, the preparation method of the stem cell extract comprises the following steps:
a. obtaining the human umbilical cord mesenchymal stem cells and culture supernatant of the human umbilical cord mesenchymal stem cells by adopting a conventional method for culturing the human umbilical cord mesenchymal stem cells; the number of the human umbilical cord mesenchymal stem cells is 3 multiplied by 108A plurality of;
b. mixing the human umbilical cord mesenchymal stem cells with normal saline, carrying out ultrasonic disruption, and collecting exosomes by adopting a conventional method;
c. collecting exosome in culture supernatant of human umbilical cord mesenchymal stem cells by a conventional method;
d. and d, mixing the exosome obtained in the step c, the exosome obtained in the step d and physiological saline to obtain a stem cell extract, wherein the concentration of exosome protein in the stem cell extract is 25-35 mg/mL.
Preferably, the exosomes obtained in the step c are subjected to BCA protein concentration measurement, and the concentration of the exosome protein is 9.5 mg/ml.
Preferably, the exosomes obtained in the step d are subjected to BCA protein concentration measurement, and the concentration of the exosome protein is 53.6 mg/ml.
The invention also provides the oral ulcer spray prepared by the technical schemeThe preparation method comprises the following steps: mixing the vitamin B2Dissolving with alkali solution, mixing with ethanol extract of flos Bombacis Cannabinae and deionized water, and sequentially mixing with antiseptic and stem cell extract to obtain oral ulcer spray.
The invention provides an oral ulcer spray which comprises the following components in parts by volume: 75-85 parts of alcohol extract of the Japanese basil, 5-15 parts of dry cell extract, 3-8 parts of deionized water, 2-6 parts of preservative and vitamin B20.1-0.5 part; the concentration of exosome protein in the stem cell extract is 25-35 mg/mL; the pH value of the oral ulcer spray is 6.6-7.11.
In the invention, the ethanol extract of the Chinese basjo flower contains a plurality of aromatic alcohol compounds such as aromatic alcohol and geraniol, and the ethanol extract of the Chinese basjo flower has the effects of resisting inflammation, relieving itching, moisturizing and repairing; the exosome protein in the stem cell extract effectively improves local inflammation of the oral cavity and promotes the growth of epithelial cells of the oral cavity so as to repair scar parts; vitamin B2 has a promoting effect on the treatment of oral ulcers. After the oral ulcer spray provided by the invention is used for curing oral ulcer, no scar is generated.
The invention has the advantages that:
the oral ulcer spray provided by the invention has good treatment effect, is mild and comfortable to use, has no scar after healing, and has a total effective rate of 97.22% for oral ulcer patients. And the raw materials are all food grade, do not contain antibiotics and hormones, are mild to mouth and throat, have no stimulation and have no toxic or side effect.
Drawings
FIG. 1 is a graph of the healing results of canker sores on day 1 of a patient using a canker sore spray;
FIG. 2 is a graph of the healing results of canker sores on day 4 with canker sore spray;
FIG. 3 is a graph of the healing results of canker sores on day 8 with canker sore spray;
figure 4 is the results of oral ulcer healing by day 10 using a canker sore spray.
Detailed Description
The invention providesThe oral ulcer spray comprises the following components in parts by volume: 75-85 parts of alcohol extract of the Japanese basil, 5-15 parts of dry cell extract, 3-8 parts of deionized water, 2-6 parts of preservative and vitamin B20.1-0.5 part; the concentration of exosome protein in the stem cell extract is 25-35 mg/mL; the pH value of the oral ulcer spray is 6.6-7.11.
The oral ulcer spray provided by the invention comprises 75-85 parts by volume of alcohol extract of guava, preferably 78-82 parts by volume of alcohol extract of guava, and more preferably 80 parts by volume of alcohol extract of guava.
In the present invention, the preparation method of the alcohol extract of guava preferably comprises the following steps:
1) mixing flos basjoliae with a first ethanol solution, extracting for 1-2 h, and filtering to obtain a first filtrate and a first filter residue; the first ethanol solution is 60-70% in volume percentage;
2) mixing the first filter residue obtained in the step 1) with a second ethanol solution, extracting for 1-2 h, and filtering to obtain a second filtrate and a second filter residue; the volume percentage content of the second ethanol solution is 55-65%;
3) mixing the second filter residue obtained in the step 2) with a third ethanol solution, extracting for 1-2 h, and filtering to obtain a third filtrate and a third filter residue; the volume percentage content of the third ethanol solution is 55-65%;
4) mixing the first filtrate obtained in the step 1), the second filtrate obtained in the step 2) and the third filtrate obtained in the step 3) to obtain a filtrate, and concentrating the filtrate under reduced pressure to obtain an alcohol extract of the basjoo flower; the volume of the alcohol extract of the guava and the weight ratio of the alcohol extract of the guava to the guava are (1-5): 1.
According to the invention, the basjoo flower and the first ethanol solution are preferably mixed and then extracted for 1-2 h, and a first filtrate and a first filter residue are obtained after filtration. In the present invention, the time for the extraction is more preferably 1.5 h. In the present invention, the basjoo flower is preferably fresh basjoo flower. In the invention, the volume percentage content of the first ethanol solution is preferably 60-70%, and more preferably 65%. In the invention, the mass ratio of the flos basjoliae to the first ethanol solution is preferably 1 (15-20), and more preferably 1: 18. In the invention, the extraction is preferably carried out by adopting a reflux extraction method commonly used by the conventional Chinese medicinal materials.
Preferably, the obtained first filter residue and the second ethanol solution are mixed and then extracted for 1-2 hours, and a second filtrate and a second filter residue are obtained after filtration. The time of extraction in the present invention is more preferably 1.5 hours. In the present invention, the volume percentage of the second ethanol solution is preferably 55 to 65%, and more preferably 60%. In the invention, the mass ratio of the first filter residue to the second ethanol solution is preferably 1 (8-12), and more preferably 1: 10.
Preferably, the obtained second filter residue is mixed with a third ethanol solution and then extracted for 1-2 hours, and a third filtrate and a third filter residue are obtained after filtration. In the present invention, the time for the extraction is more preferably 1.5 h. In the invention, the volume percentage content of the third ethanol solution is preferably 55-65%, and more preferably 60%. In the invention, the mass ratio of the second filter residue to the third ethanol solution is preferably 1 (6-8).
In the present invention, the alcohol extract of the basjoo flower contains a plurality of aromatic alcohol compounds (24.92% of the total composition), such as aromatic alcohol (about 5.97%) and geraniol (about 10.2%). In the invention, the alcohol extract of the Chinese sumac flower has the functions of anti-inflammation, itching relieving, moisturizing and repairing.
The oral ulcer spray provided by the invention comprises 5-15 parts of stem cell extract by volume fraction, preferably 8-12 parts, and more preferably 10 parts.
In the present invention, the method for preparing the stem cell extract preferably comprises the steps of:
a. obtaining the human umbilical cord mesenchymal stem cells and culture supernatant of the human umbilical cord mesenchymal stem cells by adopting a conventional method for culturing the human umbilical cord mesenchymal stem cells; the number of the human umbilical cord mesenchymal stem cells is 3 multiplied by 108A plurality of;
b. mixing the human umbilical cord mesenchymal stem cells with normal saline, carrying out ultrasonic disruption, and collecting exosomes by adopting a conventional method;
c. collecting exosome in culture supernatant of human umbilical cord mesenchymal stem cells by a conventional method;
d. and d, mixing the exosome obtained in the step c, the exosome obtained in the step d and physiological saline to obtain a stem cell extract, wherein the concentration of exosome protein in the stem cell extract is 25-35 mg/mL.
The invention preferably adopts a conventional method for culturing the human umbilical cord mesenchymal stem cells to obtain the human umbilical cord mesenchymal stem cells and culture supernatant of the human umbilical cord mesenchymal stem cells. The source of the human umbilical cord mesenchymal stem cells is not specially limited, and the method can be realized by adopting a conventional method. In the present invention, the number of the human umbilical cord mesenchymal stem cells obtained by the culture is preferably 3 × 108And (4) respectively.
The invention preferably mixes the human umbilical cord mesenchymal stem cells with normal saline and then carries out ultrasonic disruption, and an exosome is collected by adopting a conventional method. In the present invention, the ratio of the number of human umbilical cord mesenchymal stem cells to the volume of physiological saline is preferably 3 × 108300 mL.
In the present invention, the parameter setting of the ultrasonication is preferably 600W; 4 ℃; the work time is 2s, the interval is 2s, and the ultrasonic treatment is carried out for 10 min.
The invention preferentially centrifuges the obtained ultrasonic crushed material at 300g and 4 ℃ for 10min to remove cell debris, collects the supernatant, then centrifuges at 2500g and 4 ℃ for 30min to remove the cell apoptosis corpuscle, collects the supernatant, centrifuges at 13000g for 1h to remove platelet impurities, collects the supernatant, and finally centrifuges at 100000g and 4 ℃ for 70min to obtain the exosome. The BCA protein concentration of the obtained exosome is measured, and the exosome protein concentration is 9.5 mg/ml.
The invention preferably adopts a conventional method to collect exosomes in the culture supernatant of the human umbilical cord mesenchymal stem cells. The invention preferentially centrifuges culture supernatant of human umbilical cord mesenchymal stem cells for 10min at 2000g and 4 ℃, removes cell debris, collects the supernatant, centrifuges the culture supernatant for 1h at 13000g to remove platelet impurities, collects the supernatant, and finally centrifuges the culture supernatant for 70min at 100000g and 4 ℃ to obtain exosome. The BCA protein concentration of the obtained exosome is measured, and the exosome protein concentration is 53.6 mg/ml.
In the invention, exosome obtained by crushing human umbilical cord mesenchymal stem cells and exosome obtained by human umbilical cord mesenchymal stem cell culture supernatant are dissolved in normal saline respectively and then mixed in equal volume to obtain a stem cell extract.
In the invention, the concentration of the exosome protein in the stem cell extract is 25-35 mg/mL, preferably 30 mg/mL. In the invention, the stem cell extract is effective in improving local inflammation of an oral cavity and promoting growth of oral epithelial cells so as to repair scar parts.
The oral ulcer spray provided by the invention comprises 3-8 parts by volume of deionized water, preferably 5 parts.
The oral ulcer spray provided by the invention comprises 2-6 parts by volume of preservative, preferably 4 parts.
In the present invention, the preparation method of the preservative preferably includes: mixing methyl p-hydroxybenzoate and 1, 3-butanediol solution, stirring to clarify, and dissolving to obtain antiseptic. In the present invention, the ratio of the mass of methyl paraben to the volume of 1, 3-butanediol solution is preferably 15g:250 ml. The concentration of the 1, 3-butanediol solution is not particularly limited, and the concentration of the conventional dissolved methyl p-hydroxybenzoate solution is adopted.
The oral spray provided by the invention comprises 0.1-0.5 parts by volume of vitamin B2Preferably 0.3 parts. In the present invention, the vitamin B2Has an effect of promoting the treatment of oral ulcer.
In the invention, the pH value of the oral spray is 6.6-7.11. In the present invention, the agents for conditioning the mouth ulcer spray are preferably sodium hydroxide solution and citric acid. In the present invention, the concentration of the sodium hydroxide is preferably 1 mol/L.
The invention also provides a preparation method of the oral ulcer spray, which comprises the following steps: mixing the vitamin B2Dissolving with alkali solution, mixing with ethanol extract of flos Bombacis Cannabinae and deionized water, and sequentially mixing with antiseptic and stem cell extract to obtain oral ulcer spray.
In the present invention, the alkali solution is preferably a sodium hydroxide solution. In the present invention, the concentration of the sodium hydroxide is preferably 1 mol/L.
In the invention, the oral ulcer spray is slightly light yellow, transparent and clear, and slightly bitter in taste.
In the present invention, the method of using the mouth ulcer spray preferably comprises: it is administered 1 time, 5 ml/time, 4 times before sleep, and no drinking or eating within 30min after each administration.
The technical solutions provided by the present invention are further described in detail with reference to the following specific examples, which include but are not limited to the following examples.
Example 1
The preparation method of the oral ulcer spray comprises the following steps:
the method comprises the following steps: preparing a preservative: accurately weighing 15g of methyl p-hydroxybenzoate, adding 250ml of 1, 3-butanediol solution, stirring until the solution is clear and fully dissolved, standing for half an hour to observe whether insoluble substances are separated out to obtain the preservative;
step two: accurately weighing vitamin B according to actual prepared liquid quantity20.06g, adding a little sodium hydroxide solution, accurately weighing 16ml of ethanol extract of the Chinese ballflower after fully dissolving, and adding 1ml of deionized water;
step three: mixing 0.08ml of preservative into the solution, and stirring to mix uniformly;
step four: taking 2ml of the stem cell extract, stirring to mix uniformly, and finally adjusting the pH value to 7.0 by using sodium hydroxide or sodium citrate to obtain 20ml of the oral ulcer spray.
The preparation method of the alcohol extract of the Chinese paris comprises the following steps: taking fresh flowers of the Parthenia basjoo, and extracting for 3 times by reflux: adding 18 times of 65% ethanol at the 1 st time, and extracting for 1.5 hr; adding 10 times of 60% ethanol for 2 times, and extracting for 1.5 hr; adding 8 times of 60% ethanol for 3 times, and extracting for 1.5 hr. Filtering with three layers of gauze each time, mixing filtrates, and concentrating the filtrate under reduced pressure to 5 times volume of fresh flos Bombacis Cannabinae weight to obtain flos Bombacis Cannabis alcohol extract; the alcohol extract of flos Bombacis comprises various aromatic alcohol compounds (24.92% of the total component), such as aromatic alcohol (about 5.97%) and geraniol (about 10.2%).
The preparation method of the stem cell extract comprises the following steps: filling stem cells between human umbilical cords in vitro and performing in-vitro large-scale culture: selecting human umbilical cord mesenchymal stem cell seed cells according to the ratio of 1 × 105Inoculating the cells into four T175 culture bottles (GMP grade) for culture at a density, digesting, centrifuging, collecting cells, and culturing at 4 × 107Inoculating to cell factory (GMP grade), adding phenol red-free complete culture medium, culturing, changing liquid once, and harvesting to obtain 3 × 10 cells when cell fusion rate is above 95%8Collecting the culture supernatant of human umbilical cord mesenchymal stem cells;
carrying out ultrasonic disruption on the collected human umbilical cord mesenchymal stem cells: by 1 × 108Adding the cell number into 100ml of normal saline (0.9% sodium chloride aqueous solution), setting parameters of a cell disruptor as (600W; 4 ℃, 2s intermittent ultrasound for 10min in working 2 s), centrifuging for 10min at 300g and 4 ℃ to remove cell debris, collecting supernatant, centrifuging for 30min at 2500g and 4 ℃ to remove apoptotic bodies, collecting supernatant, centrifuging for 1h at 13000g to remove platelet impurities, collecting supernatant, finally centrifuging for 70min at 100000g and 4 ℃ to obtain exosome sediment, and measuring the concentration of the exosome protein by BCA protein concentration, wherein the concentration of the exosome protein is 9.5 mg/ml;
collecting mesenchymal stem cell culture supernatant, centrifuging at 2000g and 4 ℃ for 10min to remove cell debris, collecting supernatant, centrifuging at 13000g for 1h to remove platelet impurities, collecting supernatant, finally centrifuging at 100000g and 4 ℃ for 70min to obtain exosome precipitate, and measuring the concentration of the protein of the exosome by BCA (burst cutting protein) to obtain exosome precipitate, wherein the concentration of the protein of the exosome is 53.6 mg/ml;
mixing the crushed mesenchymal stem cell exosomes and the stem cell culture supernatant exosomes according to the measured protein concentration in proportion, adding the mixture into normal saline to obtain the required final protein concentration (30mg/ml), and stirring the mixture uniformly to obtain the stem cell extract.
Example 2
The preparation method of the oral ulcer spray comprises the following steps:
the method comprises the following steps: preparing a preservative: accurately weighing 15g of methyl p-hydroxybenzoate, adding 250ml of 1, 3-butanediol solution, stirring until the solution is clear and fully dissolved, standing for half an hour to observe whether insoluble substances are separated out to obtain the preservative;
step two: accurately weighing vitamin B according to actual prepared liquid quantity20.02g, adding a little sodium hydroxide solution, after fully dissolving, accurately measuring 14ml of the alcohol extract of the Japanese basil prepared in the example 1, and adding 4ml of deionized water;
step three: mixing 0.08ml of preservative into the solution, and stirring to mix uniformly;
step four: taking 1ml of the stem cell extract prepared in the example 1, stirring to mix uniformly, and finally adjusting the pH value to 7.0 by using sodium hydroxide or sodium citrate to obtain 20ml of the oral ulcer spray.
Example 3
The preparation method of the oral ulcer spray comprises the following steps:
the method comprises the following steps: preparing a preservative: accurately weighing 15g of methyl p-hydroxybenzoate, adding 250ml of 1, 3-butanediol solution, stirring until the solution is clear and fully dissolved, standing for half an hour to observe whether insoluble substances are separated out to obtain the preservative;
step two: accurately weighing vitamin B according to actual prepared liquid quantity20.1g, adding a little sodium hydroxide solution, after fully dissolving, accurately measuring 15ml of ethanol extract of the Japanese basil prepared in example 1, and adding 1.86ml of deionized water;
step three: mixing 0.04ml of antiseptic into the above solution, and stirring to mix well;
step four: taking 3ml of the stem cell extract prepared in the example 1, stirring to mix uniformly, and finally adjusting the pH value to 7.0 by using sodium hydroxide or sodium citrate to obtain 20ml of the oral ulcer spray.
The physicochemical indexes of the oral ulcer spray prepared in example 1 are as follows:
the characteristics are as follows: the product is a quantitative pressing type aerosol, and the liquid in the bottle is a pale yellow, transparent and clear oral ulcer spray with slightly bitter taste.
And (3) quality detection: (1) a small amount of oral spray liquid was dipped with pH paper and the pH was measured to be 6-7. (2) The liquid is respectively packaged in transparent spray bottles, refrigerated in a refrigerator at 4 ℃ and placed in a constant temperature cabinet at 37 ℃ for about a week to observe no layering phenomenon. Standing for 3 months, no layering phenomenon, no change in taste after use, no change in odor, and no deterioration.
And (3) detecting the irritation: animal experiments are adopted, 32 guinea pigs are taken and randomly divided into 4 groups, multiple times of administration and single administration mode are continuously administered at the same part, the administration time and the dosage are the same, and 4 days are continuously used. The skin test scoring result shows that after the spray and the ethanol solution are used for one time or multiple times, no erythema and edema (the average reaction value is less than 0.5) exist, and the oral ulcer spray is mild and has no stimulation. The scoring standard refers to the research group of subjects in the technical guidance principle of research on the irritation, allergy and hemolysis of chemical drugs, the guidance principle of the research on the irritation, allergy and hemolysis of chemical drugs, and the State food and drug administration (2005: 2-7). The results are shown in Table 1.
TABLE 1 mean scores for skin irritation of oral spray liquids
Example 4
Test of therapeutic Effect on dental ulcer
General data:
72 patients with oral ulcer treated by the oral ulcer spray prepared in example 1 in 2018, 1-2018, 12 months are used as study subjects, wherein 41 male patients, 31 female patients, 9-71 years old and average (41.6 +/-3.0) years old are selected. Inclusion criteria were: firstly, oral ulcer is confirmed in clinical diagnosis; ② the patients agree with the study and sign informed consent. Exclusion criteria: patients with oral malignant diseases and immune system diseases are excluded. The general data of both groups of patients were comparable (P > 0.05).
The method comprises the following steps:
before the test, the patient was given a compound borax solution to gargle and the surface was blotted dry with a sterile cotton swab. The patients used the canker sore spray prepared in example 1 once, 5 ml/time, 4 times each in the morning, at noon, at night and before bedtime. After all patients had been dosed, the patients were observed for 30 minutes for any possible signs of acute allergic reactions and were deprived of alcohol or food within 30 minutes after each dose.
Observing and evaluating the curative effect indexes:
ulcer site size, exudate level (see table 2) were assessed on days 1, 4, 8 and 10 of oral spray treatment. And (3) obtaining pain scores before and after treatment in patient groups and among the patient groups, and scoring the pain degree of the patients by adopting VAS pain scores, wherein the total score is 10, the score of 0 indicates no pain, and the higher the score is, the more severe the pain is.
Table 2 exudate level fractionation
| Grading | Exudate Level (enumeration Level) |
| 0 | Is free of |
| 1 | Small amount of exudate |
| 2 | Moderate exudate |
| 3 | Large amount of exudate with pseudomembrane |
Finally, the efficacy of the patients was compared between groups by means of a opinion score. The effect is shown: pain and ulcer disappeared within 3d of patient; the method has the following advantages: the pain of the patient is relieved, and the ulcer is healed within 4-7 days; and (4) invalidation: the pain of the patient worsens and the ulcer does not heal. The treatment data acquisition strictly follows the statistical principle and is analyzed and processed by software SPSS17.0, and P <0.05 indicates that the difference has statistical significance.
The results show that the oral spray can be basically cured within 4 days after being used, the scar of the ulcer part is observed to be recovered as early as the initial state by the 10 th day, and the repairing effect of the oral mucosa is good. After treatment, the total effective rate of the patients is 97.22%, and the data difference between groups has statistical significance (P < 0.05). The results are shown in tables 3 to 6.
TABLE 3 therapeutic Effect of two groups of patients [ n (%) ]
TABLE 4 reduction of ulcer size
| Time | Group of | Ulcer size (mm) | P value |
| Day 1 | Experimental group | 5.20±0.20 | 0.207 |
| Day 4 | Experimental group | 2.70±2.40 | 0.012 |
| Day 8 | Experimental group | 0.20±0.71 | 0.000 |
| Day 10 | Experimental group | 0.00±0.00 | 0.000 |
Table 5 exudate level comparison
| Time | Group of | Exudate level | P value |
| Day 1 | Experimental group | 1.40±0.10 | 0.441 |
| Day 4 | Experimental group | 0.60±0.20 | 0.030 |
| Day 8 | Experimental group | 0.00±0.00 | 0.000 |
| Day 10 | Experimental group | 0.00±0.00 | 0.000 |
TABLE 6 ulcer pain VAS score comparison
| Time | Group of | VAS score | P value |
| Day 1 | Experimental group | 7.00±0.40 | 0.081 |
| Day 4 | Experimental group | 2.50±1.36 | 0.010 |
| Day 8 | Experimental group | 0.00±0.71 | 0.000 |
| Day 10 | Experimental group | 0.00±0.00 | 0.000 |
Comparative example 1
General data:
72 patients with oral ulcer treated by the oral spray are taken as study subjects in 2018, 1-2018, 12-12 months, wherein 41 male patients and 31 female patients are 9-71 years old and the average (41.6 +/-3.0) years old. Inclusion criteria were: firstly, the recurrent oral ulcer is confirmed in clinical diagnosis; ② the diameter of the oral ulcer is less than 8cm, 1-3 oral ulcers are different, and the duration is less than 72 h. Exclusion criteria: patients with oral malignant diseases and immune system diseases are excluded.
The control group patients were sprayed with saline as placebo once each at 5 ml/time in the morning, at mid-night and before bedtime for 4 times. The rest is the same as example 3.
After treatment, the total effective rate of the control group is 66.67%, and the data difference among the groups has statistical significance (P <0.05), and the results are shown in tables 7-10.
TABLE 7 therapeutic Effect of two groups of patients [ n (%) ]
| Group of | Is remarkable in that | Is effective | Invalidation | Total effective rate |
| Control group | 20(55.56) | 4(11.11) | 12(33.33) | 24(66.67) |
| χ2Examination of | 2.130 | 2.144 | 8.881 | 9.116 |
| P value | 0.152 | 0.108 | 0.014 | 0.003 |
TABLE 8 reduction in ulcer size
| Time | Group of | Ulcer size (mm) | P value |
| Day 1 | Control group | 4.80±1.44 | 0.207 |
| Day 4 | Control group | 3.70±0.80 | 0.012 |
| Day 8 | Control group | 2.30±1.45 | 0.000 |
| Day 10 | Control group | 1.80±1.80 | 0.000 |
Table 9 exudate level comparison
| Time | Group of | Exudate level | P value |
| Day 1 | Control group | 1.80±0.30 | 0.441 |
| Day 4 | Control group | 1.50±0.70 | 0.030 |
| Day 8 | Control group | 0.60±0.30 | 0.000 |
| Day 10 | Control group | 0.50±0.22 | 0.000 |
TABLE 10 ulcer pain VAS score comparison
| Time | Group of | VAS score | P value |
| Day 1 | Control group | 6.70±0.70 | 0.081 |
| Day 4 | Control group | 4.10±1.35 | 0.010 |
| Day 8 | Control group | 2.30±0.78 | 0.000 |
| Day 10 | Control group | 0.80±0.80 | 0.000 |
The oral ulcer spray provided by the invention has good treatment effect, is mild and comfortable in use, has no scar after healing, and has a total effective rate of 97.22% for oral ulcer patients.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. The oral ulcer spray is characterized by being prepared from the following components in parts by volume:
80 parts of alcohol extract of the Japanese creeper, 10 parts of dry cell extract, 5 parts of deionized water, 4 parts of preservative and vitamin B20.3 part;
the concentration of exosome protein in the stem cell extract is 30 mg/mL;
the pH value of the oral ulcer spray is 7.0;
the oral ulcer spray is prepared by the following method: mixing the vitamin B2After being dissolved by alkali solutionMixing with ethanol extract of flos Bombacis and deionized water, and sequentially mixing with antiseptic and stem cell extract to obtain oral ulcer spray.
2. The canker sore spray according to claim 1 wherein the alcohol extract of guava is prepared by a process comprising the steps of:
1) mixing flos basjoliae with a first ethanol solution, extracting for 1-2 h, and filtering to obtain a first filtrate and a first filter residue; the first ethanol solution is 60-70% in volume percentage;
2) mixing the first filter residue obtained in the step 1) with a second ethanol solution, extracting for 1-2 h, and filtering to obtain a second filtrate and a second filter residue; the volume percentage content of the second ethanol solution is 55-65%;
3) mixing the second filter residue obtained in the step 2) with a third ethanol solution, extracting for 1-2 h, and filtering to obtain a third filtrate and a third filter residue; the volume percentage content of the third ethanol solution is 55-65%;
4) mixing the first filtrate obtained in the step 1), the second filtrate obtained in the step 2) and the third filtrate obtained in the step 3) to obtain a filtrate, and concentrating the filtrate under reduced pressure to obtain an alcohol extract of the basjoo flower; the volume of the alcohol extract of the guava and the weight ratio of the alcohol extract of the guava to the guava are (1-5): 1.
3. The oral ulcer spray according to claim 2, wherein the mass ratio of the flos bardanae in the step 1) to the first ethanol solution is 1 (15-20).
4. The oral ulcer spray according to claim 2, wherein the mass ratio of the first filter residue to the second ethanol solution in the step 2) is 1 (8-12).
5. The oral ulcer spray according to claim 2, wherein the mass ratio of the second filter residue to the third ethanol solution in the step 3) is 1 (6-8).
6. The canker sore spray according to claim 1, wherein the stem cell extract is prepared by a method comprising the steps of:
a. obtaining the human umbilical cord mesenchymal stem cells and culture supernatant of the human umbilical cord mesenchymal stem cells by adopting a conventional method for culturing the human umbilical cord mesenchymal stem cells; the number of the human umbilical cord mesenchymal stem cells is 3 multiplied by 108A plurality of;
b. mixing the human umbilical cord mesenchymal stem cells with normal saline, carrying out ultrasonic disruption, and collecting exosomes by adopting a conventional method;
c. collecting exosome in culture supernatant of human umbilical cord mesenchymal stem cells by a conventional method;
d. and d, mixing the exosome obtained in the step c, the exosome obtained in the step d and physiological saline to obtain a stem cell extract, wherein the concentration of exosome protein in the stem cell extract is 25-35 mg/mL.
7. The canker sore spray according to claim 6 wherein the exosomes obtained in step c are subjected to BCA protein concentration assay and the concentration of exosome protein is 9.5 mg/ml.
8. The canker sore spray according to claim 6 wherein the exosomes obtained in step d are subjected to BCA protein concentration assay and the concentration of exosome protein is 53.6 mg/ml.
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