Wild jujube root compound mouthwash and application thereof

Abstract

The invention discloses wild jujube root compound mouthwash and application thereof, and belongs to the technical field of biology. Through verification, the wild jujube root extracting solution has a wide prospect in the field of oral disease prevention, and the wild jujube root compound mouthwash prepared by the invention has excellent sterilization and anti-caries effects: the antibacterial effect on three bacteria, namely S.mutans, S.ureus and E.coli, is remarkable; the detection result of inhibiting bacterial biofilms shows that the wild jujube root compound mouthwash respectively inhibits the formation of 74%, 80% and 76% of bacterial biofilms of S.mutans, S.aureus and E.coli; the damage rates to three types of formed bacterial biofilms are 65%, 52% and 56% respectively; the invention provides technical support for developing green and safe oral care products.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a compound mouthwash made from jujube root and its application. Background Technology

[0002] With the improvement of living standards, people are paying more and more attention to oral health. However, oral health remains one of the important public health issues affecting human life. Dental caries not only causes physical discomfort and pain, affecting chewing and digestion, but can also lead to tooth loss, impacting speech and appearance, and threatening the patient's physical and mental health. Therefore, the prevention and treatment of dental caries are particularly important. Dental caries is a common chronic infectious disease caused by oral bacteria, which gradually destroys tooth enamel. The causes of dental caries are diverse, involving oral flora imbalance, poor dietary habits, and inadequate oral hygiene. Among these, Streptococcus mutans (S. mutans) is a key pathogen causing dental caries, damaging tooth tissue by producing acidic substances. Therefore, S. mutans is a major focus of research on dental caries prevention and treatment.

[0003] S. mutans is a major natural oral pathogen, arranged in chains, and is an acid-producing and acid-resistant bacterium. S. mutans is a primary cariogenic bacterium that secretes glucosyltransferase, an enzyme that catalyzes the cleavage of sucrose into extracellular glucans. These glucans are the main components of extracellular glucans, promoting S. mutans adhesion and enhancing microbial cohesion within oral biofilms, thereby promoting the formation of bacterial biofilms (dental plaque). Bacterial biofilms are microbial communities in which bacteria adhere to contact surfaces and embed themselves in the extracellular matrix they produce. The formation process of bacterial biofilms includes four stages: (1) the initial adhesion stage; (2) the cell aggregation stage; (3) the bacterial biofilm maturation stage; and (4) the bacterial diffusion stage. Each formation stage involves the expression of specific genes, enabling bacteria to produce extracellular matrix components such as polysaccharides and lipids, which facilitates the formation of bacterial biofilms. The presence of bacterial biofilms is a major obstacle to the treatment of dental caries. Dental plaque biofilms, primarily composed of cariogenic bacteria such as S. mutans, are dense, highly adhesive, and difficult to remove, effectively encapsulating the cariogenic bacteria. Simultaneously, the S. mutans-dominated bacterial biofilm can create a localized acidic environment (pH 4.5), leading to demineralization of the tooth hard tissues and exacerbating caries formation and progression.

[0004] Currently, the main methods for preventing and treating dental caries include physical and mechanical cleaning of dental plaque, application of protective agents, the use of xylitol products to replace sucrose, and antibiotic treatment. Among these, anti-caries products are mainly fluoride-containing chemical products such as fluoride toothpaste and fluoride mouthwash. Although fluoride plays an important role in preventing dental caries, high concentrations of fluoride are cytotoxic; long-term use of antibiotics can lead to drug resistance, disruption of the normal human flora, allergic reactions, and organ toxicity. Therefore, there is increasing interest in using green, low-toxicity plant-based natural products as novel anti-caries drugs. Compared with traditional anti-caries drugs, natural products also have good effects, but with low cytotoxicity and mild, healthy ingredients. Therefore, researching and using a mouthwash with natural plant extracts is urgently needed.

[0005] Sour jujube is an important medicinal and edible plant. Its pulp is rich in protein, dietary fiber, minerals, vitamins, cyclic nucleotides, and other nutrients and functional components, with a vitamin C content of approximately 600-1100 mg per 100 grams. Sour jujube is widely used in pharmaceuticals, health products, anti-aging and skin-whitening cosmetics, and food processing. According to the pharmacopoeia, sour jujube root, as a traditional Chinese medicine, has hemostatic, antibacterial, and anti-inflammatory effects, and is often used in the preparation of burn medications. However, research on the medicinal resources of sour jujube root is currently limited, and its antibacterial and anti-caries development is even rarer. Therefore, sour jujube root has extremely high research and application value, excellent development prospects, and high market potential. Summary of the Invention

[0006] The purpose of this invention is to provide a compound mouthwash made from jujube root and its application, in order to solve the problems existing in the prior art. It has been verified that the compound mouthwash made from jujube root prepared by this invention has excellent sterilization and anti-caries effects.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] This invention provides the application of jujube root extract in any of the following:

[0009] (1) Application in the preparation of antibacterial products;

[0010] (2) Use in the preparation of products for the prevention and / or treatment of dental caries;

[0011] The bacteria include Streptococcus mutans, Staphylococcus aureus, and Escherichia coli.

[0012] The dental caries mentioned include those caused by Streptococcus mutans;

[0013] The jujube root extract is obtained by water extraction and concentration of jujube root bark.

[0014] Optionally, the jujube root extract is prepared by the following method:

[0015] The root bark of jujube is dried and pulverized to obtain root bark powder;

[0016] Take root bark powder, add water, and then heat, filter, concentrate, and dry to obtain jujube root extract;

[0017] The root extract of jujube can be obtained by dissolving it in water.

[0018] Optionally, the heating temperature is 90°C and the heating time is 1 hour.

[0019] The present invention also provides a compound mouthwash containing jujube root extract, comprising the following ingredients: 50% jujube root extract, 25% golden hibiscus extract, 1% poloxamer, 6% sorbitol, 0.4% potassium sorbate, 0.1% citric acid, 0.25% potassium citrate, and 17.25% water;

[0020] The jujube root extract is obtained by water extraction and concentration of jujube root bark.

[0021] The golden sunflower extract was obtained by water extraction and concentration of golden sunflower.

[0022] Optionally, the jujube root extract is prepared by the following method:

[0023] The root bark of jujube is dried and pulverized to obtain root bark powder;

[0024] Take root bark powder, add water, and then heat, filter, concentrate, and dry to obtain jujube root extract;

[0025] The root extract of jujube can be obtained by dissolving it in water.

[0026] Optionally, the golden hibiscus extract is prepared by the following method:

[0027] Dry and pulverize the golden sunflower into powder;

[0028] The powder was added to water, heated, filtered, concentrated, and dried to obtain the extract of golden sunflower.

[0029] Golden flower sunflower extract is obtained by dissolving it in water.

[0030] Optionally, the heating temperature is 90°C and the heating time is 1 hour.

[0031] The present invention also provides the application of the aforementioned jujube root compound mouthwash in the preparation of antibacterial products, wherein the bacteria include Streptococcus mutans, Staphylococcus aureus, and Escherichia coli.

[0032] The present invention also provides the use of the aforementioned jujube root compound mouthwash in the preparation of products for the prevention and / or treatment of dental caries.

[0033] Optionally, the dental caries includes those caused by Streptococcus mutans.

[0034] The present invention discloses the following technical effects:

[0035] This invention focuses on jujube root extract to investigate its antibacterial and anti-caries activities, as well as the efficacy of a jujube root compound mouthwash. First, the MIC method was used to examine the antibacterial activity of the jujube root extract against three common bacteria: *S. mutans*, *S. aureus*, and *E. coli*. The results showed that the MIC values ​​against *S. mutans* and *S. aureus* were 2 mg / mL and 1 mg / mL, respectively, while there was no significant MIC value against *E. coli*, although it still exhibited antibacterial effects. Second, the jujube root extract showed a significant dynamic sterilization effect against *S. mutans*, achieving a sterilization rate of 88% within 15 minutes. The experiments demonstrate that the jujube root extract possesses good antibacterial and antimicrobial activity and has broad application value.

[0036] Further preparation of a compound mouthwash containing jujube root was conducted, and its antibacterial and bactericidal activity was tested. The mouthwash showed antibacterial activity of 100%, 91%, and 97% against *S. mutans*, *S. aureus*, and *E. coli*, respectively, demonstrating significant antibacterial effects. Dynamic sterilization testing showed that the mouthwash achieved sterilization rates of 70%, 25%, and 88% against *S. mutans*, *S. aureus*, and *E. coli* in a short time (2 minutes), with the bactericidal effect increasing significantly with increasing sterilization time. The results of bacterial biofilm inhibition tests showed that the mouthwash inhibited the formation of 74%, 80%, and 76% of the bacterial biofilms of *S. mutans*, *S. aureus*, and *E. coli*, respectively. The destruction rates of the three established bacterial biofilms were 65%, 52%, and 56%, respectively. Finally, cold and heat stability tests and pH measurements showed that the mouthwash had good stability.

[0037] In summary, this invention reveals that jujube root extract has broad prospects in the field of oral disease prevention, and the prepared jujube root compound mouthwash has excellent sterilization and anti-caries effects, possessing significant market value. This invention provides technical support for the development of green and safe oral care products. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 Images of bacterial morphology; (a): S. mutans; (b): S. aureus; (c): E. coli;

[0040] Figure 2 The minimum inhibitory concentrations of the extract were: (a) *S. mutans*; (b) *S. aureus*; (c) *E. coli*.

[0041] Figure 3 The dynamic bactericidal curve of jujube root extract against S. mutans;

[0042] Figure 4 The antibacterial activity of the compound mouthwash containing jujube root; (a): S. mutans; (b): S. aureus; (c): E. coli;

[0043] Figure 5 The sterilization activity of the compound mouthwash made from jujube root; (a): S. mutans; (b): S. aureus; (c): E. coli;

[0044] Figure 6 To inhibit bacterial biofilm activity (MBIC) in jujube root compound mouthwash; (a): crystal violet staining method; (b): MTT assay;

[0045] Figure 7 To investigate the activity of jujube root compound mouthwash in disrupting bacterial biofilm activity (MBRC); (a): crystal violet staining method; (b): MTT assay;

[0046] Figure 8 The stability status of the jujube root compound mouthwash. Detailed Implementation

[0047] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0048] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0049] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0050] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0051] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0052] Example 1

[0053] 1. Experimental Materials and Instruments

[0054] The experimental materials are shown in Table 1, and the experimental instruments are shown in Table 2.

[0055] Table 1 Experimental Materials

[0056]

[0057]

[0058] Table 2 Experimental Instruments

[0059]

[0060]

[0061] 2. Extraction of active ingredients from jujube root

[0062] Ziziphus jujuba is a typical xerophytic plant, possessing extremely strong drought resistance due to its root structure. Therefore, the cortical structure of Ziziphus jujuba roots is complex. The primary structure of Ziziphus jujuba roots includes the epidermis, cortex, and microvascular cylinder; the secondary structure includes the periderm and secondary vascular tissue, with well-developed secondary xylem accounting for 2 / 3 of the entire secondary structure. This embodiment primarily uses the primary root epidermis, cortex, and secondary root periderm as raw materials. The extraction steps are as follows.

[0063] (1) Selection and pretreatment of jujube roots: In winter, without affecting the growth of jujube, dig up the jujube plants and perform root thinning, removing lateral roots and retaining the main root to obtain raw materials, while promoting their growth. Select jujube roots, cut them short, clean them, and leave the root bark for later use;

[0064] (2) Drying and grinding: Place the root bark into a tray and put it into an electric heating drying oven at 60-80℃ for 24 hours. Then put the dried jujube root bark into a grinder to grind it into powder and pass it through a 60-mesh sieve.

[0065] (3) Dissolution and extraction: Take 30g of root bark powder and add it to a volumetric flask, then add 300mL of water, place it in a magnetic stirrer and heat it to a gentle boil. After 4 hours, while it is still hot, put it into a Buchner funnel for filtration (the bottom layer of the funnel is a layer of filter paper and then a layer of silica gel is evenly spread. Stir gently during filtration to prevent the silica gel from clumping, and then filter the filtrate thoroughly).

[0066] (4) Extraction and concentration: The filtrate is placed in a rotary evaporator to completely evaporate the solvent, thereby obtaining an extract with a concentration of 1:1.2. After drying, it is ground to obtain the jujube root extract.

[0067] (5) Preparation of mother liquor: To prepare an 8 mg / mL mother liquor of extract, take 80 mg of dried jujube root extract, add water to dissolve to 10 mL to obtain the mother liquor. After ultrasonic vibration for 30 min, filter through a filter membrane in a purification workbench, sterilize and set aside. The resulting mother liquor is the jujube root extract.

[0068] The extraction method for the golden sunflower extract used in later mouthwash products is the same as that for the jujube root extract.

[0069] 3. Microbial culture

[0070] The study selected S. mutans, a major cariogenic bacterium in the oral cavity, and two common pathogenic bacteria in daily life: the Gram-positive Staphylococcus aureus (S. aureus) and the Gram-negative Escherichia coli (E. coli) as research subjects.

[0071] 3.1 Suspension Bacterial Culture

[0072] (1) Activation: Add 10 μL of frozen bacterial culture to 1 mL of TSB medium and shake in a constant temperature shaker at 37 °C for 24 h at 125 r / min.

[0073] (2) Transfer: Add 10 μL of culture medium to 1 mL of TSB medium, place in a shaker, and incubate at 37°C for 24 h;

[0074] (3) Cryopreservation: After centrifugation at 8000 r / min, discard the supernatant, add TSB medium, then add 10 μL of bacterial solution to 1 mL of TSB medium, shake in a shaker at 37℃ (125 rpm) until the logarithmic growth phase, centrifuge to remove the supernatant, add 500 mL of fresh medium and 500 mL of 50% glycerol, resuspend, and freeze at -20℃.

[0075] 3.2 Bacterial biofilm culture

[0076] To better survive, bacteria attach to surfaces, forming a bacterial biofilm. Sucrose is a significant factor in dental caries; while providing nutrients to bacteria, it also promotes the production of extracellular polysaccharides, greatly enhancing the bacteria's ability to bind to substances. Therefore, sucrose (1% added, abbreviated as TSBS) was added to TSB for bacterial biofilm culture. The culture time was controlled at 1 day, and the specific steps are as follows:

[0077] (1) Preparation of bacterial suspension: The suspension was added to TSB medium and shaken at 37°C for 24 hours at 125 r / min. Then it was centrifuged at 8000 r / min and resuspended in TSBS.

[0078] (2) Inoculate 96-well plate: Add 100 μL of bacterial solution to each well and incubate at 37°C for 24 h (add an appropriate amount of sterile water to the outer wells to prevent the bacterial solution from evaporating and affecting the experimental results).

[0079] 4. Antibacterial activity test of jujube root extract

[0080] 4.1 MIC Method

[0081] The MIC values ​​of jujube root extract against three bacteria—S. mutans, S. aureus, and E. coli—were determined using the microdilution method. The procedure is as follows:

[0082] (1) Preparation of bacterial suspension: Take an appropriate amount of bacterial suspension into TSB medium, incubate at 37℃ with shaking (125 rpm) for 24 h, take 5 μL of bacterial suspension and dilute it with 5 mL of sterile water, shake well and set aside.

[0083] (2) Gradual dilution of jujube root extract concentration: Take 600 μL of 8 mg / mL jujube root extract and add it to 600 μL of sterile water, shake well to prepare 4 mg / mL jujube root extract for later use; take 600 μL of 4 mg / mL jujube root extract and add it to 600 μL of sterile water, shake well to prepare 2 mg / mL jujube root extract for later use; dilute and prepare 1 mg / mL and 0.5 mg / mL jujube root extracts using this method;

[0084] (3) Inoculation of 96-well plates: A blank group, a negative control group, and an experimental group were set up, and 6-7 replicates were selected for each group. 100 μL TSB and 100 μL sterile water were added to each well of the blank group; 100 μL bacterial culture and 100 μL sterile water were added to each well of the negative control group; 100 μL bacterial culture and 100 μL jujube root extract were added to each well of the experimental group. The plates were incubated at 37℃ for 24 h, and the absorbance at 600 nm was measured using a microplate reader.

[0085] (4) Data processing: The relative bacterial content was calculated according to the following formula. A graph was plotted with the concentration of jujube root as the independent variable and the relative bacterial content as the dependent variable to obtain the MIC of jujube root extract.

[0086] M = (OD) 实验 -OD 阴性 ) / OD 空白

[0087] Where M: relative bacterial content, OD 实验 : OD absorbance of the experimental group 600 Average value, OD 阴性 : Negative group absorbance OD 600 Average value, OD 空白 : Absorbance OD of blank group 600 average value.

[0088] 4.2 Plate counting method

[0089] The MIC value only indicates the inhibitory effect of jujube root extract on the growth of suspended bacteria, and cannot reflect the bactericidal rate and lethal effect of jujube root extract. Therefore, the kinetic bactericidal curve of jujube root extract against *S. mutans* was further determined by plate counting method. The specific operation steps are as follows:

[0090] (1) Preparation of bacterial culture: The steps are the same as in 4.1;

[0091] (2) Solution preparation: Take 600 μL of 8 mg / mL jujube root extract and add it to 600 μL of sterile water. Shake well and set aside.

[0092] (3) Kinetic sterilization: Control group: 0.5 mL of diluted bacterial solution was added to 0.5 mL of sterile water, shaken rapidly, and then 10 μL of the mixture was added to 990 μL of sterile water. After thorough mixing and further dilution, 20 μL of the diluted mixture was spread evenly in a petri dish and incubated at 37℃ for 24 h. Colonies were observed and counted. Experimental group: 0.5 mL of diluted bacterial solution was added to 0.5 mL of water prepared with jujube root extract and shaken rapidly. After 30 s, 2 min, 5 min, 8 min, 10 min, and 15 min, 10 μL of the mixture was added to 990 μL of sterile water, shaken thoroughly, and then diluted. 50 μL of the diluted mixture was spread evenly in a petri dish using a spreading stick and incubated at 37℃ for 24 h. Colony distribution was observed and counted.

[0093] (4) Data processing: Based on the total number of bacteria in the control group and the experimental group at each time, the bacterial survival rate at each time was calculated, and the dynamic sterilization curve of jujube root extract on bacteria was obtained.

[0094] 5. Results

[0095] 5.1 Microbial Culture

[0096] After 1 day of incubation on a constant-temperature shaker in solid TSB medium, *S. mutans* colonies were observed to be small, pale yellow, semi-transparent, round colonies, approximately 0.5-1 mm in diameter, with a smooth, glossy surface, no wrinkles, granules, or roughness, a fine and uniform texture, and regular, orderly edges without obvious defects or protrusions, arranged in a chain-like pattern. *S. aureus* colonies were medium-sized, golden yellow, semi-transparent, round colonies, approximately 1-3 mm in diameter, with neat, glossy edges, a smooth, fine, and uniform texture, and randomly arranged. *E. coli* colonies were medium-sized, grayish-white, semi-transparent, round colonies, approximately 2-3 mm in diameter, with a smooth, fine, and uniform texture, and regular edges, and randomly arranged. Detailed colony morphology is as follows: Figure 1 .

[0097] 5.2 Antibacterial activity

[0098] 5.2.1 MIC Method

[0099] The antibacterial effects of jujube root extract on *S. mutans*, *S. aureus*, and *E. coli* were obtained by incubating a gradient solution of jujube root extract with bacterial culture using a micro-dilution method and measuring absorbance. Results Figure 2As shown in the figure, the extract of jujube root showed a significant antibacterial effect against *S. mutans*, with the inhibition rate increasing with increasing extract concentration. The inhibition rate of 0.25 mg / mL jujube root extract against *S. mutans* was 17.66%; 1 mg / mL extract showed 28.30%; and 2 mg / mL extract showed 99.80%. Therefore, the MIC of jujube root extract against *S. mutans* was determined to be 2 mg / mL. Similarly, the extract also showed a very significant antibacterial effect against *S. aureus*, with the inhibition rate increasing with increasing extract concentration. The inhibition rate of 0.5 mg / mL jujube root extract against *S. aureus* was 26.15%; and 1 mg / mL extract showed 99.13%. Therefore, the MIC of jujube root extract against *S. aureus* was determined to be 1 mg / mL. The antibacterial rate of jujube root extract against E. coli also showed an increasing trend with increasing concentration, indicating a certain antibacterial effect. The antibacterial rate of 0.25 mg / mL jujube root extract against E. coli was 19.73%, and that of 4 mg / mL jujube root extract was 33.90%. However, as the concentration increased, the antibacterial rate gradually stabilized after 4 mg / mL, with no significant increase in the antibacterial rate.

[0100] 5.2.2 Plate counting method

[0101] After kinetic sterilization testing ( Figure 3 The results showed that short-time jujube root extract had a bactericidal effect on *S. mutans*, especially the extract obtained in a very short time (30 seconds), which achieved a bactericidal rate of 60.00%; at 2 minutes, the bactericidal rate was 64.48%; at 8 minutes, it reached 72.18%; and at 15 minutes, it reached approximately 88.00%. The bactericidal rate increased with increasing time. Therefore, short-time jujube root extract has a bactericidal effect on *S. mutans*.

[0102] In summary, the aqueous extract of jujube root exhibits antibacterial effects against three bacteria: *S. mutans*, *S. aureus*, and *E. coli*, with MIC values ​​of 2 mg / mL and 1 mg / mL against *S. mutans* and *S. aureus*, respectively. The jujube root extract also demonstrates significant short-term dynamic bactericidal effects against *S. mutans*, with a bactericidal rate approaching 90% after 15 minutes. Therefore, this invention selects jujube root extract as the main antibacterial and sterilizing active ingredient for the subsequent formulation of mouthwash.

[0103] Example 2: Preparation and Validation of Compound Jujube Root Anti-Caries Mouthwash

[0104] 1. Preparation of Jujube Root Compound Anti-Caries Mouthwash

[0105] Sour jujube root extract has a good antibacterial and bactericidal effect on S. mutans. Therefore, sour jujube root extract is used as the bactericidal active ingredient in mouthwash. At the same time, in order to further enhance the antibacterial and bactericidal effect of mouthwash, golden sunflower extract (preparation method is the same as sour jujube root extract) is added.

[0106] The mouthwash uses jujube root extract as the main sterilizing ingredient and golden hibiscus extract as a secondary auxiliary ingredient. Food additives such as poloxamer (a solubilizer), D-sorbitol (a sweetener), potassium sorbate (a preservative), citric acid (a pH adjuster), and potassium citrate (a buffer) are added to form the mouthwash base. The specific formulation ratios of the mouthwash base ingredients are shown in Table 3 below.

[0107] Jujube root compound mouthwash (compound mouthwash), jujube root mouthwash, golden sunflower mouthwash, and base solution were prepared separately for subsequent experimental research.

[0108] The specific preparation steps for 100mL of jujube root compound mouthwash are as follows: Measure 50mL of 8mg / mL jujube root extract (i.e., 50% by volume) and 25mL of 8mg / mL golden hibiscus extract (i.e., 25% by volume); weigh the mouthwash base ingredients separately: 1g poloxamer (emulsifier), 6g D-sorbitol (sweetener), 0.4g potassium sorbate (preservative), 0.1g citric acid (pH adjuster), and 0.25g potassium citrate (buffer); mix the above solutions with the solid reagents, and then dilute to 100mL with sterile deionized water.

[0109] The specific preparation steps for 100mL of jujube root mouthwash are as follows: Measure 75mL of 8mg / mL jujube root extract (i.e., volume percentage of 75%); weigh the mouthwash base components separately: 1g poloxamer (emulsifier), 6g D-sorbitol (sweetener), 0.4g potassium sorbate (preservative), 0.1g citric acid (pH adjuster), and 0.25g potassium citrate (buffer); mix the above solutions with the solid reagents, and then make up to 100mL with sterile deionized water.

[0110] The specific preparation steps for 100mL of golden hibiscus mouthwash are as follows: Measure 75mL of 8mg / mL golden hibiscus extract (i.e., a volume percentage of 75%); weigh the base ingredients of the mouthwash separately: 1g poloxamer (emulsifier), 6g D-sorbitol (sweetener), 0.4g potassium sorbate (preservative), 0.1g citric acid (pH adjuster), and 0.25g potassium citrate (buffer); mix the above solutions with the solid reagents, and then bring the volume to 100mL with sterile deionized water.

[0111] The specific steps for preparing 100mL of base solution are as follows: Weigh 1g poloxamer (emulsifier), 6g D-sorbitol (sweetener), 0.4g potassium sorbate (preservative), 0.1g citric acid (pH adjuster) and 0.25g potassium citrate (buffer), and bring the volume up to 100mL with sterile deionized water.

[0112] Table 3. Mouthwash base preparation ratio

[0113]

[0114] 2. Antibacterial activity test

[0115] The inhibitory effects of mouthwash on the growth of *S. mutans*, *S. aureus*, and *E. coli* were compared and determined using the microdilution method and orthogonal comparison experiments. The specific procedures are as follows:

[0116] (1) Preparation of bacterial culture: Same as 4.1 in Example 1;

[0117] (2) Preparation of compound solutions: Take jujube root compound mouthwash, jujube root mouthwash, golden hibiscus mouthwash and base solution. Under sterile conditions, prepare jujube root extract (8mg / mL) and golden hibiscus extract (8mg / mL), and set up 6 variable experimental groups;

[0118] (3) Inoculation of 96-well plates: 100 μL TSB and 100 μL compound were added to each well of the blank control group (6 groups); 100 μL bacterial culture and 100 μL sterile water were added to each well of the negative control group; and 100 μL bacterial culture and 100 μL compound were added to each well of the experimental group (6 groups). Each group had 5-6 replicates. The plates were incubated at 37℃ on a shaker for 24 h, and the OD was measured using a microplate reader. 600 ;

[0119] (4) Data processing: Calculate the relative bacterial content of each group (the calculation formula is the same as 4.1 in Example 1), plot the antibacterial activity data of each substance with the concentration of jujube root as the independent variable and the relative bacterial content as the dependent variable. Figure 4 ).

[0120] 3. Sterilization activity test

[0121] The bactericidal effects of each group against the three bacteria in a short time were tested using the plate count method. The steps are as follows:

[0122] (1) Preparation of bacterial culture: Same as 4.1 in Example 1;

[0123] (2) Kinetic sterilization: Control group: 0.5 mL of diluted bacterial solution was added to 0.5 mL of sterile water and shaken rapidly. 10 μL of the mixture was added to 990 μL of sterile water, mixed thoroughly, and then diluted again. 20 μL of the diluted mixture was evenly spread in a petri dish and incubated at 37℃ for 24 h. Colony distribution was observed and counted. Experimental group: 0.5 mL of diluted bacterial solution was added to 0.5 mL of mouthwash and shaken thoroughly. After 30 s, 1 min, 2 min, and 5 min, 10 μL of the mixture was added to 990 μL of sterile water, shaken thoroughly, and then diluted again. 50 μL of the diluted mixture was evenly spread in a petri dish and incubated at 37℃ for 24 h. Colony distribution was observed and counted.

[0124] (3) Data processing: Based on the total number of bacteria in the control group and the experimental group at each time, the bacterial survival rate at each time was calculated to obtain the dynamic short-time sterilization effect of each group on bacteria.

[0125] 4. Antibacterial biofilm activity

[0126] To better simulate the dental plaque film environment, this embodiment used 96-well plates to construct 1-day bacterial biofilms in vitro. The inhibitory and destructive activities of the jujube root compound mouthwash on the formation of S. mutans, S. aureus, and E. coli biofilms were investigated by crystal violet staining and MTT staining methods, respectively, from two stages: inhibiting bacterial biofilm formation and destroying the activity of mature biofilms.

[0127] 4.1 Detection of Inhibitory Bacterial Biofilm Activity

[0128] Crystal violet staining: Based on the Gram staining principle, crystal violet stains bacteria blue-violet. Crystal violet is a basic dye; during ionization, the stained areas become positively charged, while the bacterial surface becomes negatively charged. Therefore, crystal violet staining was chosen as the method for detecting the total bacterial count. The experimental steps are as follows:

[0129] (1) Preparation of bacterial culture: Same as 4.1 in Example 1;

[0130] (2) Inoculation of 96-well plates: A blank control group, a negative control group, and an experimental group were set up, with 6-7 replicates in each group. 100 μL TSB and 100 μL sterile water were added to each well of the blank control group; 100 μL bacterial culture and 100 μL sterile water were added to each well of the negative control group; 100 μL bacterial culture and 100 μL Ziziphus jujuba root compound mouthwash were added to each well of the experimental group. The plates were placed in a constant temperature incubator at 37℃ and incubated for 24 h.

[0131] (3) Crystal violet staining: Discard the supernatant, wash away unattached bacteria with sterile water, add 50 μL of methanol to each well, fix at room temperature for 15 minutes, stain with crystal violet (0.1%, 50 μL / well) at 37℃ for 15 minutes, wash away excess staining, remove the supernatant, add 200 μL of 33% acetic acid solution to each well to dissolve the dye, and then measure the OD using a microplate reader. 595 ;

[0132] (4) Data processing: The relative content of biofilm was calculated according to the following formula, and a plot was drawn with mouthwash bacteria as the independent variable and the relative content of biofilm as the dependent variable.

[0133] M = (OD) 实验 -OD 空白 ) / (OD 阴性 -OD 空白 )

[0134] Where M: relative content of bacterial biofilm, OD 实验 : Average absorbance of the experimental group, OD 阴性 : Average absorbance of the negative group, OD 空白 : Average absorbance of the blank group.

[0135] The survival rate of residual biofilm bacteria was further determined using the MTT assay. The experimental steps are as follows:

[0136] (1) Preparation of bacterial culture and inoculation of 96-well plates are the same as the crystal violet staining method described above.

[0137] (2) MTT staining: Remove suspended bacteria from the 96-well plate with sterile water, then add 50 μL of 0.5 mg / mL MTT solution, incubate the plate at 37°C in the dark for 15 min, add 100 μL of DMSO to each well to completely dissolve it, and measure the absorbance at 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0138] (3) Data processing: Same as the crystal violet staining method described above.

[0139] 4.2 Detection of bacterial biofilm disruption activity

[0140] Crystal violet staining method: (1) Preparation of bacterial culture: Same as 4.1 in Example 1;

[0141] (2) Inoculation of 96-well plates: 100 μL of jujube root compound mouthwash was added to each well of the experimental group, 100 μL of TSBS was added to each well of the negative control group, and 100 μL of TSBS was added to each well of the blank control group. The blank control group did not contain any biofilm. Each group was set up in parallel with 6-7 wells and incubated in a constant temperature incubator at 37℃ for 24 h.

[0142] (3) Crystal violet staining and data processing: Same as the operation in 4.1 Crystal violet staining method above.

[0143] MTT method: After completing the first two steps of the experiment according to the crystal violet staining method described above, perform MTT staining to determine the viable bacterial count in the residual biofilm and process the data according to the MTT method in section 4.1 above.

[0144] 5. Stability testing

[0145] To determine the stability of the jujube root compound mouthwash, three temperature gradients (low, medium, and high) were established. The low-temperature sample was stored at -4℃; the medium-temperature sample was stored at 25℃; and the high-temperature sample was stored in an oven at 40℃. 3 mL samples from each temperature gradient were stored in sterile, transparent sample bottles. The samples were observed daily for 90 days, recording any precipitation, pH levels, and other solution conditions.

[0146] 6. Sensory evaluation

[0147] A panel of 30 people was randomly selected to taste the mouthwash samples in turn. The panel scored the mouthwash samples on three aspects: appearance, aroma, and taste. The total score of each individual was calculated. The highest and lowest scores were removed, and the average of the 28 scores was taken as the final evaluation score. (After the evaluators completed the sample evaluation, they rinsed their mouths thoroughly with drinking water and then gave a second evaluation.)

[0148] Total score = Appearance score × 33% + Aroma score × 33% + Taste score 34%.

[0149] The sensory evaluation criteria for jujube root compound mouthwash are shown in Table 4.

[0150] Table 4 Sensory Indicators and Scoring Criteria for Jujube Root Compound Mouthwash

[0151]

[0152]

[0153] 7. Results

[0154] 1. Sensory evaluation of jujube root compound mouthwash

[0155] Sensory rating: 90.50±1.51. The mouthwash is light brownish-yellow in appearance, the liquid is clear and transparent, with a pure and uniform luster, and no insoluble suspended particles or turbid substances; the mouthwash has a pine scent, no off-flavors from other additives, no irritating odor, and the scent is harmonious; the mouthwash tastes cool and refreshing, with a pleasant mouthfeel and a harmonious flavor without any special odor.

[0156] 2. The antibacterial activity of jujube root compound mouthwash

[0157] After incubating the jujube root compound mouthwash with bacterial solution at 37℃, the absorbance was read using an ELISA reader, and an orthogonal control experiment was conducted to compare the antibacterial activities of each group against *S. mutans*, *S. aureus*, and *E. coli*. Figure 4 ).

[0158] For *S. mutans*, the base solution showed some antibacterial activity, with an inhibition rate of approximately 38.00%. *Hippophae rhamnoides* extract showed an inhibition rate of 98.80% against *S. mutans*, but the inhibition rate of *Hippophae rhamnoides* mouthwash was only about 33.50%, indicating a significantly lower antibacterial effect compared to the extract. *Ziziphus jujuba* root extract showed an inhibition rate of approximately 99.40% against *S. mutans*, and *Ziziphus jujuba* root mouthwash showed an inhibition rate of approximately 99.90%, demonstrating significant antibacterial activity in both the mouthwash and the extract. The compound *Ziziphus jujuba* root mouthwash showed even better antibacterial activity against *S. mutans*, nearly reaching 100%.

[0159] For *S. aureus*, the base solution showed an antibacterial rate of approximately 44.00%; the extract of *Hippophae rhamnoides* showed an antibacterial rate of approximately 42.00%; and the mouthwash showed an antibacterial rate of approximately 66.80%, indicating that the formulated mouthwash had a higher antibacterial effect than the extract. The extract of *Ziziphus jujuba* root showed an antibacterial activity of 99.07% against *S. aureus*; and the mouthwash showed an antibacterial rate of approximately 52.00%, indicating that the formulated mouthwash had lower antibacterial activity than the extract. The compound mouthwash containing *Ziziphus jujuba* root showed good antibacterial activity against *S. aureus*, with an antibacterial rate of 91.00%.

[0160] For E. coli, the base solution showed significant anti-E. coli activity, with an inhibition rate of approximately 64.50%. Golden hibiscus extract had no effect on E. coli, but golden hibiscus mouthwash showed an inhibition rate of approximately 81.60%, with the formulated golden hibiscus mouthwash showing a significantly increased antibacterial effect compared to the golden hibiscus extract. Ziziphus jujuba root extract showed weak anti-E. coli activity, with an inhibition rate of 5.50%, while the ziziphus jujuba root mouthwash showed a significant inhibition rate of 86.36%, with the formulated ziziphus jujuba root mouthwash showing a substantial increase in antibacterial activity compared to the ziziphus jujuba root extract. The ziziphus jujuba root compound mouthwash showed the most significant antibacterial effect against E. coli, with an inhibition rate reaching 97.10%.

[0161] In summary and through comparison, the compound mouthwash containing jujube root showed significant activity against three bacteria: S. mutans, S. aureus, and E. coli, especially against the major cariogenic bacterium S. mutans, with an inhibition rate of 100%. The compound mouthwash containing jujube root has a good antibacterial effect.

[0162] 3. The antibacterial activity of jujube root compound mouthwash

[0163] The results are as follows Figure 5 As shown.

[0164] Short-term bactericidal activity against S. mutans: The bactericidal effect increases significantly with increasing sterilization time. The sterilization rate of mouthwash against S. mutans reaches 46.64% after 30 seconds; about 52.00% after 1 minute; and about 70.00% after 2 minutes.

[0165] Short-term bactericidal activity against S. aureus: The sterilization rate of mouthwash against S. aureus is only about 9.00% after 30 seconds; about 25.00% after 2 minutes. The bactericidal effect is slightly improved with increasing sterilization time, but the effect is still average.

[0166] Short-term bactericidal activity against E. coli: The bactericidal effect increases significantly with the increase of sterilization time. The sterilization rate of mouthwash against E. coli is only 14.50% after 30 seconds; it is about 87.70% after 2 minutes.

[0167] Overall, the jujube root compound mouthwash is effective, showing good bactericidal effect against common oral bacteria such as S. mutans, slightly weaker effect against S. aureus, and also good bactericidal effect against E. coli.

[0168] 4. Jujube root compound mouthwash has antibacterial biofilm activity.

[0169] 4.1 Jujube root compound mouthwash inhibits bacterial biofilm activity.

[0170] Residual biofilm was measured using crystal violet staining and residual viable bacteria was determined using the MTT assay. The effect of the jujube root compound mouthwash on inhibiting biofilm activity was then assessed. Results are as follows: Figure 6 As shown.

[0171] The jujube root compound mouthwash showed a 74% inhibition rate against S. mutans bacterial biofilm. Among the 26% bacterial biofilm formed, the MTT assay showed that 35% were dead bacteria and 65% were live bacteria. The jujube root compound mouthwash has a good inhibitory effect on S. mutans bacterial biofilm.

[0172] The jujube root compound mouthwash showed an 80% inhibition rate against S. aureus bacterial biofilm. Among the 20% bacterial biofilm formed, 61% were live bacteria and 39% were dead bacteria. The jujube root compound mouthwash also had a good inhibitory effect on S. aureus bacterial biofilm.

[0173] The jujube root compound mouthwash inhibited the growth of E. coli bacterial biofilm by 76%. Among the 24% of the bacterial biofilm that grew, 55% were live bacteria and 45% were dead bacteria. The jujube root compound mouthwash also had a good inhibitory effect on E. coli biofilm.

[0174] Overall, the jujube root compound mouthwash has a good overall effect against biofilm formation and has a good inhibitory effect on the formation of biofilms of common oral bacteria.

[0175] 4.2 Jujube root compound mouthwash disrupts bacterial biofilm activity.

[0176] The residual amount of bacteria on the bacterial biofilm disrupted by the mouthwash and the live and dead bacteria in the residual biofilm were obtained by crystal violet staining and MTT assay, respectively. Figure 7 ).

[0177] Regarding the S. mutans bacterial biofilm: The jujube root compound mouthwash destroyed 65% of the formed biofilm. Of the remaining 35% of the bacterial biofilm, 27% were dead bacteria and 73% were live bacteria. The jujube root compound mouthwash was effective in destroying the S. mutans bacterial biofilm.

[0178] Regarding the biofilm of S. aureus bacteria: the compound mouthwash containing jujube root has a destruction rate of 52%. Among the 48% of the remaining bacterial biofilm, 86% are live bacteria and 14% are dead bacteria, which has a bactericidal and destructive effect on the biofilm of S. mutans bacteria.

[0179] Regarding E. coli bacterial biofilm: The compound mouthwash containing jujube root had a 56% destruction rate of E. coli bacterial biofilm. Among the 44% of undamaged bacterial biofilm, 20% were dead bacteria and 80% were live bacteria. The compound mouthwash containing jujube root has a destructive effect on E. coli bacterial biofilm.

[0180] In summary, the jujube root compound mouthwash has a destructive effect on the biofilm of three common bacteria and a bactericidal effect on the biofilm of common oral bacteria.

[0181] 5. Stability test of jujube root compound mouthwash using sensory evaluation

[0182] After regular, fixed-point observation, the mouthwash remained clear and free of suspended matter under low, medium, and high temperature conditions for 5 days, demonstrating good stability. After 10 days, the mouthwash maintained good stability under all three temperature conditions. The mouthwash also maintained good stability after 30, 60, and 90 days. The pH value remained neutral, which is within the normal range for the human oral cavity. The mouthwash's stability is satisfactory; room temperature storage is recommended. Figure 8 ).

[0183] In summary, the jujube root compound mouthwash prepared in this invention achieved antibacterial efficiencies of 100%, 91%, and 97% against S. mutans, S. aureus, and E. coli, respectively, demonstrating significant antibacterial effects. Dynamic sterilization testing showed that the mouthwash achieved sterilization rates of 70%, 25%, and 88% against S. mutans, S. aureus, and E. coli in a short time of 2 minutes, respectively, with the sterilization effect increasing significantly with increasing sterilization time. The results of bacterial biofilm inhibition testing showed that the jujube root compound mouthwash inhibited the formation of 74%, 80%, and 76% of the bacterial biofilms of S. mutans, S. aureus, and E. coli, respectively; the destruction rates of the three established bacterial biofilms were 65%, 52%, and 56%, respectively. Stability testing showed normal cold and heat stability within 90 days, indicating that the jujube root compound mouthwash is stable and effective.

[0184] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of jujube root extract in any of the following categories: (1) Application in the preparation of antibacterial products; (2) Use in the preparation of products for the prevention and / or treatment of dental caries; The bacteria include Streptococcus mutans, Staphylococcus aureus, and Escherichia coli. The dental caries mentioned include those caused by Streptococcus mutans; The jujube root extract is obtained by water extraction and concentration of jujube root bark.

2. The application as described in claim 1, characterized in that, The jujube root extract was prepared by the following method: The root bark of jujube is dried and pulverized to obtain root bark powder; Take root bark powder, add water, and then heat, filter, concentrate, and dry to obtain jujube root extract; The root extract of jujube can be obtained by dissolving it in water.

3. The application as described in claim 2, characterized in that, The heating temperature is 90°C and the time is 1 hour.

4. A compound mouthwash containing jujube root, characterized in that, The ingredients include: 50% jujube root extract, 25% golden hibiscus extract, 1% poloxamer, 6% sorbitol, 0.4% potassium sorbate, 0.1% citric acid, 0.25% potassium citrate, and 17.25% water; The jujube root extract is obtained by water extraction and concentration of jujube root bark. The golden sunflower extract was obtained by water extraction and concentration of golden sunflower.

5. The jujube root compound mouthwash as described in claim 4, characterized in that, The jujube root extract was prepared by the following method: The root bark of jujube is dried and pulverized to obtain root bark powder; Take root bark powder, add water, and then heat, filter, concentrate, and dry to obtain jujube root extract; The root extract of jujube can be obtained by dissolving it in water.

6. The jujube root compound mouthwash as described in claim 4, characterized in that, The golden hibiscus extract was prepared by the following method: Dry and pulverize the golden sunflower into powder; The powder was added to water, heated, filtered, concentrated, and dried to obtain the extract of golden sunflower. Golden flower sunflower extract is obtained by dissolving it in water.

7. The jujube root compound mouthwash as described in claim 5 or 6, characterized in that, The heating temperature was 90℃ and the heating time was 1 hour.

8. The application of the jujube root compound mouthwash as described in claim 4 in the preparation of antibacterial products, characterized in that, The bacteria include Streptococcus mutans, Staphylococcus aureus, and Escherichia coli.

9. The use of the jujube root compound mouthwash as described in claim 4 in the preparation of products for the prevention and / or treatment of dental caries.

10. The application as described in claim 9, characterized in that, The dental caries mentioned include those caused by Streptococcus mutans.

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