New application of artemisinin compound

Abstract

The invention relates to the technical field of oral medicines, and particularly discloses a novel application of an artemisinin compound, and the artemisinin compound is composed of artemisinic acid and dihydroartemisinic acid. The concentration of the arteannuic acid is 50 to 400 mg / L; the concentration of the dihydroarteannuic acid is 50 to 400 mg / L. The arteannuic acid and the dihydroarteannuic acid in the artemisinin compound can effectively inhibit cariogenic toxicity factors of candida albicans and streptococcus mutans, can inhibit formation of single-strain and double-strain biological membranes, can inhibit acid production of the biological membranes and formation of extracellular polysaccharides, and can reduce the number of viable bacteria in the biological membranes.

Description

Technical Field

[0001] The present invention relates to the technical field of oral drugs, and specifically to a new application of artemisinin compounds. Background Art

[0002] Dental caries is a disease in which the hard tissues of teeth are chronically and progressively damaged under the influence of multiple factors mainly microbial infection, and dental plaque biofilm is the initiating factor for the onset of dental caries. The fourth national oral health epidemiological survey in 2018 showed that the prevalence of dental caries in primary teeth of 5-year-old children was as high as 70.9%, and the prevalence of root caries in permanent teeth of the elderly aged 65-74 was as high as 61.9%, showing an increasing trend year by year. [1] . Streptococcus mutans ( Streptococcus mutans , S. mutans ) is the main cariogenic bacterium, which secretes glucosyltransferase (Gtf) to produce extracellular polysaccharide (EPS) using sucrose, thereby adhering to the tooth surface to form dental plaque biofilm, metabolizing carbohydrates to produce acid, causing tooth demineralization, and leading to the occurrence of dental caries. Candida albicans ( Candida albicans , C. albicans ) is the most common opportunistic pathogenic fungus in the oral cavity, including yeast phase and hyphal phase, which can cause oral mucosal candidiasis; Candida albicans has a weak adhesion ability to the tooth surface, but can interact with a variety of cariogenic bacteria to promote the occurrence and development of dental caries. In early childhood caries and root caries, the co-detection rate of Candida albicans and Streptococcus mutans is high, and the two have strong synergistic cariogenic ability in vivo. The specific mechanism can be manifested as follows: Streptococcus mutans secretes Gtf, which binds to Candida albicans and promotes its adhesion to the tooth surface; Candida albicans can promote the expression of the Gtf gene of Streptococcus mutans, increase the production of EPS, and further enhance the adhesion ability of the biofilm; the dual-species biofilm can promote the colonization of Streptococcus mutans and Candida albicans, enhance the cariogenic virulence; the hyphae of Candida albicans can promote the cariogenicity of Streptococcus mutans.

[0003] Drug therapy is one of the main ways for the treatment of early caries. Fluoride is the most commonly used anti-caries drug, and its anti-caries efficacy has been fully affirmed. However, improper use can lead to the occurrence of dental fluorosis and the emergence of fluoride-resistant strains. Antibiotic drugs are also one of the more common anti-caries drugs, but long-term application can lead to bacterial resistance and seriously disrupt the oral microecological balance. Therefore, the development of safer and more effective anti-caries drugs is the focus of caries prevention and treatment research. Natural drugs have received extensive attention due to their wide sources and low toxic and side effects. Many natural drugs such as Magnolia officinalis, Chinese gallnut, Nidus Vespae, and tea polyphenols can achieve anti-caries effects by inhibiting the growth and virulence factors of cariogenic microorganisms. However, current research on the anti-caries effects of natural drugs is mostly limited to single cariogenic bacteria and biofilms, and there is still a lack of drugs that can effectively inhibit the cross-kingdom interaction and synergistic caries-causing effects of Candida albicans and Streptococcus mutans. Summary of the Invention

[0004] The purpose of the present invention is to address the deficiencies of the prior art and provide a new application of artemisinin compounds to solve the problems raised in the above background art.

[0005] To achieve the above purpose, the present invention provides the following technical solution: The application of artemisinin compounds in the preparation of drugs for the treatment and / or prevention of caries, wherein the artemisinin compounds are composed of artemisinic acid and dihydroartemisinic acid; the concentration of artemisinic acid is 50 - 400 mg / L; the concentration of dihydroartemisinic acid is 50 - 400 mg / L.

[0006] Further, the concentration of artemisinic acid is 100 - 400 mg / L; the concentration of dihydroartemisinic acid is 100 - 400 mg / L.

[0007] Further, the preparation method of the artemisinin compounds: Dissolve artemisinic acid and dihydroartemisinic acid in DMSO, and dilute with sterile deionized water to 40 g / L, and store at 4°C.

[0008] Further, the caries is caused by Streptococcus mutans and / or Candida albicans.

[0009] Further, the drug is a drug containing an effective dose of artemisinin compounds and pharmaceutically acceptable excipients.

[0010] Further, the drug is a tablet, powder, gargle, spray, or gel.

[0011] Further, the tablet is an oral tablet.

[0012] Application of artemisinin compounds in preparing oral care products for improving dental caries, wherein the artemisinin compounds are composed of artemisinic acid and dihydroartemisinic acid; the concentration of artemisinic acid is 50 - 400 mg / L; the concentration of dihydroartemisinic acid is 50 - 400 mg / L.

[0013] Furthermore, the oral care products contain artemisinin compounds and excipients acceptable in oral care products.

[0014] Furthermore, the oral care products are toothpaste, chewing gum or mouthwash Compared with the prior art, the beneficial effects of the present invention are as follows: Artemisinic acid and dihydroartemisinic acid in the artemisinin compounds of the present invention can effectively inhibit the cariogenic virulence factors of Candida albicans and Streptococcus mutans, inhibit the formation of single-species and dual-strain biofilms, inhibit biofilm acid production and extracellular polysaccharide formation, and reduce the number of viable bacteria in the biofilm. In a rat dental caries model, the colonization of Streptococcus mutans and Candida albicans in the dental plaque of rats was reduced, the incidence of dental caries in rats was decreased, the severity of dental caries in rats was alleviated, and the present invention has a good preventive and therapeutic effect on rat dental caries. Therefore, artemisinic acid and dihydroartemisinic acid can block the cross-kingdom interaction between Candida albicans and Streptococcus mutans and effectively inhibit the synergistic cariogenic ability of Candida albicans and Streptococcus mutans, which indicates that it has good application prospects in the prevention and treatment of dental caries, especially in the prevention and treatment of early childhood caries and root caries. Description of the Drawings

[0015] Figure 1 It is a diagram of the experimental results of the inhibition of biofilm formation by the artemisinin compounds of the present invention; S.m : Streptococcus mutans; C.a : Candida albicans; (* P < 0.05; ** P < 0.01; *** P < 0.001) Figure 2 It is a statistical result diagram of the reduction of the number of viable bacteria in the biofilm by the artemisinin compounds of the present invention; (A) Colonization amount of Candida albicans in single-species and dual-strain biofilms; (B) Colonization amount of Streptococcus mutans in single-species and dual-strain biofilms. (* P < 0.05; ** P < 0.01) Figure 3 It is a diagram of the experimental results of the inhibition of biofilm acid production by the artemisinin compounds of the present invention; S.m: Streptococcus mutans; C.a: Candida albicans. (*P < 0.05; **P < 0.01) Figure 4This is the experimental result graph of artemisinin compounds of the present invention inhibiting the formation of biofilm EPS; S.m: Streptococcus mutans; C.a: Candida albicans. (*P < 0.05; **P < 0.01); Figure 5 This is the statistical result graph of the colonization amounts of Streptococcus mutans and Candida albicans in the dental plaque of rats in the present invention; S.m : Streptococcus mutans; C.a : Candida albicans. (** P <0.01); Figure 6 This is the statistical result graph of the caries lesion scores of rats in the present invention; S.m : Streptococcus mutans; C.a : Candida albicans. (* P <0.05); The E-level lesion is limited within the enamel; The Ds-level lesion involves about 1 / 4 of the dentin between the enamel and the pulp chamber; The Dm-level lesion involves about 1 / 4 - 3 / 4 of the dentin; The Dx-level involves more than 3 / 4 of the dentin area. Detailed implementation manners

[0016] The following elaborates on the preferred embodiments of the present invention in conjunction with the attached drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making the protection scope of the present invention more clearly defined.

[0017] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0018] Streptococcus mutans: Serial number ATCC 700610, purchased from the American Type Culture Collection (ATCC).

[0019] Candida albicans: Serial number ATCC MYA - 2876, purchased from the American Type Culture Collection (ATCC).

[0020] Example 1: Preparation and preservation of artemisinin compounds Artemisinin compounds (artemisinic acid, dihydroartemisinic acid) were purchased from Solarbio Science & Technology Co., Ltd. in Beijing, China, dissolved in DMSO, and diluted with sterile deionized water to 40 g / L, and stored at low temperature of 4°C.

[0021] Example 2: Artemisinin compounds inhibiting biofilm formation The amount of biofilm formation was evaluated by the crystal violet staining experiment of biofilm to explore the inhibitory effect of artemisinin compounds on the growth of single-species and dual-species biofilms of Candida albicans and Streptococcus mutans. A single colony of Streptococcus mutans was picked and inoculated into BHI liquid medium, cultured overnight in a 37 °C incubator, centrifuged at 4 °C, the supernatant was removed, washed with PBS, resuspended in YNBB, and the bacterial concentration was adjusted to 2×10 6 CFU / mL. A single colony of Candida albicans was picked and inoculated into YPD liquid medium, cultured in a 37 °C incubator for 24 hours, centrifuged at 4 °C, the supernatant was discarded, washed with PBS, resuspended in YNBB, and the bacterial concentration was adjusted to 2×10 4 CFU / mL. Each drug treatment group was divided into a single-species biofilm group of Candida albicans, a single-species biofilm group of Streptococcus mutans, and a dual-species biofilm group of Candida albicans - Streptococcus mutans. For the dual-species biofilm experimental group, 50 μL of Candida albicans and Streptococcus mutans bacterial solutions were added to a 96-well plate respectively; for the single-species biofilm experimental group, 100 μL of Candida albicans or Streptococcus mutans bacterial solution was added. Different mass concentration groups of artemisinin compounds (400 mg / L, 200 mg / L, 100 mg / L, 50 mg / L) and a negative control group (DMSO) were set, and each group was repeated with 3 wells. The 96-well plate was cultured at 37 °C and 5% CO2 for 24 h, the liquid in the wells was discarded, the biofilm was gently washed 3 times with PBS, fixed with absolute methanol for 15 min, dried at room temperature, 100 μL of 0.1% crystal violet solution was added to each well for staining for 15 min, then the staining solution was aspirated, the remaining dye was gently washed away with PBS, dried, 200 μL of absolute ethanol was added to each well and shaken at room temperature for 20 min, 100 μL of the eluate was aspirated and transferred to a new 96-well plate, and the absorbance value was measured at a wavelength of 595 nm using a microplate reader.

[0022] The results are as Figure 1 shown. For the single-species biofilm of Candida albicans, the absorbance values of all concentration drug treatment groups were lower than those of the control group, indicating that artemisinin compounds at 50 - 400 mg / L could significantly inhibit the formation of Candida albicans biofilm, and this inhibitory effect was concentration-dependent. For the single-species biofilm of Streptococcus mutans, artemisinic acid could effectively reduce the amount of biofilm formation in the concentration range of 50 - 400 mg / L. Dihydroartemisinic acid could effectively reduce the amount of biofilm formation in the concentration range of 100 - 400 mg / L. For the dual-species biofilm of Candida albicans - Streptococcus mutans, both drugs could significantly reduce the absorbance value and inhibit biofilm formation at all concentrations.

[0023] Example 3: Artemisinin compounds reduce the number of viable bacteria in biofilms The methods of Example 2 were used to culture single-species and dual-species biofilms of Candida albicans and Streptococcus mutans. After culturing the single-species and dual-species biofilms for 24 h, the 96-well plates were taken out, the supernatant was aspirated and discarded, the biofilms were gently washed 3 times with sterile PBS, the biofilms at the bottom of the plates were scraped off with a sterile pipette tip, transferred to a 1.5-ml sterile centrifuge tube, 1 ml of sterile PBS was added, sonicated for 5 minutes, gradient diluted with PBS, 100 μL of the bacterial solution was taken and spread on CHROMagar medium and MSB plates respectively, and 3 parallel groups were set for each group. The plates were incubated at 37 °C for 24 h, and the colony counts of Candida albicans and Streptococcus mutans were performed respectively.

[0024] The results were as Figure 2 shown that both drugs at concentrations of 50 - 400 mg / L could reduce the colonization amount of Candida albicans in single-species and dual-species biofilms. At the same time, both drugs at concentrations of 50 - 400 mg / L could also reduce the colonization amount of Streptococcus mutans in single-species and dual-species biofilms.

[0025] Example 4: Inhibition of acid production by artemisinin compounds The methods of Example 2 were used to adjust the concentration of Streptococcus mutans bacterial solution to 2×10 6 CFU / mL and the concentration of Candida albicans bacterial solution to 2×10 4 CFU / mL. In the dual-species biofilm experimental group, 1 ml of Candida albicans and Streptococcus mutans bacterial solutions were added to each well of the 12-well plate; in the single-species biofilm experimental group, 2 ml of Candida albicans or Streptococcus mutans bacterial solution was added. Different concentration groups of artemisinin compounds (400 mg / L, 200 mg / L, 100 mg / L, 50 mg / L) and a negative control group (DMSO) were set, and 3 wells were repeated for each group. After culturing the biofilms for 24 h, the supernatant was discarded, the biofilms were gently washed 3 times with PBS, fresh YNBB medium was added, and after 24 h, the pH values of the supernatants of each group were detected using a pH meter.

[0026] The results were as Figure 3 shown that the Candida albicans biofilm group had only a relatively weak acid production ability, and there was no significant difference in the pH value of the supernatant of the drug-treated group and the control group. In the Streptococcus mutans biofilm and the Candida albicans - Streptococcus mutans dual-species biofilm, the pH values of the supernatants of the negative control groups without drugs were acidic. Artesunic acid could significantly inhibit the acid production ability of Streptococcus mutans biofilm and Candida albicans - Streptococcus mutans dual-species biofilm in the concentration range of 100 - 400 mg / L, and this inhibitory effect was concentration-dependent. Dihydroartemisinin could significantly inhibit the acid production ability of Streptococcus mutans biofilm and Candida albicans - Streptococcus mutans dual-species biofilm in the concentration range of 50 - 400 mg / L, and this inhibitory effect was concentration-dependent.

[0027] Example 5: Artemisinin Compounds Inhibit the Formation of Biofilm EPS The sulfuric acid - anthrone method was used to detect the effect of artemisinin compounds on the formation of water - insoluble EPS in Candida albicans - Streptococcus mutans biofilms. Anthrone powder was added to concentrated sulfuric acid and mixed evenly to make the mass concentration of anthrone 0.2%. According to the method of Example 2, 24 - hour biofilms were prepared using a 96 - well plate. The supernatant was discarded, and the biofilms were gently rinsed twice. The biofilms in the wells were scraped off with a pipette tip, resuspended in PBS, transferred to a 1.5 - ml centrifuge tube, centrifuged, and the supernatant was discarded. The precipitate was gently rinsed twice with PBS. 300 μL of 0.4 mol / L NaOH and 900 μL of 0.2% anthrone solution were added to the precipitate, and it was incubated in a 95°C water bath for 10 min. 100 μL of the liquid from each well was transferred to another 96 - well plate, and the absorbance value was measured at 625 nm using a microplate reader.

[0028] The results are as Figure 4 shown. For the single - species biofilm of Candida albicans, at concentrations of 50 - 400 mg / L, both artemisinin compounds reduced the absorbance value of the experimental group, significantly inhibiting the production of biofilm EPS. For the single - species biofilm of Streptococcus mutans, both artemisinin compounds at 50 - 400 mg / L could significantly inhibit the production of biofilm EPS. For the dual - species biofilm, both artemisinin compounds could significantly inhibit the formation of biofilm EPS at concentrations of 100 - 400 mg / L.

[0029] Example 6: Artemisinin Compounds Prevent and Treat Dental Caries in Rats A dental caries model was constructed by inoculating Candida albicans, Streptococcus mutans or a mixed bacterial solution of both into the oral cavity of rats and a high - sugar diet, and the preventive and therapeutic effects of artemisinin compounds on dental caries in rats were explored. The specific experimental methods are as follows: 1. Preparation of bacterial suspension: (1) Candida albicans: A single colony of Candida albicans was picked from a YPD solid medium and cultured overnight in a YPD liquid medium at 37°C and 5% CO2. After centrifugation, the supernatant was discarded, and the bacterial suspension was resuspended in PBS and adjusted to a concentration of 5×10 4 CFU / mL. (2) Streptococcus mutans: A single colony of Streptococcus mutans was picked from a BHI solid medium and cultured overnight in a BHI liquid medium at 37°C and 5% CO2. After centrifugation, the supernatant was discarded, and the bacterial suspension was resuspended in PBS and adjusted to a concentration of 5×10 6 CFU / mL. (3) Candida albicans + Streptococcus mutans: Equal volumes of 5×10 4 CFU / mL Candida albicans and 5×10 6 CFU / mL Streptococcus mutans were mixed evenly.

[0030] 2. Construction of the rat dental caries model: 2.1 Establishment of animal model A total of 63 female Sprague-Dawley (SD) rats cultured for 21 days were used. From day 22 to 24, the rats were fed with normal feed, and 0.1% ampicillin, 0.1% carbenicillin, and 0.1% chloramphenicol were added to the drinking water to inhibit bacteria. At day 24, it was determined that they were not infected with Candida albicans and Streptococcus mutans by swabbing the oral cavity on selective medium. At day 25, they were rinsed with sterile deionized water. From day 26 to 30, oral inoculation was performed with the bacterial solution. The rats were randomly divided into three major groups: the group infected with Candida albicans alone (1 ml / rat), the group infected with Streptococcus mutans alone (1 ml / rat), and the group co-infected with Candida albicans + Streptococcus mutans (1 ml / rat). Each major group was further divided into 2 drug experimental groups (400 mg / L artemisinic acid / dihydroartemisinic acid) and a vehicle control group (n = 7). Ear tags were given to the rats and their body weights were recorded. From day 30 to 31, rat saliva (100 μl / rat) was collected to detect the colonization of Candida albicans and Streptococcus mutans. From day 26 until the end of the experiment, the rats were fed with Diet 2000 cariogenic model feed and 5% sucrose aqueous solution.

[0031] 2.2 Oral treatment with drugs Starting from day 31 of the rat age, 400 mg / L artemisinic acid, dihydroartemisinic acid, and vehicle control (0.1% DMSO) were used to clean the rats' oral cavities. Take 1.5 ml of each drug solution and dispense it into 2 ml EP tubes, one tube for each rat. Dip a sterile cotton swab into the experimental drug solution until it is completely saturated, and then wipe the rats' teeth and various parts of the oral mucosa in sequence. The remaining drug solution in the EP tube was used to rinse the rats' oral cavities with a 5 ml blunt syringe. The rats were fasted for 1 hour after treatment. This treatment was carried out continuously for 5 weeks, once in the morning and once in the evening. The body weights of the rats were measured once a week, and the feeding, activities, and oral mucosa health conditions of each rat were observed every day.

[0032] 2.3 Specimen collection At day 66, the rats were sacrificed with anhydrous ether, and the mandible specimens of the rats were taken using sterile surgical instruments. The mandible samples of each rat were placed into 5 ml of sterile physiological saline respectively, and ultrasonic oscillation was performed for 10 minutes to remove the plaque biofilm. The obtained suspension was serially diluted with sterile physiological saline. 100 μL of the liquid diluted by different multiples was evenly spread on the MSB solid medium and CHROMagar medium using a sterile spreading rod. Colony counting was performed after culturing at 37 °C for 24 hours, and 3 replicates were set for each sample. The mandibles were washed, dried, stained with 0.4% murexide solution for 12 hours, rinsed and dried, and then sagittally sectioned along the mesial-distal direction of the mandibular molars to prepare mandibular molar specimens.

[0033] 2.4 Cariogenic score The caries situation of rat molars was observed under a stereomicroscope. According to the Keyes scoring method, the degree of caries was divided into enamel layer (E grade), superficial dentin layer (Ds grade), middle dentin layer (Dm grade), and deep dentin layer (Dx grade), and the occlusal fissure caries lesions of rat molars were scored. The scores of each grade of each molar of the rats were added up respectively for statistical analysis. Since the caries lesion starts from the enamel area and gradually develops into the dentin area, the E grade score represents the incidence of caries, and the Ds, Dm, and Dx scores represent the severity of caries.

[0034] The experimental results are as follows: 1. Artemisinin compounds reduce the colonization of Candida albicans and Streptococcus mutans After diluting and plating the rat dental plaque biofilm, CFU counting was performed on Streptococcus mutans and Candida albicans respectively. The results are as Figure 5 shown. In the group infected with Streptococcus mutans alone and the group co-infected with Candida albicans - Streptococcus mutans, artemisinic acid and dihydroartemisinic acid can significantly reduce the colonization amount of Streptococcus mutans. In the group infected with Candida albicans alone and the group co-infected with Candida albicans - Streptococcus mutans, artemisinic acid and dihydroartemisinic acid can significantly reduce the colonization amount of Candida albicans.

[0035] 2. Artemisinin compounds alleviate the degree of rat caries lesions The longitudinal sections of rat molars were observed under a stereomicroscope. The Keyes scoring results of caries are as Figure 6 shown. Caries occurred in rats in the Candida albicans infection group, Streptococcus mutans infection group, and Candida albicans - Streptococcus mutans co-infection group. The two artemisinin compounds have good inhibitory effects on the formation and severity of rat occlusal fissure caries. In rats infected with Streptococcus mutans, artemisinic acid and dihydroartemisinic acid can reduce the caries scores of E grade, Ds grade, Dm grade, and Dx grade in rats. In rats infected with Candida albicans, the caries scores of E grade, Ds grade, and Dm grade in the artemisinic acid and dihydroartemisinic acid treatment groups were significantly lower than those in the control group. In rats co-infected with Candida albicans - Streptococcus mutans, the scores of E grade, Ds grade, Dm grade, and Dx grade in the artemisinic acid and dihydroartemisinic acid treatment groups were significantly reduced compared with the control group. In summary, the two artemisinin compounds have good preventive and therapeutic effects on rat caries and show obvious anti-caries efficacy.

[0036] The above embodiments only express the implementation modes of the present invention, and the description is relatively specific and detailed, but it should not be understood as a limitation to the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several deformations and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. Use of artemisinin compounds in the preparation of a medicament for treating and / or preventing dental caries, characterized in that, The artemisinin compound is composed of artemisinic acid and dihydroartemisinic acid; the concentration of artemisinic acid is 50 - 400 mg / L; the concentration of dihydroartemisinic acid is 50 - 400 mg / L.

2. The application according to claim 1, characterized in that: The concentration of artemisinic acid is 100 - 400 mg / L; the concentration of dihydroartemisinic acid is 100 - 400 mg / L.

3. The application according to claim 2, wherein: The preparation method of the artemisinin compound: Dissolve artemisinic acid and dihydroartemisinic acid in DMSO, and dilute with sterile deionized water to 40 g / L, and store at 4 °C.

4. The application according to claim 1, characterized in that: The dental caries is dental caries caused by Streptococcus mutans and / or Candida albicans.

5. The application according to claim 1, wherein: The drug contains an effective dose of artemisinin compound and pharmaceutically acceptable excipients.

6. The application according to claim 1, characterized in that: The drug is tablets, powders, mouthwashes, sprays, gels.

7. The application according to claim 6, wherein: The tablets are oral tablets.

8. Use of artemisinin compounds in the preparation of oral care products for improving dental caries, characterized in that, The artemisinin compound is composed of artemisinic acid and dihydroartemisinic acid; the concentration of artemisinic acid is 50 - 400 mg / L; the concentration of dihydroartemisinic acid is 50 - 400 mg / L.

9. The application according to claim 8, wherein: The oral care product contains artemisinin compound and excipients acceptable for oral care products.

10. The application according to claim 8, characterized in that: The oral care product is toothpaste, chewing gum or mouthwash.

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