A volatile oil from Clausena lansium seeds for treating dental caries, its extraction method, analysis method and application

Abstract

The present invention discloses a volatile oil from Clausena lansium seeds for treating dental caries. The present invention also discloses an extraction method, an analysis method of the volatile oil from Clausena lansium seeds and its applications in the preparation of anti-caries drugs, the preparation of anti-Streptococcus mutans drugs, and the preparation of anti-dental biofilm drugs. The present invention makes full use of Clausena lansium seeds which are rich in resources and mostly discarded. The volatile oil from Clausena lansium seeds is extracted by a volatile oil analyzer, and its components are analyzed by GC-MS. The paper disk diffusion method and the microdilution method are used to determine that it has a certain inhibitory effect on Streptococcus mutans. The XTT method confirms that the volatile oil from Clausena lansium seeds has the effect of anti-biofilm formation. This provides a feasibility for the current difficult problem of dental caries treatment. Natural products have the advantages of being effective, safe, and not easily developing drug resistance, etc., providing an experimental basis for their research as natural anti-caries drugs.

Description

Technical Field

[0001] The present invention relates to a volatile oil from Clausena lansium seeds for treating dental caries, and its extraction method, analysis method and application, belonging to the technical field of volatile oils. Background Art

[0002] The oral microecology maintains a dynamic balance under normal physiological conditions. When the balance is disrupted, some normal microorganisms in the oral cavity will turn into pathogenic microorganisms, leading to the occurrence of related oral diseases. Dental caries is a multi-factorial disease in the oral cavity and is listed as one of the three major chronic non-communicable diseases that need to be key-prevented and treated together with cardiovascular diseases and cancers. Although the course of dental caries develops relatively slowly, its high incidence rate and wide prevalence area cannot be ignored. Streptococcus mutans is currently recognized as the main pathogenic bacterium causing dental caries. It often adheres to the tooth surface to form a biofilm, and the dental plaque biofilm is the causative factor for the occurrence of dental caries. After its formation, it can bind to the receptors on the bacterial surface, resist the penetration of drugs, and can also enhance the adhesion of microorganisms to tooth enamel.

[0003] Aiming at the role and mechanism of causing dental caries, using drugs to control the number of Streptococcus mutans and inhibit the formation of biofilms is of great significance for the prevention and treatment of dental caries. At present, the use of antibiotics, fluorides, and immunoprophylaxis against dental caries has been proven to effectively inhibit cariogenic bacteria in the oral cavity. However, long-term use of antibiotics is likely to cause dysbacteriosis and the emergence of drug-resistant strains. Fluorides have problems such as toxic side effects and mucosal irritation, and immunoprophylaxis against dental caries faces problems of enhancing immunogenicity and safety. Therefore, finding effective, safe, and long-term usable natural anti-caries drugs has become the research focus for preventing and treating dental caries.

[0004] Clausena lansium (Lour.) Skeels is a plant of the genus Clausena in the Rutaceae family and is widely distributed in tropical and subtropical regions of the Eastern Hemisphere. In China, Clausena lansium is mainly distributed in Hainan, Guangxi, Yunnan, Fujian, Guangdong, Taiwan and other regions. Clausena lansium mainly contains volatile oils, alkaloids, coumarins, flavonoids and other components, and has a wide range of pharmacological activities, with effects such as promoting qi flow, relieving stagnation, relieving exterior syndrome, dissipating heat, relieving pain, and resolving phlegm. All parts of Clausena lansium can be used as medicine, but their effects are different, which is directly related to the different chemical components in different parts. Among them, the volatile components also have obvious differences. With the development of various new analysis and research technologies, in addition, the pulp of Clausena lansium can be eaten fresh or made into preserved fruits or jams for consumption, but its seeds account for a high mass in the fruit and are by-products of pulp processing, which is both a waste of resources and environmental pollution. How to utilize waste to achieve its maximum application value has become a hot topic of concern. Summary of the Invention

[0005] The object of the present invention is to provide a volatile oil from Clausena lansium seeds for treating dental caries, and this volatile oil has strong activity against Streptococcus mutans.

[0006] Meanwhile, the present invention provides a method for extracting the volatile oil from Clausena lansium seeds for treating dental caries. This method has a high yield of volatile oil, is simple and easy to operate, and the obtained volatile oil has strong activities against Streptococcus mutans and dental caries.

[0007] Meanwhile, the present invention provides an analytical method for the volatile oil from Clausena lansium seeds for treating dental caries. This method has a short analysis time, good resolution, and accurate and reliable analysis results.

[0008] Meanwhile, the present invention provides a new application of the volatile oil from Clausena lansium seeds for treating dental caries.

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[0010] A volatile oil from Clausena lansium seeds for treating dental caries, comprising the following components:

[0011]

[0012]

[0013]

[0014] A method for extracting the volatile oil from Clausena lansium seeds for treating dental caries, comprising the following steps: collecting Clausena lansium fruits, collecting Clausena lansium seeds, removing the pulp, washing the Clausena lansium seeds with distilled water, and drying them in an oven; weighing after pulverization, soaking with distilled water at a material ratio of 1:8 for at least 5 h, extracting with a volatile oil extractor for at least 3 h, stopping heating and standing for at least 1 h, and then collecting the volatile oil. The obtained volatile oil is white and transparent in color, and the oil yield is 0.69 ± 0.05%; sealing the obtained volatile oil and storing it in a refrigerator for standby.

[0015] The drying temperature of the oven is 35 - 40°C.

[0016] The storage temperature in the refrigerator is 1 - 5°C.

[0017] An analytical method for the volatile oil from Clausena lansium seeds for treating dental caries, comprising the following steps:

[0018] Precisely pipette 100 μL of the volatile oil from Clausena lansium seeds, place it in a 10 mL volumetric flask, dissolve and make up the volume with chromatographic pure n - hexane, and analyze it under the following GC - MS conditions:

[0019] GC conditions: DB - 5MS capillary column, column specification is 30 m × 0.25 mm × 0.25 μm; the column temperature adopts programmed temperature rise, the initial temperature is 60°C and is maintained for 3 min, 5°C·min-1 Raise to 140 °C, hold for 2 min, and then at 10 °C·min -1 Raise to 210 °C and hold for 7 min; The carrier gas is helium, and the column flow rate is 1.0 mL·min -1 ; The injection volume is 1.0 μL, split injection is used, and the split ratio is 20:1; The purge flow rate is 3.0 mL·min -1 ; The inlet temperature is 250 °C;

[0020] MS conditions: EI source, ion source temperature 250 °C; Interface temperature 280 °C; Solvent delay time 2.0 min; Mass scanning time 3.00 min to 16 min; Detector voltage 0.86 kv, scanning range m / z 50 - 500; The mass spectrometry library is NIST08 and NIST08s.

[0021] Use of volatile oil from Clausena lansium seeds for treating dental caries in the preparation of drugs against Streptococcus mutans.

[0022] Use of volatile oil from Clausena lansium seeds for treating dental caries in the preparation of drugs against dental biofilm.

[0023] Use of volatile oil from Clausena lansium seeds for treating dental caries in the preparation of drugs against dental caries.

[0024] The volatile oil from Clausena lansium seeds for treating dental caries provided by the present invention, its extraction method, analysis method and application have the following beneficial effects: The present invention takes Clausena lansium seeds as the research object, extracts the volatile oil from Clausena lansium seeds by a volatile oil analyzer, analyzes and identifies the components and relative content differences of its volatile oil by gas chromatography-mass spectrometry (GC-MS), and conducts antibacterial activity research against Streptococcus mutans by disk diffusion method drug susceptibility test, evaluates its antibacterial activity against Streptococcus mutans by the size of the inhibition zone, and uses the XTT method to study the anti-biofilm ability of the volatile oil from Clausena lansium seeds, providing a basis for the volatile oil from Clausena lansium seeds as a natural drug against dental caries.

[0025] The present invention makes full use of Clausena lansium seeds which are rich in resources and mostly discarded, extracts the volatile oil from Clausena lansium seeds by a volatile oil analyzer, analyzes its components by GC-MS, determines that it has a certain inhibitory effect on Streptococcus mutans by disk diffusion method and microdilution method, and the XTT method confirms that the volatile oil from Clausena lansium seeds has the effect of anti-biofilm formation, which provides a feasibility for the current difficult problem of dental caries treatment. Natural products have the advantages of being effective, safe, and not easily developing drug resistance, providing an experimental basis for its research as a natural anti-caries drug. Description of the Drawings

[0026] Figure 1 is the flow chart of the present invention;

[0027] Figure 2 It is the chromatogram of the volatile oil from Clausena lansium seeds in the present invention. Specific Embodiments

[0028] The present invention will be further described below in conjunction with the accompanying drawings. The following examples are only used to more clearly illustrate the technical solutions of the present invention and should not be used to limit the protection scope of the present invention.

[0029] Example 1

[0030] As Figure 1 shown, a method for extracting the volatile oil from Clausena lansium seeds for treating dental caries includes the following steps: Collect Clausena lansium fruits from Rulin, Haikou City, Hainan Province, collect the seeds, remove the pulp, wash the seeds with distilled water, and dry them in an oven at 40 °C. After rough pulverization, weigh the materials, soak them for 5 h at a material ratio of 1:8, extract them with a volatile oil detector for 3 h, stop heating and let it stand for 1 h, then collect the volatile oil. The obtained volatile oil is white and transparent in color, and the oil yield is calculated to be 0.69 ± 0.05%. Seal the obtained volatile oil and store it in a refrigerator at 4 °C for later use.

[0031] A method for analyzing the volatile oil from Clausena lansium seeds for treating dental caries includes the following steps:

[0032] Precisely pipette 100 μL of the volatile oil from Clausena lansium seeds, place it in a 10 mL volumetric flask, dissolve and make up the volume with chromatographic n-hexane, and analyze it under the following GC-MS conditions:

[0033] GC conditions: DB-5MS capillary column, column specifications: 30 m × 0.25 mm × 0.25 μm; The column temperature uses programmed temperature rise. The initial temperature is maintained at 60 °C for 3 min, and it is raised to 140 °C at 5 °C·min -1 and maintained for 2 min, then raised to 210 °C at 10 °C·min -1 and maintained for 7 min; The carrier gas is helium, and the column flow rate is 1.0 mL·min -1 ; The injection volume is 1.0 μL, split injection, split ratio 20:1; The purge flow rate is 3.0 mL·min -1 ; The injection port temperature is 250 °C;

[0034] MS conditions: EI source, ion source temperature 250 °C; Interface temperature 280 °C; Solvent delay time 2.0 min; Mass scanning time 3.00 min - 16 min; Detector voltage 0.86 kv, scanning range m / z 50 - 500; Mass spectrometry library is NIST08 and NIST08s.

[0035] Under the above GC-MS conditions, the chromatogram of the volatile oil from Clausena lansium seeds is shown in Figure 2, combined with computer spectrum retrieval for qualitative identification and peak area normalization method for analyzing each chemical component and its relative content, and the specific results are shown in Table 1. A total of 34 components were identified in the volatile oil of Clausena lansium seeds, accounting for 98.43% of the total volatile oil. The components with relatively high contents were monoterpenoid components such as sabinene (49.85%), β-phellandrene (14.97%), 4-terpineol (9.37%), etc., and there were also sesquiterpenes, alcohols, esters and other compounds.

[0036] Table 1 GC-MS analysis results of the volatile oil of Clausena lansium seeds

[0037]

[0038]

[0039] The antibacterial activity of the volatile oil of Clausena lansium seeds against Streptococcus mutans was determined by the disc diffusion method. Streptococcus mutans ATCC700610 (Shanghai Ruichu Biotechnology Co., Ltd.) was selected. The freeze-dried powder was routinely resuscitated and cultured to the second generation in an anaerobic incubator at 37 °C. Fresh colonies subcultured in brain heart infusion (BHI) medium were picked, and colonies with a size of 1 mm were picked into 5.0 mL of physiological saline, and the turbidity of the bacterial suspension was adjusted to 0.5 McFarland turbidity, about 1-2×10 8 CFU / mL. The adjusted turbid bacterial suspension was evenly spread on a Mueller-Hinton (MH) agar plate, left for 3 min - 5 min, not exceeding 15 min, and filter papers were placed using sterile forceps. 10 μL of the volatile oil of Clausena lansium seeds and the positive control (3 mg / mL chloramphenicol solution) were added respectively. It was cultured in an incubator at 37 °C for 16 - 18 h, the results were observed, the diameter of the inhibition zone was measured, and each experiment was repeated 3 times, and the average value was taken. The sensitivity of the volatile oil to Streptococcus mutans was judged by the size of the inhibition zone. The results showed that the inhibition zone of the control drug chloramphenicol was 25.6 ± 0.2 mm, and the inhibition zone of the volatile oil of Clausena lansium seeds was 18.5 ± 0.2 mm. The volatile oil of Clausena lansium seeds had strong activity against Streptococcus mutans. By relating it to its specific components, the high contents of sabinene, β-phellandrene, 4-terpineol, etc. in Clausena lansium seeds may have better antibacterial effects, but the strength of antibacterial activity may not entirely depend on the main chemical components, and other chemical components with smaller contents may also have antibacterial activity or different components may have a synergistic effect.

[0040] The minimum inhibitory concentration (MIC) of the volatile oil from Clausena lansium seeds against Streptococcus mutans was determined by the microdilution method: Take a sterile 96-well culture plate, add 100 μL of BHI culture medium to each well. After adding a certain concentration of the volatile oil solution from Clausena lansium seeds to the first well, serially dilute it to the tenth well, then inoculate 100 μL of the bacterial suspension. Add 100 μL of the bacterial suspension to the 11th well as the growth control well, and add only 200 μL of BHI culture medium to the 12th well as the blank control. Incubate the Streptococcus mutans culture plate in a CO 2 anaerobic incubator at 37 °C for 48 h. The MIC value was determined by comparing with the drug-free control well cultured for 48 h to reach the lowest drug concentration with 50% inhibition. Each experiment was repeated 3 times, and the average value of the MIC was taken. The result showed that the MIC of the volatile oil from Clausena lansium seeds against Streptococcus mutans was 1 mg / mL.

[0041] By searching the existing studies on the antibacterial effects of extracted components of Chinese herbal medicines against Streptococcus mutans, it was found that the MIC value of ellagic acid against Streptococcus mutans was 12.5 mg / mL; the MIC value of tea tree essential oil against Streptococcus mutans was 0.125%, that is, 1.25 mg / mL; the MIC value of the extract of Lonicera japonica against Streptococcus mutans was 12.5 mg / mL. In this invention, the MIC of the volatile oil from Clausena lansium seeds against Streptococcus mutans was 1 mg / mL, with a low antibacterial concentration and more obvious effects.

[0042] The effect of the volatile oil from Clausena lansium seeds on the biofilm formation of Streptococcus mutans was studied by the XTT method: Take a 96-well culture plate, add 100 μL of BHI culture medium to each well. After adding a certain concentration of the volatile oil solution from Clausena lansium to the first well, serially dilute it to the tenth well, then inoculate 100 μL of the bacterial suspension. Add 100 μL of the bacterial suspension to the 11th well as the growth control well, and add only 200 μL of BHI culture medium to the 12th well as the blank control. Incubate the Streptococcus mutans culture plate in a CO 2 anaerobic incubator at 37 °C for 48 h, wash it 2 - 3 times with sterile PBS, add 100 μL of XTT solution to each well, incubate in the dark for 4 h, measure OD490. The experiment was repeated 3 times, and calculate the lowest drug concentration that can inhibit more than 90% of biofilm formation, namely MBIC 90 . The result showed that the MBIC 90 of the volatile oil from Clausena lansium seeds against Streptococcus mutans was 4 mg / mL.

[0043] The research on the extraction, analysis, antibacterial activity against Streptococcus mutans by the disk diffusion method and microdilution method, and the research on anti-biofilm formation by the XTT method of the volatile oil from Clausena lansium seeds provided in this invention provided an experimental basis for the volatile oil from Clausena lansium seeds as a natural drug for anti-caries.

[0044] Application of a volatile oil from Clausena lansium seeds for treating dental caries in the preparation of drugs against Streptococcus mutans.

[0045] Use of volatile oil from Clausena lansium seeds for treating dental caries in the preparation of drugs against dental biofilm.

[0046] Use of volatile oil from Clausena lansium seeds for treating dental caries in the preparation of drugs against dental caries.

[0047] In this example, the explanations of relevant technical terms are as follows:

[0048] Streptococcus mutans;

[0049] Gas chromatography-mass spectrometry (GC-MS);

[0050] Mueller-Hinton (MH) casein hydrolysate;

[0051] Brain heart infusion (BHI) medium;

[0052] Minimum inhibitory concentration (MIC);

[0053] XTT method [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide)];

[0054] Minimum biofilm inhibitory concentration (MBIC).

[0055] Example 2

[0056] The difference between this example and Example 1 is only that: the oven drying temperature is 35 °C; the storage temperature in the refrigerator is 1 °C.

[0057] Meanwhile, in this example, Streptococcus mutans ATCC700610 (Shanghai Ruichu Biotechnology Co., Ltd.) was selected to determine the antibacterial activity of volatile oil from Clausena lansium seeds by the disc diffusion method. The freeze-dried powder of Streptococcus mutans was routinely resuscitated and cultured to the second generation at 37 °C in an anaerobic incubator. Fresh colonies subcultured in brain heart infusion (BHI) medium were picked, and colonies with a size of 1 mm were placed in 5.0 mL of physiological saline, and the turbidity of the bacterial suspension was adjusted to 0.5 McFarland turbidity, about 1 - 2×10 8CFU / mL. The adjusted turbidity of the bacterial suspension was evenly spread on a Mueller-Hinton (MH) agar plate, left for 3 min to 5 min, not exceeding 15 min. Sterile forceps were used to place the paper discs, and 10 μL of the volatile oil from Clausena lansium seeds and the positive control (1.2 mg / mL chlorhexidine acetate solution) were added respectively. It was cultured in an incubator at 37 °C for 16 - 18 h, the results were observed, and the diameter of the inhibition zone was measured. Each test was repeated 3 times, and the average value was taken. The sensitivity of the volatile oil to Streptococcus mutans was judged by the size of the inhibition zone. The results showed that the inhibition zone of the control drug chlorhexidine acetate was 20.6 ± 0.2 mm, and the inhibition zone of the volatile oil from Clausena lansium seeds was 18.5 ± 0.2 mm. The volatile oil from Clausena lansium seeds had strong activity against Streptococcus mutans.

[0058] Example 3

[0059] The difference between this example and Example 1 is only that: the storage temperature in the refrigerator is 5 °C.

[0060] In the specification provided here, a large number of specific details are described. However, it can be understood that the embodiments of the present invention can be practiced without these specific details. In some instances, well-known methods and technologies are not shown in detail so as not to obscure the understanding of this specification.

[0061] Similarly, it should be understood that, in order to streamline this disclosure and assist in understanding one or more of the various inventive aspects, in the above description of the exemplary embodiments of the present invention, the various features of the present invention are sometimes grouped together into a single embodiment or description thereof. However, the disclosed method should not be construed as reflecting the intention that: the claimed invention requires more features than those expressly recited in each claim. Rather, as reflected by the claims, the inventive aspects lie in less than all the features of the single foregoing disclosed embodiment. Thus, the claims following the detailed description are hereby expressly incorporated into this detailed description, where each claim itself serves as a separate embodiment of the present invention.

[0062] Those skilled in the art can understand that, although some of the embodiments described herein include certain features included in other embodiments but not other features, the combination of the features of different embodiments means that it is within the scope of the present invention and forms different embodiments. For example, in the claims, any one of the claimed embodiments can be used in any combination.

[0063] Although the present invention has been described in terms of a limited number of embodiments, those skilled in the art, having the benefit of the foregoing description, will appreciate that other embodiments can be contemplated within the scope of the invention as thus described. In addition, it should be noted that the language used in this specification has been principally selected for readability and instructional purposes and not to limit or circumscribe the inventive subject matter. Accordingly, many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the appended claims. For the scope of the present invention, the disclosure herein is illustrative, and not restrictive, the scope of the invention being defined by the appended claims.

Claims

1. Use of volatile oil from Clausena lansium seeds in the preparation of a drug against Streptococcus mutans, characterized in that, the method for preparing the volatile oil from Clausena lansium seeds comprises the following steps: Collect Clausena lansium fruits, collect the seeds, remove the pulp, wash the seeds with distilled water, and place them in an oven for drying; weigh after pulverization, soak with distilled water at a material ratio of 1:8 for at least 5 h, extract with a volatile oil extractor for at least 3 h, stop heating and let stand for at least 1 h, then collect the volatile oil. The obtained volatile oil is white and transparent in color, and the oil yield is 0.69±0.05%; seal the obtained volatile oil and store it in a refrigerator for standby.

2. The use of the volatile oil from Clausena lansium seeds according to claim 1 in the preparation of a drug against Streptococcus mutans, characterized in that, the drying temperature of the oven is 35-40°C.

3. The use of the volatile oil from Clausena lansium seeds according to claim 1 in the preparation of a drug against Streptococcus mutans, characterized in that, the storage temperature in the refrigerator is 1-5°C.

4. Use of volatile oil from Clausena lansium seeds in the preparation of a drug against dental caries caused by Streptococcus mutans, characterized in that, the method for preparing the volatile oil from Clausena lansium seeds comprises the following steps: Collect Clausena lansium fruits, collect the seeds, remove the pulp, wash the seeds with distilled water, and dry them in an oven at 35-40°C; weigh after pulverization, soak with distilled water at a material ratio of 1:8 for at least 5 h, extract with a volatile oil extractor for at least 3 h, stop heating and let stand for at least 1 h, then collect the volatile oil. The obtained volatile oil is white and transparent in color, and the oil yield is 0.69±0.05%; seal the obtained volatile oil and store it in a refrigerator for standby.

5. The use according to claim 4, characterized in that, the storage temperature in the refrigerator is 1-5°C.

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