Sophora flower extract for inhibiting formation of oral biofilm and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a sophora flower extract for inhibiting formation of an oral biofilm and application thereof. According to the method, the sophora flower extract obtained after alcohol extraction is subjected to specific macroporous resin column elution conditions, the sophora flower extract obtained through enrichment has a very excellent effect of inhibiting oral biofilm formation, and further elution is carried out under specific silica gel column conditions, so that the effect of the obtained sophora flower extract is further improved. The inhibition rate of the obtained sophora flower extract is obviously higher than that of sophora flower extracts prepared by traditional alcohol extraction or other processes, and the sophora flower extract has important application value in preparation of products for inhibiting and / or killing oral microorganisms or biological membranes thereof, and can be used for preventing and / or treating dental bacterial diseases including decayed teeth.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a Sophora japonica extract that inhibits the formation of oral biofilms and its applications. Background Technology

[0002] Sophora japonica flowers, the dried flowers and buds of the Sophora japonica plant (a legume), possess both medicinal and edible properties. They are believed to have cooling and hemostatic effects, as well as clearing liver heat. Due to their pleasant aroma and high safety profile, they are widely used in various toothpastes and oral care products. For example, a Chinese patent application discloses a herbal tooth powder containing Sophora japonica flower extract, claiming it can prevent and treat periodontitis, gingivitis, cavities, oral mucosal inflammation, and halitosis. However, the Sophora japonica flower extract used in this tooth powder is obtained only through simple alcohol extraction and extraction processes, and Sophora japonica flowers are only one of the compound ingredients, resulting in a low content of effective active ingredients and difficulty in achieving full efficacy. Furthermore, the patent application does not provide any experimental data to support its claimed preventive and therapeutic effects, making its actual application lack scientific basis.

[0003] Currently, evaluations of the antibacterial effects of traditional Chinese medicine (TCM) primarily focus on planktonic bacteria, with common methods including the determination of inhibition zone diameter, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC). However, oral pathogens mainly exist in a biofilm state, and their physiological characteristics, drug resistance, and pathogenic mechanisms differ fundamentally from those of planktonic bacteria (Gao Z, Chen X, Wang C, et al. New strategies and mechanisms for targeting Streptococcus mutans biofilm formation to prevent dental caries: a review[J]. Microbiological Research, 2023: 127526.). Therefore, antibacterial TCM extracts screened based on planktonic bacterial models often fail to effectively inhibit oral biofilm formation in practical applications. Existing oral care products based on Sophora japonica flowers have not been optimized for biofilm characteristics in terms of ingredient preparation, active content, and mechanism of action, resulting in limited efficacy in preventing and treating oral biofilm-related diseases.

[0004] In summary, there is an important and urgent need to develop an oral care product that can effectively inhibit the formation of oral biofilms. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the limitations of conventional oral products in inhibiting oral biofilms and their inability to effectively remove oral microorganisms within the oral biofilm. The primary objective is to provide a Sophora japonica extract that can effectively inhibit the formation of oral biofilms with a high inhibition rate (>84%).

[0006] Another object of the present invention is to provide the application of the aforementioned Sophora japonica extract.

[0007] Another object of the present invention is to provide a product that inhibits and / or kills oral microorganisms or their biofilms.

[0008] Another object of the present invention is to provide the use of the Sophora japonica extract or the product in the preparation of medicaments for the prevention and / or treatment of dental bacterial diseases. The above-mentioned objective of this invention is achieved through the following technical solution: This invention protects a Sophora japonica flower extract, which is prepared by the following steps: Sophora japonica flowers are extracted with alcohol and then treated with a non-polar macroporous resin. After full adsorption, the extract is first eluted with a 5 vol% to 8 vol% ethanol aqueous solution to remove impurities, and then eluted a second time with a 20 vol% to 30 vol% ethanol aqueous solution. The eluent obtained from the second elution is collected, concentrated, and dried to obtain the Sophora japonica flower extract.

[0009] This invention protects a Sophora japonica flower extract, which is mainly prepared by the following steps: (1) The extract of Sophora japonica flowers prepared by alcohol extraction was treated with non-polar macroporous resin. After full adsorption, it was first eluted with 5 vol%~8 vol% ethanol aqueous solution to remove impurities, and then eluted with 20 vol%~30 vol% ethanol aqueous solution for a second time. The eluent obtained from the second elution was collected, concentrated and dried to obtain the macroporous resin eluted fraction of Sophora japonica flowers. (2) Elute the macroporous resin elution portion of Sophora japonica obtained in step (1) onto a silica gel column. First, perform a first elution to remove impurities with a chloroform-methanol-water solution with a volume ratio of (85~95):(5~10):1. Then, perform a second elution with a methanol-water solution with a volume ratio of (1~2):1. Collect the eluent obtained from the second elution, concentrate and dry it to obtain the Sophora japonica extract.

[0010] Addressing the limitation of existing sophora japonica-based oral care or treatment products in terms of ingredient preparation, active ingredient content, and mechanism of action, which fail to optimize for the characteristics of biofilms and thus have limited efficacy in preventing and treating oral biofilm-related diseases, the inventors, after extensive creative work, discovered that the sophora japonica extract obtained through the aforementioned method can effectively inhibit oral biofilm formation, with an inhibition rate significantly higher than that obtained through traditional alcohol extraction or other processes. The macroporous resin column elution and silica gel column conditions are crucial in this invention; the effects of the sophora japonica extracts prepared using different macroporous resin column elution conditions and silica gel column elution conditions vary significantly.

[0011] Furthermore, the non-polar macroporous resin treatment includes one or more of the following (1) to (4): (1) The non-polar macroporous resin includes any one of the following: D101 type macroporous resin column, HPD-100 type macroporous resin column, and HP-20 type macroporous resin column; (1) The time for complete adsorption is 2-4 h; (2) The amount of eluent used for the first elution to remove impurities is 3 to 5 times the column volume; (3) The amount of eluent used for the second elution to remove impurities is 3 to 5 times the column volume.

[0012] Specifically, as a preferred feasible method, the non-polar macroporous resin treatment includes the following steps: loading the extract of Sophora japonica flowers prepared by alcohol extraction onto a non-polar macroporous resin column for adsorption for 2-4 hours; then performing a first elution to remove impurities using 3-5 column volumes (of the non-polar macroporous resin column) of 5 vol%-8 vol% ethanol aqueous solution; followed by a second elution using 3-5 column volumes of 20 vol%-30 vol% ethanol aqueous solution, and collecting the eluent obtained from the second elution.

[0013] Preferably, in the non-polar macroporous resin treatment step, the eluent for the first elution and impurity removal is a 5 vol% to 7 vol% aqueous ethanol solution, more preferably a 5.5 vol% to 6.5 vol% aqueous ethanol solution.

[0014] Preferably, in the non-polar macroporous resin treatment step, the eluent for the second elution is a 20 vol% to 25 vol% aqueous ethanol solution, more preferably a 22 vol% to 24 vol% aqueous ethanol solution.

[0015] Preferably, in the non-polar macroporous resin treatment step, the time for sufficient adsorption is 2.5~3.5 h, more preferably 2.8~3.2 h.

[0016] Preferably, in the non-polar macroporous resin treatment step, the amount of eluent used for the first elution and impurity removal is 3.5 to 4.5 times the column volume, more preferably 3.8 to 4.2 times the column volume.

[0017] Preferably, in the nonpolar macroporous resin treatment step, the amount of eluent used in the second elution is 3.5 to 4.5 times the column volume, more preferably 3.8 to 4.2 times the column volume.

[0018] Furthermore, the conditions for elution of the silica gel column include one or more of the following (1) to (5): (1) The method of attaching the silicone column is a dry column attachment; (2) The mass ratio of the macroporous resin elution portion of the Sophora japonica to the silica gel is 1:(20~40); (3) The silica gel filling the silica gel column has a mesh size of 200~300 mesh; (4) The amount of eluent used for the first elution to remove impurities is 2 to 4 times the column (silica gel column) volume; (5) The amount of eluent used in the second elution is 3 to 5 times the column volume.

[0019] Preferably, the silica gel is column chromatography silica gel.

[0020] Preferably, in the silica gel column elution step, the eluent for the first elution to remove impurities is a chloroform-methanol-water solution with a volume ratio of (88~92):(8~10):1, more preferably a chloroform-methanol-water solution with a volume ratio of (89~91):(9~10):1.

[0021] Preferably, in the silica gel column elution step, the eluent for the second elution is a methanol-water solution with a volume ratio of (1.2~1.8):1, more preferably a methanol-water solution with a volume ratio of (1.4~1.6):1.

[0022] Preferably, in the silica gel column elution step, the mass ratio of the macroporous resin elution portion to the silica gel is 1:(25~35), more preferably 1:(28~32).

[0023] Preferably, in the silica gel column elution step, the amount of eluent used in the first elution is 2.5 to 3.5 times the column volume, more preferably 2.8 to 3.2 times the column volume.

[0024] Preferably, in the silica gel column elution step, the amount of eluent used in the second elution is 3.5 to 4.5 times the column volume, more preferably 3.8 to 4.2 times the column volume.

[0025] Specifically, as a preferred feasible method, the silica gel column elution step includes the following steps: the macroporous resin elution portion obtained in step (1) is loaded onto a silica gel column (the mass ratio of the macroporous resin elution portion to silica gel is 1:(28~32)), and a first elution is performed using a chloroform-methanol-water solution with a volume ratio of (89~91):(9~10):1, which is 2.8~3.2 times the column volume; then a second elution is performed using a methanol-water solution with a volume ratio of (1.4~1.6):1, which is 3.8~4.2 times the column volume, and the eluent obtained from the second elution is collected.

[0026] Furthermore, the preparation method of the extract made from Sophora japonica flowers by alcohol extraction includes the following steps: fully extracting Sophora japonica flowers with a 50 vol% to 70 vol% aqueous ethanol solution, combining the extracts, concentrating them, and obtaining the extract.

[0027] Furthermore, the ethanol aqueous solution is a 60 vol% to 65 vol% ethanol aqueous solution.

[0028] Furthermore, the mass ratio of the Sophora japonica flowers to the ethanol aqueous solution is 1:(8~15) g / mL, preferably 1:(10~12) g / mL.

[0029] Furthermore, the complete alcohol extraction is performed using ultrasound-assisted alcohol extraction.

[0030] Preferably, the ultrasonic extraction is performed at an ultrasonic power of 80~120 kW (more preferably 90~110 kW) for 20~40 min (more preferably 25~35 min).

[0031] Preferably, the full alcohol extraction is performed 1 to 3 times, more preferably 2 times.

[0032] Furthermore, all concentrations mentioned are vacuum concentrations.

[0033] Preferably, the temperature for vacuum concentration is 40~50 °C.

[0034] Furthermore, the concentration of the concentrated extract is 0.5~2 g / mL, preferably 0.8~1.2 g / mL.

[0035] Furthermore, the drying process is freeze-drying, and the freeze-drying temperature is -70 to -90 °C. The freeze-drying time depends on the sample quantity, typically 2 to 5 days.

[0036] This invention protects the use of the sophora japonica extract in the preparation of products that inhibit and / or kill oral microorganisms or their biofilms.

[0037] Furthermore, the oral microorganisms include one or more of Streptococcus mutans, Porphyromonas gingivalis, Staphylococcus aureus, Candida albicans, Prevotella intermedius, Actinomyces negrius, Actinomyces viscous, Actinomyces esculenta, Actinomyces mesenteriae, Actinomyces odontolytica, and Rhizoctonia solani.

[0038] Preferably, the oral microorganisms are selected from one or more of Streptococcus mutans, Porphyromonas gingivalis, Staphylococcus aureus, and Candida albicans.

[0039] This invention also protects a product that inhibits and / or kills oral microorganisms or their biofilms, the active ingredient of which includes one or more of the Sophora japonica extract.

[0040] Furthermore, the product is an oral care daily chemical product or an oral treatment drug.

[0041] Furthermore, the oral care products include toothpaste, tooth powder, mouthwash, or breath freshener.

[0042] This invention also protects the use of the sophora flower extract or the product in the preparation of medicaments for the prevention and / or treatment of bacterial dental diseases.

[0043] Furthermore, the bacterial dental diseases include one or more of dental caries, pulpitis, periodontitis, periapical periodontitis, and pericoronitis of wisdom teeth.

[0044] Furthermore, the caries is caused by bacteria, including Streptococcus mutans.

[0045] Compared with the prior art, the present invention has the following beneficial effects: This invention involves eluting a Sophora japonica flower extract obtained after alcohol extraction using specific macroporous resin column conditions. The resulting extract exhibits excellent inhibitory effects on oral biofilm formation. Further elution using specific silica gel column conditions further enhances the efficacy of the extracted Sophora japonica flower. The inhibition rate of the obtained extract is significantly higher than that obtained through traditional alcohol extraction or other processes. This makes it valuable for the preparation of products that inhibit and / or kill oral microorganisms or their biofilms, and it can be used for the prevention and / or treatment of dental bacterial diseases, including dental caries. Detailed Implementation

[0046] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0047] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0048] The powdered Sophora japonica flowers was purchased from a traditional Chinese medicine clinic in Nanjing.

[0049] Streptococcus mutans: ATCC 25175, purchased from Shanghai Ruichu Biotechnology Co., Ltd.; D101 type macroporous resin: purchased from Shanghai Yuanye Biotechnology Co., Ltd.; Silica gel column: The silica gel used for filling is column chromatography silica gel (200-300 mesh), purchased from Qingdao Ocean Chemical Co., Ltd.

[0050] Example 1: Sophora japonica flower extract and its preparation Sophora japonica flower extract was prepared by the following steps: (1) Take Sophora japonica powder and extract it with 60 vol% ethanol aqueous solution at 100 kw ultrasonic power for 30 min. After extraction, separate the solid and liquid to obtain Sophora japonica residue and extract A. The ratio of Sophora japonica powder to ethanol is 1 g: 12 mL. (2) The Sophora japonica residue obtained in step (1) is further extracted with 60 vol% ethanol aqueous solution under ultrasonic power of 100 kw for 30 min. After the extraction is completed, solid-liquid separation is performed to obtain extract B; wherein, the ratio of Sophora japonica powder to ethanol is 1 g: 10 mL. (3) Combine the extract A obtained in step (1) and the extract B obtained in step (2) and concentrate them under reduced pressure (40 °C) to 1 g / mL to obtain Sophora japonica extract; (4) The Sophora japonica extract obtained in step (3) is loaded onto a D101 macroporous resin column and adsorbed for 3 hours. Then, it is eluted with 4 column volumes of 6 vol% ethanol aqueous solution to remove impurities. Next, it is eluted with 4 column volumes of 23 vol% ethanol aqueous solution. The eluent eluted from the 23 vol% ethanol aqueous solution is collected and concentrated under reduced pressure (40 °C) to dryness to obtain the macroporous resin eluted part of Sophora japonica, which is the Sophora japonica extract.

[0051] Example 2: Sophora japonica flower extract and its preparation The preparation of Sophora japonica flower extract includes the following steps: (1) Take Sophora japonica powder and extract it with 60 vol% ethanol aqueous solution at 100 kw ultrasonic power for 30 min. After extraction, separate the solid and liquid to obtain Sophora japonica residue and extract A. The ratio of Sophora japonica powder to ethanol is 1 g: 12 mL. (2) The Sophora japonica residue obtained in step (1) is further extracted by ultrasonic extraction with 60 vol% ethanol aqueous solution at 100 kw ultrasonic power for 30 min. After extraction, solid-liquid separation is performed to obtain extract B; wherein, the ratio of Sophora japonica powder to ethanol is 1 g: 10 mL. (3) Combine the extract A obtained in step (1) and the extract B obtained in step (2) and concentrate them under reduced pressure (40 °C) to 1 g / mL to obtain Sophora japonica extract; (4) The Sophora japonica extract obtained in step (3) is loaded onto a D101 macroporous resin column and adsorbed for 3 hours. Then, it is eluted with 4 column volumes of 6 vol% ethanol aqueous solution to remove impurities. Next, it is eluted with 4 column volumes of 23 vol% ethanol aqueous solution. The eluent eluted from the 23 vol% ethanol aqueous solution is collected and concentrated under reduced pressure (40 °C) to dryness to obtain the Sophora japonica macroporous resin eluted fraction. (5) Load the macroporous resin eluent obtained in step (4) onto a silica gel column (the mass ratio of the macroporous resin eluent to silica gel is 1:30, and the column is loaded using this ratio and the loading is done by dry method). First, use 3 column volumes of a mixed organic solvent consisting of chloroform, methanol and water in a volume ratio of 90:10:1 to remove impurities. Then, use 4 column volumes of a methanol-water solution in a volume ratio of 60:40 as the eluent. Collect the eluent obtained by the mixed organic solvent consisting of methanol and water in a volume ratio of 60:40, concentrate it under reduced pressure (40 °C) to extract, and then freeze-dry it (-80 °C, the time depends on the amount of sample, usually 2 to 5 days) to obtain the Sophora japonica extract.

[0052] Example 3: Sophora japonica flower extract and its preparation The difference from Example 2 is that the elution conditions of the macroporous resin column in step (4) are different. Specifically, the column is eluted with 6.5 vol% ethanol aqueous solution to remove impurities, and then eluted with 24 vol% ethanol aqueous solution. The eluent eluted from the 24 vol% ethanol aqueous solution is collected.

[0053] The other steps and parameters are the same as in Example 2.

[0054] Example 4: Sophora japonica flower extract and its preparation The difference from Example 1 is that the elution conditions of the macroporous resin column in step (4) are different. Specifically, it is eluted with 5.5 vol% ethanol aqueous solution to remove impurities, and then eluted with 22 vol% ethanol aqueous solution. The eluent eluted from the 22 vol% ethanol aqueous solution is collected.

[0055] The other steps and parameters are the same as in Example 2.

[0056] Example 5 Preparation of Sophora japonica flower extract The difference from Example 1 is that the elution conditions of the silica gel column in step (5) are different. Specifically, the column is eluted with a mixed organic solvent consisting of chloroform, methanol and water in a volume ratio of 88:10:1. Then, the column is eluted with a methanol-water solution in a volume ratio of 16:10, and the eluent obtained by elution with the mixed organic solvent consisting of methanol and water in a volume ratio of 16:10 is collected.

[0057] The other steps and parameters are the same as in Example 2.

[0058] Example 6 Preparation of Sophora japonica flower extract The difference from Example 1 is that the elution conditions of the silica gel column in step (5) are different. Specifically, the column is eluted with a mixed organic solvent consisting of chloroform, methanol and water in a volume ratio of 92:8:1 to remove impurities. Then, the column is eluted with a methanol-water solution in a volume ratio of 14:10 and the eluent is collected.

[0059] The other steps and parameters are the same as in Example 2.

[0060] The properties of the Sophora japonica extracts obtained in Examples 3-6 are similar to those in Example 2. Subsequent experiments used the Sophora japonica extract obtained in Example 2 as a representative to conduct specific tests on the inhibition of biofilm formation.

[0061] Comparative Example 1: Sophora japonica flower extract and its preparation The difference from Example 1 is that step (4) is omitted, that is, the Sophora japonica extract obtained in step (3) is the Sophora japonica extract.

[0062] The other steps and parameters are the same as in Example 1.

[0063] Comparative Example 2: Preparation of Sophora japonica flower extract The difference from Example 1 is that the elution conditions of the macroporous resin column are different. Specifically, the column is eluted with a 6 vol% ethanol aqueous solution to remove impurities, and then eluted with a 60 vol% ethanol aqueous solution. The eluent obtained from the 60 vol% ethanol aqueous solution is collected.

[0064] The other steps and parameters are the same as in Example 1.

[0065] Comparative Example 3: Preparation of Sophora japonica flower extract The difference from Example 1 is that the elution conditions of the macroporous resin column are different. Specifically, the column is eluted with a 50 vol% ethanol aqueous solution to remove impurities, followed by elution with a 70 vol% ethanol aqueous solution, and the eluent eluted from the 70 vol% ethanol aqueous solution is collected.

[0066] The other steps and parameters are the same as in Example 1.

[0067] Comparative Example 4: Preparation of Sophora japonica flower extract The difference from Example 1 is that the elution conditions of the macroporous resin column are different. Specifically, the column is eluted with a 75 vol% ethanol aqueous solution to remove impurities, followed by elution with a 90 vol% ethanol aqueous solution, and the eluent eluted from the 90 vol% ethanol aqueous solution is collected.

[0068] The other steps and parameters are the same as in Example 1.

[0069] Comparative Example 5: Preparation of Sophora japonica flower extract The difference from Example 2 is that the elution conditions of the silica gel column are different. Specifically, elution is performed with chloroform to remove impurities, followed by elution with an organic solvent consisting of chloroform, methanol and water in a volume ratio of 100:25:5. The eluent eluted by the organic solvent consisting of chloroform, methanol and water in a volume ratio of 100:25:5 is collected.

[0070] The other steps and parameters are the same as in Example 2.

[0071] Comparative Example 6: Preparation of Sophora japonica flower extract The difference from Example 2 is that the elution conditions of the silica gel column are different. Specifically, the column is eluted with an organic solvent consisting of chloroform-methanol-water in a volume ratio of 90:10:1 to remove impurities. Then, it is eluted again with an organic solvent consisting of chloroform, methanol and water in a volume ratio of 70:30:5. The eluent eluted with the organic solvent consisting of chloroform and methanol in a volume ratio of 70:30:5 is collected.

[0072] The other steps and parameters are the same as in Example 2.

[0073] Experimental Example: Sophora japonica flower extract inhibits biofilm formation. The Sophora japonica extracts obtained in Examples 1-2 and Comparative Examples 1-6 were selected for experiments on inhibiting biofilm formation.

[0074] Experimental method: In a 96-well plate, 20 μL of artificial saliva was added to each well. Then, 180 μL of culture medium was added to the blank control group, and 160 μL of culture medium and 20 μL of 1×10⁻⁶ solution were added to the model group. 6 Streptococcus mutans suspension at CFU / mL; experimental group added 160 μL of drug-containing culture medium and 20 μL of 1×10⁻⁶ CFU / mL. 6A CFU / mL suspension of *Streptococcus mutans* was prepared and incubated at 37 °C for 24 h in an anaerobic incubator. After incubation, the supernatant was discarded, and the plate was gently washed three times with PBS to remove airborne bacteria and residual liquid was aspirated. 200 μL of 4% paraformaldehyde was added to each well for fixation for 10 min, followed by drying. 200 μL of 1% crystal violet was added for staining for 5 min, and the stain was removed. The plate was then washed three times with PBS, and residual PBS was aspirated. The plate was dried, and 200 μL of 33% glacial acetic acid solution was added to each well. The plate was incubated at 37 °C for 30 min, and the OD was measured using a microplate reader. 570 The absorbance value is calculated according to the formula "I 生物膜抑制率 = (A 模型组 -A 样品 ) / (A 模型组 -A 空白组 The biofilm inhibition rate was calculated by multiplying the result by 100%; the test results are shown in Table 1. Each experiment was independently repeated 3 times, with 6 replicates per group, and the average of the multiple measurements was taken.

[0075] The culture medium used in this experiment was prepared as follows: 3.85 g of brain heart extract (BHI) broth and 0.600 g of sucrose were accurately weighed and dissolved in 100 mL of distilled water. The mixture was heated and stirred, cooled to room temperature, and the pH was adjusted to 6.5. The mixture was then autoclaved at 121 °C for 30 min to obtain BHI culture medium containing 6% sugar (w / v).

[0076] The method for preparing the drug-containing culture medium is as follows: the sample to be tested is added to the BHI culture medium containing 6% sugar (W / V) prepared above to prepare a drug-containing culture medium with a drug content of 1 mg / mL.

[0077] Table 1. Experimental results of Sophora japonica flower extract inhibiting biofilm formation.

[0078] As shown in Table 1, the Sophora japonica extracts prepared in Examples 1-2 achieved a biofilm inhibition rate of over 84%, with Example 2 reaching over 90%, significantly higher than the Sophora japonica extracts obtained in Comparative Examples 1-6. This indicates that the Sophora japonica extract obtained after ethanol extraction, enriched under specific macroporous resin column elution conditions (eluting with 5-8 vol% ethanol aqueous solution as the eluent, collecting the fraction eluted with 20-30 vol% ethanol aqueous solution), has an excellent inhibitory effect on oral biofilm formation. Further elution under specific silica gel column elution conditions (eluting with a volume ratio of (85-95):(1-10):1) of chloroform-methanol-water solution as the eluent, collecting the fraction eluted with a volume ratio of (1-2):1 of methanol-water solution) further enhances the effect of the obtained Sophora japonica extract.

[0079] Compared with the Sophora japonica extract prepared by the traditional method in Comparative Example 1, the Sophora japonica extract obtained by alcohol extraction and macroporous resin adsorption in Comparative Example 2 showed a certain degree of improvement in biofilm inhibition rate, but the inhibition rate was still below 80%, and the improvement was limited.

[0080] Compared with the Sophora japonica extract prepared by the conventional method in Comparative Example 1, the biofilm inhibition rate of the Sophora japonica extracts prepared in Comparative Example 3 (elution conditions of macroporous resin: elution with 50 vol% ethanol aqueous solution as eluent, collection of the fraction eluted with 70 vol% ethanol aqueous solution) and Comparative Example 4 (elution with 75 vol% ethanol aqueous solution as eluent, collection of the fraction eluted with 90 vol% ethanol aqueous solution) was not only not improved, but was significantly reduced; this shows that the elution conditions of the macroporous resin column in this invention are very critical.

[0081] Compared with the Sophora japonica extract prepared in Comparative Example 1, the biofilm inhibition rates of the Sophora japonica extracts prepared in Comparative Examples 5 and 6 were not improved, especially the inhibition rate of the Sophora japonica extract obtained in Comparative Example 5 was significantly reduced. This indicates that the silica gel column elution conditions of the present invention are also crucial; the types and contents of active ingredients enriched in the Sophora japonica extracts prepared by further using different silica gel column elution conditions on the macroporous resin elution fraction of Sophora japonica are completely different; only the Sophora japonica extract enriched under the silica gel column elution conditions of the present invention can significantly improve the inhibitory effect on oral biofilm formation compared with the macroporous resin elution fraction of Sophora japonica; while the Sophora japonica extracts enriched under other silica gel column elution conditions do not significantly improve the inhibitory effect on oral biofilm formation compared with the Sophora japonica extract obtained in Example 2.

[0082] In summary, the types and contents of active ingredients enriched in the macroporous resin elution fractions of Sophora japonica prepared using different macroporous resin and silica gel column elution conditions are completely different, resulting in significant differences in the efficacy of Sophora japonica extracts prepared under different elution conditions. Only the Sophora japonica extract enriched under the macroporous resin and silica gel column elution conditions described in this invention shows a significant improvement in its inhibitory effect on oral biofilm formation compared to the Sophora japonica extract obtained after traditional alcohol extraction; while the Sophora japonica extracts enriched under other macroporous resin column elution and / or silica gel column conditions do not show a significant improvement in their inhibitory effect on oral biofilm formation compared to the Sophora japonica extract obtained after alcohol extraction.

[0083] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A Sophora japonica flower extract, characterized in that, The extract was prepared by the following steps: Sophora japonica flowers were extracted with alcohol and treated with a non-polar macroporous resin. After full adsorption, the extract was first eluted with a 5 vol% to 8 vol% ethanol aqueous solution to remove impurities, and then eluted a second time with a 20 vol% to 30 vol% ethanol aqueous solution. The eluent obtained from the second elution was collected, concentrated, and dried to obtain the Sophora japonica flower extract.

2. A Sophora japonica flower extract, characterized in that, The preparation mainly includes the following steps: (1) The extract of Sophora japonica flowers prepared by alcohol extraction was treated with non-polar macroporous resin. After full adsorption, it was first eluted with 5 vol%~8 vol% ethanol aqueous solution to remove impurities, and then eluted with 20 vol%~30 vol% ethanol aqueous solution for a second time. The eluent obtained from the second elution was collected, concentrated and dried to obtain the macroporous resin eluted fraction of Sophora japonica flowers. (2) Elute the macroporous resin elution portion of Sophora japonica obtained in step (1) onto a silica gel column. First, perform a first elution to remove impurities with a chloroform-methanol-water solution with a volume ratio of (85~95):(5~10):

1. Then, perform a second elution with a methanol-water solution with a volume ratio of (1~2):

1. Collect the eluent obtained from the second elution, concentrate and dry it to obtain the Sophora japonica extract.

3. The Sophora japonica extract according to claim 1 or 2, characterized in that, The non-polar macroporous resin treatment includes one or more of the following (1) to (4): (1) The non-polar macroporous resin includes any one of the following: D101 type macroporous resin column, HPD-100 type macroporous resin column, and HP-20 type macroporous resin column; (2) The time for complete adsorption is 2-4 h; (3) The amount of eluent used for the first elution to remove impurities is 3 to 5 times the column volume; (4) The amount of eluent used in the second elution is 3 to 5 times the column volume.

4. The Sophora japonica extract according to claim 2, characterized in that, The conditions for elution of the silica gel column include one or more of the following (1) to (5): (1) The method of attaching the silicone column is a dry column attachment; (2) The mass ratio of the macroporous resin elution portion of the Sophora japonica to the silica gel is 1:(20~40); (3) The silica gel filling the silica gel column has a mesh size of 200~300 mesh; (4) The amount of eluent used for the first elution to remove impurities is 3 to 5 times the column volume; (5) The amount of eluent used in the second elution is 3 to 5 times the column volume.

5. The Sophora japonica extract according to claim 1 or 2, characterized in that, The preparation method of the extract made from Sophora japonica flowers by alcohol extraction includes the following steps: fully extracting Sophora japonica flowers with a 50 vol% to 70 vol% aqueous ethanol solution, combining the extracts, concentrating them, and obtaining the extract.

6. The use of the Sophora japonica extract according to any one of claims 1 to 5 in the preparation of products that inhibit and / or kill oral microorganisms or their biofilms.

7. The application according to claim 6, characterized in that, The oral microorganisms include one or more of Streptococcus mutans, Porphyromonas gingivalis, Staphylococcus aureus, Candida albicans, Prevotella intermedius, Actinomyces negrius, Actinomyces viscous, Actinomyces esculenta, Actinomyces mesenteriae, Actinomyces odontolytica, and Corynebacterium cariesii.

8. A product that inhibits and / or kills oral microorganisms or their biofilms, characterized in that, The active ingredients of the product include one or more of the Sophora japonica extracts described in any one of claims 1 to 5.

9. The use of the Sophora japonica extract according to any one of claims 1 to 5 or the product according to claim 8 in the preparation of a medicament for the prevention and / or treatment of bacterial dental diseases.

10. The application according to claim 9, characterized in that, The aforementioned bacterial dental diseases include one or more of the following: dental caries, pulpitis, periodontitis, periapical periodontitis, and pericoronitis of wisdom teeth.

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