CN116531495A - Mouth wash containing lysozyme - Google Patents

Abstract

The invention relates to a mouthwash for preventing or treating dental ulcers, which comprises lysozyme and pulsatilla chinensis extract as active ingredients, wherein the weight ratio of the lysozyme to the pulsatilla chinensis extract is 1:1-1:9. When the pulsatilla root extract and the lysozyme in the mouthwash are combined, the two active ingredients can exert respective effects and simultaneously generate a synergistic effect, so that the oral ulcer, in particular to recurrent oral ulcer, can be obviously prevented or treated. The mouthwash disclosed by the invention is simple in preparation method, safe and nontoxic in active ingredients and free of side effects.

Description

Mouth wash containing lysozyme

Technical Field

The invention relates to the field of medicines, in particular to mouthwash for treating dental ulcers.

Background

In daily life, oral mucosa diseases such as dental pulp diseases, periodontal diseases and dental ulcers are common oral problems, and the cause of these oral diseases is mostly related to bacteria. About 710 more bacteria are commonly found in the oral cavity, wherein about 99% of the total harmful bacteria are present, which can cause inflammatory reactions in the human body and the onset of bacteremia.

Canker sores are the most common ulcerated diseases of the oral mucosa, the etiology mechanism of which is not yet elucidated, and modern medicine considers that pathogenesis is related to local wounds, pressure, food and drug, hormone secretion disorder and vitamin and trace element deficiency. Oral ulcers in traditional Chinese medicine belong to the categories of aphtha, aphtha and the like, and are mostly caused by viscera dysfunction caused by factors such as improper diet, internal injury, seven emotions, tiredness, internal injury, exogenous six exogenous pathogenic factors and the like, and are caused by internal excessive fire or deficient fire fumigating of the mouth and tongue. The onset of canker sores is characterized by ulceration of gums, oral mucosa, throat or tongue, localized burning pain, affecting the speaking and eating of the patient, and even causing insomnia, which brings a lot of inconveniences to the patient.

Recurrent oral ulcers, also known as recurrent aphtha ulcers (recurrent aphthous ulcer, RAU), are chronic inflammatory diseases, and may be induced by factors of local trauma, stress, immune disorders, and gastrointestinal tract. Studies show that the induction factor is related to DNA polymorphism in individual genome, especially IL-1 beta, IL-6, TNF-alpha and other cell factor metabolism. IL-1β is an effective pro-inflammatory factor produced by the innate immune system and is critical for infection and damage to the host, mediating the biological activity of IL-1β, participating in the immunomodulation of the host and mediating the inflammatory response in the body. The host can rapidly and transiently produce IL-6 after infection or tissue injury, stimulates acute phase and immune response, achieves the effect of helping host defense, and has continuous synthesis and important pathological effects on chronic inflammatory diseases and autoimmunity. TNF-alpha has various biological functions and complex mechanism, and can activate vascular endothelial cells, promote blood coagulation, shrink blood vessels, change vascular permeability, up regulate adhesion molecules, cause inflammatory cells to infiltrate, stimulate neutral granules and monocytes, and release various inflammatory mediators.

The daily tooth brushing can only remove about 45% -60% of dental plaque, and the problem is solved by the presence of the mouthwash. The mouthwash has strong fluidity, can clean all parts of the oral cavity, reduces the number of dental plaque and harmful bacteria in the oral cavity, and keeps the oral cavity fresh and odorless. In recent years, mouthwashes are also used as therapeutic aids for treating stomatopathy such as gingivitis, oral mucosa diseases, periodontitis, etc.

The mouthwashes in the market are generally divided into two main categories, western medicine mouthwashes and Chinese herbal medicine mouthwashes. The western medicine mouthwash consists of a plurality of chemical medicines, and adverse reactions such as dry mouth, taste change, staining of teeth and tongue, dysbacteriosis of the oral cavity and the like can be generated after long-term use. The Chinese herbal medicine mouthwash has the advantages of small side effect of active ingredients, higher safety, no adverse reaction to human bodies, mild effect, and capability of well inhibiting the growth of pathogenic microorganisms in the oral cavity and improving the comfort level of the oral cavity of a patient. Thus, people are increasingly concerned with mouthwashes containing Chinese herbal medicines, so as to seek better use degree and safety.

The root of pulsatilla chinensis belonging to family Ranunculaceae is dried. The pulsatilla chinensis is originally loaded in Shennong Ben Cao Jing, has the effects of clearing heat and detoxicating, cooling blood and stopping diarrhea, drying dampness and killing parasites, and is mainly used for treating bacillary dysentery, amebic dysentery, gynecological vaginitis and the like clinically. Modern researches have considered that pulsatilla chinensis has the effects of resisting cancer, killing sperm, treating endotoxemia and the like besides the antibacterial and antiprotozoal effects.

Lysozyme is widely found in tissues and secretions of higher animals, protozoa, insects, plants and various microorganisms. The lysozyme consists of 18 kinds of 129 amino acids, has the molecular weight of 14.6kD, and has the advantages of good biocompatibility, no stimulation to tissues, no toxicity and the like. Lysozyme is an important antibacterial component in human saliva, and is involved in the non-immune defense of a host against bacteria, and has a certain relationship with the regulation of the steady state balance of the oral environment and the resistance to the occurrence and development of oral diseases.

Lysozyme is used as one of the nonspecific immune factors of the organism and participates in the immune reaction of the organism, thereby maintaining the physiological balance of the organism. It can also improve and enhance phagocytic and digestive functions of macrophages, and activate phagocytic functions of leukocytes, thereby enhancing the immunity of the body. Lysozyme also has the function of activating platelets, and can improve local blood circulation of tissues and strengthen local defense function. As the lysozyme has good curative effects on preventing and treating oral diseases, the lysozyme lozenge is marketed in China at present, and the lysozyme has very broad application prospect in oral diseases.

At present, the related report of combining lysozyme and traditional Chinese medicine extracts as medicines for treating canker sore is not seen.

Disclosure of Invention

In order to solve the technical problems, the invention comprises the following aspects:

in a first aspect, the present invention provides a mouthwash for preventing or treating canker sores, the mouthwash comprising lysozyme and pulsatilla chinensis extract as active ingredients in a weight ratio of 1:1 to 1:9.

Preferably, the weight ratio of lysozyme to pulsatilla chinensis extract is 1:2-1:5.

More preferably, the weight ratio of lysozyme to pulsatilla root extract is 1:3.

Preferably, the lysozyme is egg white lysozyme.

Preferably, the weight percentage of the lysozyme in the mouthwash is 0.1-3%; more preferably, the weight percentage of lysozyme in the mouthwash is 0.2-1%; further preferred, the lysozyme is present in the mouthwash in an amount of 0.5% by weight.

Preferably, the weight percentage of the pulsatilla chinensis extract in the mouthwash is 0.5-5%; more preferably, the weight percentage of the pulsatilla root extract in the mouthwash is 1-3%; further preferably, the weight percentage of the pulsatilla chinensis extract in the mouthwash is 1.5%.

Preferably, the preparation method of the pulsatilla chinensis bunge extract comprises the following steps: extracting radix Pulsatillae coarse powder with 3-6 times of organic solvent under reflux for 1-3 times, each time for 1-3 hr, mixing filtrates, standing for 12 hr, recovering organic solvent, concentrating, and drying under reduced pressure to obtain radix Pulsatillae extract.

Preferably, the organic solvent is added in an amount of 5 times by weight in the extraction process, and the extraction is carried out for 2 times each time for 2 hours.

Preferably, the organic solvent is selected from one or more of ethanol aqueous solution, isopropanol and n-butanol.

Preferably, the volume percentage of ethanol in the ethanol aqueous solution is 90%.

Preferably, the organic solvent is isopropanol.

More preferably, the preparation method of the pulsatilla root extract comprises the following steps: adding 5 times of isopropanol into the coarse powder of Chinese pulsatilla root, extracting for 2 times under reflux for 2 hours each time, mixing filtrates, standing for 12 hours, recovering solvent, concentrating, and drying under reduced pressure at 80deg.C.

Preferably, the mouthwash further comprises pharmaceutically acceptable adjuvants.

Preferably, the mouthwash further comprises one or more of a humectant, a flavoring agent, and a solvent. More preferably, the mouthwash further comprises a humectant, a flavoring agent, and a solvent.

Preferably, the weight percentage of the humectant in the mouthwash is 1-10%; more preferably, the humectant is 2-8% by weight of the mouthwash; further preferred, the humectant is present in the mouthwash in an amount of 5% by weight.

Preferably, the weight percentage of the flavoring agent in the mouthwash is 0.01-0.5%; more preferably, the flavoring agent is 0.05-0.2% by weight of the mouthwash; further preferred, the flavoring agent is present in the mouthwash in an amount of 0.1% by weight.

Preferably, the humectant is selected from glycerin or sorbitol.

Preferably, the flavoring agent is selected from peppermint essence or green tea essence.

Preferably, the solvent is deionized water.

Preferably, the mouthwash further comprises sorbitol, peppermint flavor and deionized water.

Preferably, the mouthwash is prepared from the following ingredients in parts by weight per 100 parts by weight: 0.1-3 parts by weight of lysozyme, 0.5-5 parts by weight of pulsatilla chinensis extract, 1-10 parts by weight of humectant, 0.01-0.5 part by weight of flavoring agent and the balance of deionized water.

More preferably, the mouthwash is made of the following ingredients in parts by weight per 100 parts by weight: 0.2-1 part by weight of lysozyme, 1-3 parts by weight of pulsatilla chinensis extract, 2-8 parts by weight of humectant, 0.05-0.2 part by weight of flavoring agent and the balance of deionized water.

Further preferably, the mouthwash is prepared from the following ingredients in parts by weight per 100 parts by weight: 0.5 weight part of lysozyme, 1.5 weight parts of pulsatilla chinensis extract, 5 weight parts of humectant, 0.1 weight part of flavoring agent and the balance of deionized water.

Most preferably, the mouthwash is made of the following ingredients in parts by weight per 100 parts by weight: 0.5 part by weight of lysozyme, 1.5 parts by weight of pulsatilla chinensis extract, 5 parts by weight of sorbitol, 0.1 part by weight of mint essence and the balance of deionized water.

A second aspect of the present invention provides a method of preparing the above mouthwash comprising the steps of:

(1) Adding lysozyme, pulsatilla root extract, humectant and flavoring agent in proper amount in proper solvent in sequence, stirring until the materials are fully dissolved;

(2) Adding the rest solvent to the prescription amount to obtain the final product.

Preferably, the preparation method of the mouthwash comprises the following steps:

(1) Sequentially adding lysozyme, pulsatilla root extract, sorbitol and peppermint essence with a prescription amount into a proper amount of deionized water, and stirring until the lysozyme, the pulsatilla root extract, sorbitol and peppermint essence are fully dissolved;

(2) Adding the rest deionized water to the prescription amount to obtain the product.

In a third aspect, the present invention provides the use of a mouthwash as described above in the manufacture of a medicament for the prevention or treatment of canker sores.

Preferably, the oral ulcer is a recurrent oral ulcer.

In a fourth aspect the invention provides the use of a mouthwash as described above for the manufacture of a medicament for reducing the amount of an inflammatory factor in plasma, the inflammatory factor being selected from one or more of IL-1 beta, IL-6, TNF-alpha.

Preferably, the present invention provides the use of a mouthwash as described above for the manufacture of a medicament for simultaneously reducing the levels of inflammatory factors IL-1 beta, IL-6 and TNF-alpha in plasma.

The invention has the technical effects that:

1. according to the invention, the pulsatilla chinensis extract with good treatment effect on the oral ulcer is obtained by optimizing the extraction process of the Chinese pulsatilla chinensis.

2. According to the invention, when the pulsatilla chinensis extract and lysozyme are combined, the two active ingredients can exert respective effects and simultaneously generate a synergistic effect, so that the oral ulcer, particularly recurrent oral ulcer, can be remarkably prevented or treated.

3. The mouthwash disclosed by the invention is simple in preparation method, and the lysozyme and the pulsatilla chinensis extract serving as active ingredients are safe and nontoxic and have no side effects.

Detailed Description

The present invention will be further described with reference to examples, but the embodiments of the present invention are not limited thereto. The experimental methods used in the following examples are conventional methods unless otherwise specified. The lysozyme used in the examples and test examples of the present invention was egg white lysozyme unless otherwise specified.

Preparation example 1 (isopropanol extract E1)

Adding 5 times of isopropanol into the coarse powder of Chinese pulsatilla root, extracting for 2 times under reflux for 2 hours each time, mixing filtrates, standing for 12 hours, recovering solvent, concentrating, and drying under reduced pressure at 80deg.C to obtain extract E1 of radix Pulsatillae Weng Yi propanol.

Preparation example 2 (90% ethanol extract E2)

Adding ethanol water solution with the ethanol volume percentage content of 90% into the Chinese pulsatilla chinensis coarse powder, performing thermal reflux extraction for 2 times and 2 hours each time, combining the filtrates, standing for 12 hours, recovering the solvent, concentrating, and drying under reduced pressure at 80 ℃ to obtain pulsatilla chinensis ethanol extract E2.

Preparation example 3 (n-butanol extract E3)

Adding 5 times of n-butanol into the coarse powder of radix Pulsatillae, extracting under reflux for 2 hr for 2 times, mixing filtrates, standing for 12 hr, recovering solvent, concentrating, and drying under reduced pressure at 80deg.C to obtain radix Pulsatillae n-butanol extract E3.

Example 1 (mouthwash W1 containing lysozyme and pulsatilla extract)

0.5g of lysozyme, 11.5g of pulsatilla chinensis extract E obtained in preparation example 1, 5g of sorbitol and 0.1g of peppermint essence are sequentially added into 30g of deionized water, stirred until the components are fully dissolved, and the rest of deionized water is added into 100g, so that the mouthwash W1 is obtained.

Example 2 (mouthwash W2 containing lysozyme and pulsatilla extract)

0.5g of lysozyme, 21.5g of pulsatilla chinensis extract E obtained in preparation example 2, 5g of sorbitol and 0.1g of peppermint essence are sequentially added into 30g of deionized water, stirred until the components are fully dissolved, and the rest of deionized water is added into 100g, so that the mouthwash W2 is obtained.

Example 3 (mouthwash W3 containing lysozyme and pulsatilla extract)

0.5g of lysozyme, 31.5g of pulsatilla chinensis extract E, 5g of sorbitol and 0.1g of peppermint essence obtained in preparation example 3 are sequentially added into 30g of deionized water, stirred until the components are fully dissolved, and the rest of deionized water is added into 100g, so that the mouthwash W3 is obtained.

Example 4 (mouthwash W4 containing lysozyme and pulsatilla extract)

1g of lysozyme, 11g of pulsatilla chinensis extract E obtained in preparation example 1, 5g of sorbitol and 0.1g of peppermint essence are sequentially added into 30g of deionized water, stirred until the components are fully dissolved, and the rest of deionized water is added into 100g, so that the mouthwash W4 is obtained.

Example 5 (mouthwash W5 containing lysozyme and pulsatilla extract)

0.2g of lysozyme, 11.8g of pulsatilla chinensis extract E obtained in preparation example 1, 5g of sorbitol and 0.1g of peppermint essence are sequentially added into 30g of deionized water, stirred until the components are fully dissolved, and the rest of deionized water is added into 100g, so that the mouthwash W5 is obtained.

Comparative example 1 (mouthwash D1 containing lysozyme only)

2g of lysozyme, 5g of sorbitol and 0.1g of mint essence are sequentially added into 30g of deionized water, stirring is carried out until the lysozyme, the sorbitol and the mint essence are fully dissolved, and the rest deionized water is added into 100g, so that the comparative mouthwash D1 is obtained.

Comparative example 2 (mouthwash D2 containing pulsatilla root extract alone)

And (3) sequentially adding 12g of the pulsatilla chinensis extract E, 5g of sorbitol and 0.1g of mint essence obtained in preparation example 1 into 30g of deionized water, stirring until the components are fully dissolved, and adding the rest of deionized water to 100g to obtain the comparative mouthwash D2.

Test example 1 therapeutic Effect of the mouthwash of the present invention on recurrent aphtha in rats

1. Test method

After 1 week of adaptive feeding, 80 SD rats were randomly divided into model, inventive examples 1-5 and comparative examples 1-2, 10 rats per group, each test group was anesthetized with 1% sodium pentobarbital (50 mg/kg) and fixed, and after drying the right cheek mucosa with sterile cotton balls, the right cheek mucosa was isolated with sterile cotton balls, and the rats were subjected to 9mm humidification ² Immersing square filter paper with a size into 50% glacial acetic acid for 5s, rapidly placing the filter paper in cleaned cheek mucosa, waiting for about 1min to remove the filter paper, wiping with physiological saline sterile cotton balls, and removing redundant glacial acetic acid; after 24 hours, the rat was observed for ulceration. The ulcer wound surface model is successfully formed in the mouth of the rat, the diameter is about 5-6mm, the surface is covered with a false membrane, the center is concave, the edge of the ulcer is red and swollen, and the stinging response is obvious.

The mouthwashes prepared in examples 1-5 and comparative examples 1-2 were administered by gavage at a dose of 10g of mouthwash per kg of body weight in the inventive and comparative examples 1-5 and comparative examples 1-2, respectively, and an equivalent amount of physiological saline was administered by gavage in the model group. Each test group was dosed 1 time daily for 10 days.

After the end of the administration, after the rats were anesthetized with 1% sodium pentobarbital (50 mg/kg), the negative pressure tube heart was taken about 5mL of blood, which was injected into the EP tube, allowed to stand at 4℃for 4 hours, and centrifuged at 3000r/min for 10min in a high-speed centrifuge at 4 ℃. After centrifugation, the supernatant was lifted using a pipette and dispensed into enzyme-free EP tubes, and ELISA procedures were performed according to the instructions to determine the effect of the drug in each test group on the levels of inflammatory factors IL-1 beta, IL-6 and TNF-alpha in serum of RAU rats.

2. Test results

The effect of mouthwashes prepared in accordance with various examples of the present invention on the levels of inflammatory factors (IL-1. Beta., IL-6 and TNF-. Alpha.) in serum of RAU rats is shown in Table 1 below.

TABLE 1 Effect of the mouthwashes of the present invention on the amount of inflammatory factors in serum of RAU rats

Compared with rats in a model group, the serum IL-1 beta, IL-6 and TNF-alpha contents of the rats in the examples 1-5 and the comparative examples 1-2 are obviously reduced, and the mouthwashes of the examples and the comparative examples have different degrees of pro-inflammatory factor regulation and control effects, so that the healing of ulcer wound surfaces of RAU rats is promoted.

In addition, the reduction in IL-1. Beta., IL-6 and TNF-. Alpha.levels in the serum of rats in the groups of examples 1-5 was more pronounced (the differences were statistically significant) compared to the groups of comparative examples 1-2, resulting in a pronounced pro-inflammatory factor modulating effect. Namely, under the condition that the total amount of the active ingredients is the same, the pro-inflammatory factor regulation effect of the combination of lysozyme and pulsatilla chinensis extracts in the mouthwash of the examples 1-5 is obviously better than that of the mouthwash of the single active ingredient (lysozyme or pulsatilla chinensis extracts) in the comparative examples 1-2, and the combination of lysozyme and pulsatilla chinensis extracts is proved to produce a synergistic effect in preventing or treating canker sore. Wherein the IL-1 beta, IL-6 and TNF-alpha levels in the serum of the group of rats of example 1 were down-regulated to the maximum extent, resulting in unexpected excellent technical effects. The above test results indicate that the mouthwash of the present invention is clinically useful for preventing or treating canker sores, particularly recurrent canker sores.

Although specific embodiments of the invention have been described, those skilled in the art will recognize that many changes and modifications may be made thereto without departing from the scope or spirit of the invention. Accordingly, the present invention is intended to embrace all such alterations and modifications that fall within the scope of the appended claims and equivalents thereof.

Claims (10)

1. A mouthwash for preventing or treating canker sores, characterized in that the mouthwash comprises lysozyme and pulsatilla chinensis extract as active ingredients in a weight ratio of 1:1 to 1:9.

2. The mouthwash of claim 1, wherein the weight ratio of lysozyme to pulsatilla extract is from 1:2 to 1:5.

3. The mouthwash of claim 2, wherein the weight ratio of lysozyme to pulsatilla extract is 1:3.

4. The mouthwash of claim 1, wherein the pulsatilla extract is prepared by the following method: extracting radix Pulsatillae coarse powder with 3-6 times of organic solvent under reflux for 1-3 times, each time for 1-3 hr, mixing filtrates, standing for 12 hr, recovering organic solvent, concentrating, and drying under reduced pressure to obtain radix Pulsatillae extract.

5. The mouthwash of claim 4, wherein the organic solvent in the pulsatilla extract preparation method is selected from one or more of an aqueous ethanol solution, isopropyl alcohol and n-butanol.

6. The mouthwash of any of claims 1 to 5, further comprising one or more of a humectant, a flavoring agent, and a solvent.

7. The mouthwash of claim 6, wherein the mouthwash is prepared from the following ingredients in parts by weight per 100 parts by weight: 0.1-3 parts by weight of lysozyme, 0.5-5 parts by weight of pulsatilla chinensis extract, 1-10 parts by weight of humectant, 0.01-0.5 part by weight of flavoring agent and the balance of deionized water.

8. The mouthwash of claim 7, wherein the mouthwash is prepared from the following ingredients in parts by weight per 100 parts by weight: 0.5 part by weight of lysozyme, 1.5 parts by weight of pulsatilla chinensis extract, 5 parts by weight of sorbitol, 0.1 part by weight of mint essence and the balance of deionized water.

9. A method of preparing a mouthwash according to any one of claims 1 to 8, comprising the steps of:

(1) Adding lysozyme, pulsatilla root extract, humectant and flavoring agent in proper amount in proper solvent in sequence, stirring until the materials are fully dissolved;

(2) Adding the rest solvent to the prescription amount to obtain the final product.

10. Use of a mouthwash according to any one of claims 1 to 8 in the manufacture of a medicament for the prevention or treatment of canker sores.