Camellia nitidissima leaf extract with oral care and bacteriostatic effects as well as preparation method and application of camellia nitidissima leaf extract

Abstract

The invention provides a golden camellia leaf extract with oral care and bacteriostatic effects as well as a preparation method and application thereof, and relates to the technical field of dental oral products. The preparation method comprises the following steps: (1) crushing golden camellia leaves to obtain golden camellia leaf powder; (2) extracting: mixing the golden camellia leaf powder obtained in the step (1) with an ethanol solution with the volume fraction of 75-95%, extracting at 70-100 DEG C, filtering, and collecting an extracting solution; and (3) purification: concentrating the extracting solution obtained in the step (2), carrying out purification treatment, eluting by using an ethanol solution with the volume fraction of 60-80%, and drying to obtain the golden camellia leaf extract. The camellia nitidissima leaf extract is high in flavone content, excellent in DPPH free radical scavenging rate and COX-2 inhibition rate, remarkable in antibacterial effect on candida albicans and malassezia and capable of being applied to preparation of oral care products.

Description

Technical Field

[0001] This invention belongs to the field of dental and oral care products technology, and relates to the extract of golden chrysanthemum tea with oral care and antibacterial effects, its preparation method and application. Background Art

[0002] Golden camellia (scientific name: Camellia nitidissima Who It is a plant of the Theaceae family, growing in acidic soil at altitudes of 200-500 meters, and prefers a warm and humid climate. Its plants are mostly evergreen shrubs or small trees, with hairless young branches and leathery, oblong leaves. The flowers are solitary in the leaf axils, mostly cup-shaped or bowl-shaped, with waxy, transparent, golden-yellow petals that have a unique luster.

[0003] Golden camellia contains over 400 active ingredients, mainly including flavonoids, polysaccharides, tea polyphenols, and saponins. It is a traditional medicinal and edible plant. Studies have shown that golden camellia has effects such as clearing heat and detoxifying, regulating blood pressure and lipids, promoting diuresis and removing dampness, treating dysentery, and preventing tumors. Its polysaccharides and tea pigments can scavenge free radicals and enhance immunity; research has confirmed its significant antioxidant capacity. The "Guangxi Standard for Traditional Chinese Medicine" records that golden camellia tea leaves can treat pharyngitis, nephritis, and ascites due to liver cirrhosis. Traditional Chinese medicine uses it for clearing heat and detoxifying, moistening the lungs and relieving cough. Modern research supports its antibacterial and anti-inflammatory effects, and it is effective against respiratory and digestive tract inflammation.

[0004] Chinese invention patent CN109464541A discloses a method for extracting *Tea laurentii* extract: *Tea laurentii* leaves are dried and pulverized, and a crude extract is obtained by ultrasonic-assisted extraction with an ethanol solution. The crude extract is then further extracted with hexane and ethyl acetate, and the solvent is removed by rotary evaporation. The concentrate is then dried at low temperature to obtain the *Tea laurentii* extract. This extraction method requires large amounts of hexane and ethyl acetate for extraction, using a large amount of organic solvents, making waste treatment difficult. Furthermore, the final extract yields a low total flavonoid content, which is detrimental to the development and utilization of *Tea laurentii*.

[0005] Chinese invention patent CN110201061A discloses a method for extracting flavonoids from Camellia chrysantha: Camellia chrysantha is dried after removing impurities, boiled in water, filtered, and the filtrate is concentrated and then placed on a macroporous resin column for adsorption. The column is eluted sequentially with purified water and an ethyl acetate-ethanol mixture, and the eluent is collected and concentrated under reduced pressure to obtain the Camellia chrysantha extract. This extraction method targets flavonoid glycosides, requiring ethyl acetate extraction to obtain total flavonoids. However, this method results in residual organic solvents such as ethyl acetate in the macroporous resin, and the increased number of extractions may raise production costs, hindering the development and utilization of Camellia chrysantha.

[0006] Golden flower tea extract is widely used in cosmetics and health products due to its unique bioactivity. Existing technologies mainly focus on the application of golden flower tea extract in skin care products such as whitening and moisturizing, with little research on its application in oral care products. Summary of the Invention

[0007] This invention addresses the problems existing in the prior art by providing an oral care and antibacterial extract of *Tea laurentii*, its preparation method, and its application. Through a specific preparation process, the flavonoid content, DPPH free radical scavenging rate, and COX-2 inhibition rate of the *Tea laurentii* extract are significantly improved, and the extract exhibits significant antibacterial effects against *Candida albicans* and *Malassezia*.

[0008] This invention applies golden chrysanthemum tea extract to oral care products, developing an oral care product with a high concentration of golden chrysanthemum tea extract, clear color, good taste, high foaming, low irritation, satisfactory cleaning power, and minimal bitterness.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows: First, this invention provides a method for preparing an extract of *Tea japonica* with oral care and antibacterial effects, comprising the following steps: (1) The golden flower tea leaves are crushed to obtain golden flower tea powder; (2) Mix the golden flower tea powder obtained in step (1) with an ethanol solution with a volume fraction of 75%-95%, extract at 70-100℃, filter, and collect the extract; (3) Purification: The extract obtained in step (2) is concentrated and purified by elution with 60%-80% ethanol solution and dried to obtain the extract of golden flower tea leaves.

[0010] Preferably, in step (1), the particle size of the powder is 80-120 mesh; more preferably 100 mesh.

[0011] Preferably, in step (2), the volume fraction of the ethanol solution is 75%.

[0012] Preferably, in step (2), the extraction temperature is 70-80℃; more preferably 80℃.

[0013] Preferably, in step (2), the extraction is performed 3-5 times, and more preferably 3 times.

[0014] Preferably, in step (2), the extraction time is 2-4 hours each time, and more preferably 3.5 hours.

[0015] Preferably, in step (2), the ratio of the extractant powder of golden chrysanthemum tea to the ethanol solution is 1g:20-30mL, and more preferably 1g:20mL.

[0016] Preferably, in step (3), the concentration specifically involves: concentrating the extract until the solid content is 1-3g:1mL to obtain a concentrated solution.

[0017] More preferably, in step (3), the concentration specifically involves: concentrating the extract until the solid content is 1g:1mL to obtain a concentrated solution.

[0018] Preferably, in step (3), the purification process specifically includes the following steps: packing the pretreated resin into a column, with the weight ratio of the concentrate to the resin being 1:5-20; loading the sample at a rate of 2-4 mL / min, allowing it to stand for 2-3 hours after adsorption for 1-3 hours; rinsing away impurities at a rate of 2-4 mL / min, and then eluting with 2-5 column volumes of 60%-80% ethanol at a rate of 2-4 mL / min and an elution volume of 2-3 BV; collecting the ethanol wash solution and concentrating it to 8%-15% of the total volume.

[0019] More preferably, in step (3), the purification process specifically includes the following steps: packing the pretreated resin into a column, with the weight ratio of the concentrate to the resin being 1:10, loading the sample at a rate of 2 mL / min, allowing it to stand for 3 hours after adsorption for 2 hours; rinsing with water at a rate of 2 mL / min to remove impurities, and then eluting with 2-5 column volumes of 60% ethanol at a rate of 2 mL / min and an elution volume of 2.5 BV; collecting the ethanol rinse solution and concentrating it to 10% of the total volume.

[0020] More preferably, the resin is AB-8 resin.

[0021] Preferably, in step (3), the drying is freeze drying, and the drying time is 40-50 hours; more preferably, it is 48 hours.

[0022] Furthermore, the present invention provides a golden flower tea extract, which is prepared by the above-described preparation method.

[0023] Then, the present invention provides the application of the above-mentioned golden flower tea extract in the preparation of oral care products.

[0024] Finally, the present invention provides an oral care product containing extracts from golden chrysanthemum tea leaves.

[0025] Preferably, the oral care product is mouthwash or toothpaste.

[0026] Preferably, the oral care product is a mouthwash, comprising the following components: cocamidopropyl betaine, tea extract of golden chrysanthemum, glycerin, citric acid, tripotassium citrate, zinc citrate, potassium dihydrogen phosphate, poloxamer 407, propylene glycol, menthol lactate, methyl diisopropylpropionamide, borneol, sodium saccharin, xylitol, trichlorogalactose, sodium benzoate, and water.

[0027] More preferably, the oral care product, by weight percentage, comprises the following components: cocamidopropyl betaine 0.2%-0.4%, *Tea laurel* extract 0.1%-0.3%, glycerin 5%-15%, citric acid 0.1%-0.3%, tripotassium citrate 1%-2%, zinc citrate 0.5%-1.5%, potassium dihydrogen phosphate (to adjust pH to 7.2-7.4), poloxamer 407 1%-3%, propylene glycol 1%-3%, menthol lactate 0.05%-0.15%, methyl diisopropyl propionamide 0.05%-0.15%, borneol 0.1%-0.2%, sodium saccharin 0.02%-0.04%, xylitol 4%-6%, trichlorogalactose 0.01%-0.03%, sodium benzoate 0.1%-0.3%, and the balance being water.

[0028] More preferably, the oral care product, by weight percentage, comprises the following components: 0.30% cocamidopropyl betaine, 0.20% *Tea japonica* extract, 10.00% glycerin, 0.20% citric acid, 1.50% tripotassium citrate, 1.00% zinc citrate, appropriate amount of potassium dihydrogen phosphate (to adjust pH to 7.3), 2.00% poloxamer 407, 2.00% propylene glycol, 0.10% menthol lactate, 0.10% methyl diisopropyl propionamide, 0.15% borneol, 0.03% sodium saccharin, 5.00% xylitol, 0.02% trichlorogalactose, 0.20% sodium benzoate, and the balance being water.

[0029] More preferably, the oral care product may also contain flavoring to adjust the taste and smell.

[0030] Compared with the prior art, the present invention has the following beneficial effects: 1. The preparation method of the present invention involves extracting golden chrysanthemum tea leaves with a specific concentration of ethanol solution, followed by elution and purification using a specific purification method. This significantly increases the flavonoid content, while also significantly improving the DPPH free radical scavenging rate and COX-2 inhibition rate. The resulting golden chrysanthemum tea leaf extract exhibits significant antibacterial effects against Candida albicans and Malassezia.

[0031] 2. This invention applies golden tea extract to oral care products. By compounding it with surfactants, buffers, and preservatives, an oral care product with low irritation, satisfactory cleaning power, good foaming and cleaning performance, and minimal bitterness is obtained. DETAILED DESCRIPTION

[0032] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0033] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0034] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all chemical reagents used in the embodiments of the present invention were obtained through conventional commercial means. All contents mentioned below refer to mass content.

[0035] In the following examples, the golden flower tea leaves were purchased from Hegui Group Company.

[0036] In a specific embodiment of the present invention, the resin used is pretreated by the following methods: (a) taking the resin and soaking it in 90% ethanol overnight; (b) washing it with water until there is no ethanol smell; (c) soaking it in 5% HCl for 8 hours; (d) washing it with water until there is no smell; (e) soaking it in 5% NaOH for 8 hours; (f) washing it with water until there is no smell; and (g) packing it into a column.

[0037] Example 1 A method for preparing an extract of *Tea japonica* with oral care and antibacterial effects: (1) After crushing the golden flower tea leaves with a pulverizer, collect the powder and pass it through a 100-mesh sieve to obtain golden flower tea powder; (2) 20g of golden flower tea powder, 400mL of 75% ethanol, material-to-liquid ratio 1:20, temperature 80℃, hot reflux extraction for 3.5h, repeated extraction 3 times, filter, and collect the extract; (3) The extract was concentrated to 1 g / mL and packed into a column with pretreated AB-8 resin. The weight ratio of the concentrate to the resin was 1:10. The sample was loaded at a rate of 2 mL / min. After adsorption for 2 h, the sample was allowed to stand for 3 h. The sample was washed with water at a rate of 2 mL / min and then eluted with 3 times the volume of 60% ethanol at a rate of 2 mL / min and an elution volume of 2.5 BV. The washing solution was collected, concentrated, concentrated by rotary evaporation, and freeze-dried for 48 h to obtain the extract of golden flower tea leaves.

[0038] Example 2 A method for preparing an extract of *Tea japonica* with oral care and antibacterial effects: (1) Same as Example 1; (2) 20g of golden flower tea powder, 600mL of 90% ethanol, material-liquid ratio 1:30 g:mL, temperature 80℃, hot reflux extraction for 2h, repeated extraction 5 times, filter, and collect the extract; (3) The extract was concentrated to 1 g / mL and packed into a column with pretreated AB-8 resin. The weight ratio of the concentrate to the resin was 1:5 g:mL. The sample was loaded at a rate of 2 mL / min. After adsorption for 1 h, the sample was allowed to stand for 3 h. The sample was washed with water at a rate of 2 mL / min and then eluted with 3 times the volume of 60% ethanol at a rate of 2 mL / min and an elution volume of 2.5 BV. The washing solution was collected, concentrated by rotary evaporation, and freeze-dried for 40 h to obtain the extract of golden flower tea leaves.

[0039] Example 3 A method for preparing an extract of *Tea japonica* with oral care and antibacterial effects: (1) Same as Example 1; (2) 20g of golden flower tea powder, 400mL of 95% ethanol, material-liquid ratio 1:20 g:mL, temperature 70℃, hot reflux extraction for 4h, repeated extraction 3 times, filter, and collect the extract; (3) The extract was concentrated to 1 g / mL and packed into a column with pretreated AB-8 resin. The weight ratio of the concentrate to the resin was 1:20 g:mL. The sample was loaded at a rate of 4 mL / min. After adsorption for 3 h, the sample was allowed to stand for 2 h. The sample was washed with water at a rate of 4 mL / min and then eluted with 5 times the volume of 80% ethanol at a rate of 4 mL / min and an elution volume of 3 BV. The washing solution was collected, concentrated, concentrated by rotary evaporation, and freeze-dried for 50 h to obtain the extract of golden flower tea leaves.

[0040] Comparative Example 1 The difference from Example 1 is that ethanol is replaced with water. Everything else is the same as in Example 1.

[0041] Comparative Example 2 Unlike Example 1, the purification step (3) was not performed. The extract obtained in step (2) was concentrated, the ethanol was evaporated, and the extract was freeze-dried in a freeze dryer for 48 hours. The freeze-dried powder was then collected.

[0042] Comparative Example 3 Unlike Example 1, step (2) is repeated twice. Everything else is the same as in Example 1.

[0043] Comparative Example 4 Unlike Example 1, the volume concentration of ethanol in step (2) is 60%. Everything else is the same as in Example 1.

[0044] Comparative Example 5 Unlike Example 1, the purification steps in step (3) are different.

[0045] (1) Same as Example 1; (2) Same as Example 1; (3) The extract was concentrated to 1 g / mL and packed into a column with pretreated AB-8 resin. The weight ratio of the concentrate to the resin was 1:10. The sample was loaded at a rate of 2 mL / min. After adsorption for 2 h, the sample was allowed to stand for 3 h. The sample was washed with water at a rate of 2 mL / min and then eluted with 3 times the volume of 90% ethanol at a rate of 2 mL / min and an elution volume of 2.5 BV. The washing solution was collected, concentrated, concentrated by rotary evaporation, and freeze-dried to obtain the extract of golden flower tea leaves.

[0046] Comparative Example 6 The preparation process is the same as CN104027477A.

[0047] Golden flower tea leaves were pulverized and passed through a 100-mesh sieve to obtain golden flower tea powder. 1000g of the golden flower tea powder was soaked overnight in 5 times its volume of 70% ethanol, and then extracted three times by heating and reflux. The first extraction was for 3 hours, and the second and third extractions were for 2.5 hours each. The extracts were combined, the solvent was recovered, and a crude extract was obtained. The filtrate had a specific gravity of 1.05-1.25. The obtained filtrate was separated by D101 macroporous resin column chromatography, and eluted sequentially with 10 column volumes of 10%, 30%, 80%, and 95% ethanol and water. The eluates were collected separately, and the solvent of the 80% ethanol and water eluate was recovered. The eluate was then freeze-dried, and the freeze-dried powder was collected.

[0048] I. Results of Detection of Golden Flower Tea Extract 1. Flavonoid content determination experiment (1) Reagent preparation 5% sodium nitrite: Take 10g of sodium nitrite (NaNO2), add deionized water to 200g, and stir well; 10% aluminum nitrate: Take 35.27g of aluminum nitrate nonahydrate (Al(NO3)3·9H2O), add deionized water to 200g, stir well, and let stand; 1 mol / L sodium hydroxide: Take 4 g of sodium hydroxide, add 100 mL of deionized water, and stir well.

[0049] (2) Standard curve determination Accurately weigh 0.1 g of rutin reference standard, dissolve it in 75% ethanol, and dilute to 100 mL in a volumetric flask. The mass concentration of this solution is 1.0 g / L (it can be dissolved by sonication). Accurately pipette 0.0 mL, 2.0 mL, 4.0 mL, 6.0 mL, 8.0 mL, and 10.0 mL into 10 mL volumetric flasks, dilute to the mark with 75% ethanol (v / v), and shake well to obtain reference solutions with mass concentrations of 0.0 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, and 1.0 mg / mL, respectively. Take 1 mL of rutin reference solutions of different concentrations into 10 mL volumetric flasks, add 4 mL of deionized water and 0.3 mL of 5% sodium nitrite aqueous solution in sequence, add 0.3 mL of 10% aluminum nitrate aqueous solution after 5 min, react for 6 min, add 2 mL of 1 mol / L sodium hydroxide aqueous solution and 2.4 mL of deionized water in sequence, measure the absorbance at 510 nm after 10 min, and use 75% ethanol as a blank control; Plot a standard curve with absorbance value A as the ordinate and rutin concentration as the abscissa.

[0050] (3) Sample determination Take 1 mg of the freeze-dried powder of the golden chrysanthemum tea extract from each example and comparative example, dissolve it in pure water to prepare a solution with a mass concentration of 1 mg / mL, add 4 mL of deionized water and 0.3 mL of 5% sodium nitrite aqueous solution in sequence, add 0.3 mL of 10% aluminum nitrate aqueous solution after 5 min, react for 6 min, add 2 mL of 1 mol / L sodium hydroxide aqueous solution and 2.4 mL of deionized water in sequence, and measure the absorbance at 510 nm after 10 min, using pure water as a blank control.

[0051] (4) Results of flavonoid content determination experiment The flavonoid content results for each group are shown in Table 1.

[0052] Table 1 Results of Flavonoid Content Determination Experiment

[0053] Table 1 shows that the highest flavonoid content was obtained in Example 6 after hot reflux extraction with 75% ethanol followed by macroporous resin column chromatography. Compared with the comparative method that changed the extraction and purification parameters, the preparation method of this invention can significantly improve the total flavonoid content in the extract of *Tea japonica*.

[0054] 2. In vitro antioxidant effect experiment DPPH free radical inhibition experiment (1) Preparation of DPPH ethanol solution Weigh 20 mg of DPPH, dissolve it in anhydrous ethanol, and dilute to a final volume of 250 mL in a volumetric flask. The DPPH concentration is prepared to be 2 × 10⁻⁶ mg / mL. -4 mol / L; store protected from light at 0-4℃, prepare and use immediately, effective within 4 hours (positive controls include vitamin C: 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL).

[0055] (2) Preparation of the test solution The freeze-dried powders of golden chrysanthemum tea extract prepared in each example and comparative example were diluted with pure water to prepare test solutions of 1 mg / mL, 0.5 mg / mL, and 0.25 mg / mL, respectively.

[0056] (3) Experimental steps Add the reagents according to Table 2.

[0057] ① Take 1 mL of the test solution and 1 mL of 2×10 -4 Mix the mol / L DPPH solution thoroughly (tube A). ② Take 1 mL of solvent and 1 mL of 2×10 -4 Mix the mol / L DPPH solution thoroughly (tube B). ③ Take 1 mL of solvent and mix it with 1 mL of the test solution (tube C). ④ After reacting in the dark for 30 minutes, measure the absorbance values ​​of tubes A, B, and C at 517 nm.

[0058] Table 2 Reagent Proportioning Table

[0059] (4) Calculation formula for DPPH free radical inhibition rate: DPPH inhibition rate (%) = (B+CA) / B×100% (A, B, and C represent the absorbance of each tube respectively).

[0060] (5) Results of DPPH free radical inhibition experiment are shown in Table 3.

[0061] Table 3. Experimental results of DPPH free radical inhibition

[0062] Table 3 (continued)

[0063] The results showed that the DPPH radical scavenging rate was the highest in Example 1 when the material was extracted by hot reflux with 75% ethanol and then passed through an AB-8 macroporous resin column.

[0064] 3. Evaluation of anti-inflammatory efficacy COX-2 inhibition experiment (1) Sample preparation: The freeze-dried powder of golden tea extract prepared in the examples and comparative examples was diluted with pure water to 0.2 mg / mL and 0.1 mg / mL, respectively.

[0065] (2) Reagent preparation Dissolve all reagents except rhCOX-2 at room temperature, centrifuge to allow the solution to settle to the bottom of the tube, and then mix well before use. COX-2 Probe, COX-2 Cofactor (50x), and COX-2 Substrate (50x) are prepared in dimethyl sulfoxide (DMSO) and can be dissolved in a 37°C water bath for 0.5–2 min to promote dissolution. After use, store immediately at -20°C protected from light.

[0066] Preparation of COX-2 Cofactor working solution: Prepare an appropriate amount of COX-2 Cofactor working solution according to the ratio of 5 μL of COX-2 Cofactor working solution required for each sample. Take an appropriate amount of COX-2 Cofactor (50x) and dilute it with COX-2 Assay Buffer at a ratio of 1:49. For example, add 4 μL of COX-2 Cofactor (50x) to 196 μL of COX-2 Assay Buffer to prepare 200 μL of COX-2 Cofactor working solution. The prepared COX-2 Cofactor working solution can be stored at 4°C and should only be used on the same day.

[0067] Preparation of COX-2 working solution: Prepare an appropriate amount of COX-2 working solution according to the ratio of 5 μL of COX-2 working solution required for each sample. Take an appropriate amount of rhCOX-2 (25x) and dilute it with COX-2 Assay Buffer at a ratio of 1:24. For example, add 8 μL of rhCOX-2 (25x) to 192 μL of COX-2 Assay Buffer to prepare 200 μL of COX-2 working solution. The prepared COX-2 working solution can be temporarily stored on ice; the enzyme activity is basically stable within 1 hour. Note: All operations involving COX-2 should be performed on ice.

[0068] Tyrosinase solution: Dissolve tyrosinase powder with a total enzyme activity of 25 kU in 125 mL of PBS buffer, mix well, and prepare a tyrosinase solution with an enzyme activity of 200 U / mL. Aliquot the solution into 1 mL EP tubes and store at -40°C.

[0069] Preparation of COX-2 Substrate Working Solution: Prepare an appropriate amount of COX-2 Substrate working solution according to the ratio of 5 μL of COX-2 Substrate working solution required for each sample. Take an appropriate amount of COX-2 Substrate (50x), add an equal volume of Substrate Buffer, and vortex thoroughly. Dilute this mixture with Milli-Q grade pure water or redistilled water at a ratio of 1:24, and vortex thoroughly. For example, add 20 μL of COX-2 Substrate (50x) to 20 μL of Substrate Buffer, vortex thoroughly, then add 960 μL of Milli-Q grade pure water or redistilled water, and vortex thoroughly again to obtain 1 mL of COX-2 Substrate working solution. The prepared COX-2 Substrate working solution can be temporarily stored on ice and is relatively stable for up to 1 hour. Note: The COX-2 Substrate working solution can also be prepared during the sample detection process by incubating at 37°C for 10 minutes.

[0070] Preparation of the positive control inhibitor Celecoxib solution: The positive control inhibitor Celecoxib provided in this kit is at a concentration of 100 µM, prepared in DMSO. It can be diluted to the desired concentration or concentration gradient using the same solvent as the analyte inhibitor, as needed. Typically, the IC50 of Celecoxib is... 50 It is approximately 10nM-100nM.

[0071] (3) Sample testing ① Referring to Table 4, set up control wells and sample wells using a 96-well blackboard, and add the sample and each solution in the order shown in Table 4. After adding the sample to be tested, mix well and incubate at 37°C for 10 minutes.

[0072] Table 4 Reagent Proportioning Table

[0073] ② Add 5 μL of COX-2 Probe to each well.

[0074] ③ Quickly add 5 μL of COX-2 Substrate working solution to each well and mix well. Note: The reaction will start immediately after adding the COX-2 Substrate working solution. If there are many wells, you can operate at a low temperature or use a multi-pipette to reduce the error caused by the time difference in adding the COX-2 Substrate working solution between wells. Mixing can also be done on a culture plate shaker.

[0075] ④ After incubating at 37℃ in the dark for 5 minutes, perform fluorescence measurement. The excitation wavelength is 560nm and the emission wavelength is 590nm. If the fluorescence reading is low, the incubation time can be appropriately extended to 10-20 minutes.

[0076] (4) Calculation ① Calculate the average fluorescence value of each sample well and blank control well, which can be recorded as RFU (Relative Fluorescence Unit) blank control, RFU 100% enzyme activity control, RFU positive inhibitor control and RFU sample, respectively.

[0077] ② Calculate the inhibition percentage for each sample. The calculation formula is as follows: Inhibition rate (%) = (RFU 100% enzyme activity control - RFU sample) / (RFU 100% enzyme activity control - RFU blank control) × 100%.

[0078] (5) The results of the COX-2 inhibition experiment are shown in Table 5.

[0079] Table 5. Results of COX-2 Inhibition Experiment

[0080] The results showed that the COX-2 inhibition rate was highest in Example 1 when the COX-2 was extracted by hot reflux with 75% ethanol and then passed through a macroporous resin column.

[0081] 4. Evaluation of antibacterial efficacy Evaluation of the efficacy of Candida albicans and Malassezia (1) Culture of Candida albicans and Malassezia Shake the culture medium with Candida albicans and Malassezia thoroughly (until no sterile clumps settle at the bottom). Using a sterile syringe pipette, transfer 200-800 μL of the bacterial suspension into a new culture medium and incubate at 32°C in a biochemical incubator. After 3-5 days of incubation, subculture.

[0082] (2) Microbroth method for testing the antibacterial activity of samples (2.1) Sample preparation Prepare high-concentration stock solutions according to the required concentration. For solutions insoluble in water, use DMSO; for solutions soluble in water, use PBS.

[0083] (2.2) Preparation of fresh culture medium Transfer sterile Sabouraud / malt extract medium into a sterile 50 mL centrifuge tube. Centrifuge the medium at 25°C and 3500 rpm for 10 min. Transfer the supernatant to another sterile 50 mL centrifuge tube for later use. For each sample, dilute the fresh medium to the desired concentration.

[0084] (2.3) Extraction of Candida albicans and Malassezia Gently mix the culture medium with Candida albicans and Malassezia, and transfer to a sterile 50 mL centrifuge tube. Centrifuge at 25°C and 1000 rpm for 30 seconds. Transfer the supernatant to a sterile 50 mL centrifuge tube and centrifuge at 25°C and 3500 rpm for 10 minutes.

[0085] (2.4) Determination of bacterial concentration Turn on the microplate reader and set the wavelength to 600 nm. Transfer 400 μL of the bacterial culture to a 1.5 mL sterile centrifuge tube. Transfer 200 μL of the bacterial culture from the 1.5 mL centrifuge tube containing the 400 μL culture to a standard 96-well plate. Measure the OD of the bacterial culture at 600 nm. 600 Value. If OD 600 If the OD value is >1, dilute with sterile PBS. When the OD value equals 1, proceed to the next step of the experiment.

[0086] (2.5) Dilution of bacterial culture Calculate the required volume of bacterial suspension. Add the bacterial suspension (OD) 600 =1) Dilute with fresh culture medium 160 times (Candida albicans, Malassezia).

[0087] (2.6) Paving In sterile 96-well plates, 100 μL of diluted bacterial suspension was added to each well, followed by 100 μL of samples of different concentrations. A control group consisted of 100 μL of samples of different concentrations plus 100 μL of Sabouraud / malt extract medium. A blank control was prepared using 200 μL of Sabouraud / malt extract medium without Candida albicans or Malassezia. A negative control was prepared using 100 μL of diluted bacterial suspension plus 100 μL of Sabouraud / malt extract medium. A positive control was prepared using 100 μL of diluted bacterial suspension plus 100 μL of 16 µg / mL ketoconazole. Each control group was prepared in triplicate. After plating, the plates were incubated at 32°C for 48 h and 72 h, and the OD value at 600 nm was measured.

[0088] (2.7) Inhibition rates of Candida albicans and Malassezia:

[0089] The inhibition rate is calculated using the OD formula.

[0090] (2.8) The inhibition rate test results of Candida albicans and Malassezia are shown in Tables 6 and 7.

[0091] Table 6. Inhibition rate (%) of *Candida albicans* extract by *Tea laurentii*

[0092] Table 6 (continued)

[0093] Examples 1-3 showed the ability to inhibit Candida albicans at concentrations above 0.625 mg / mL, and the inhibition rate against Candida albicans was significantly better than that of the comparative example.

[0094] Table 7. Inhibition rate (%) of *Tea laurel* extract against *Malassezia*.

[0095] Table 7 (continued)

[0096] The experimental results show that Examples 1-3 have the ability to inhibit Malassezia at concentrations above 0.625 mg / mL, and the inhibitory effect is better than that of the comparative example; Example 1 has the ability to inhibit Malassezia at concentrations above 2.5 mg / mL, and its MIC50 is 1.99 mg / mL.

[0097] II. Formula for mouthwash containing golden flower tea extract Application Examples A mouthwash containing golden chrysanthemum tea extract, the formula of which is shown in Table 8 by weight percentage.

[0098] Table 8

[0099] Comparative Experiment 1 Unlike Application Example 1, the surfactants are different, with cocamidopropyl betaine (Group 1) being replaced by sodium lauryl sulfate (Group 2) or sophorolipid (Group 3).

[0100] The taste, foaming properties, and cleaning power of mouthwash were evaluated using the Roche foam analysis method.

[0101] The results show that: Taste: Groups 1-2 had no obvious taste sensation, while Group 3 had a milk candy taste, slightly leaning towards the beverage, creating the illusion of improper cleaning.

[0102] Foaming properties: Group 1 > Group 2 > Group 3.

[0103] Cleaning power: Group 1 >> Group 2 = Group 3. Group 1 produces a noticeable astringent feeling after rinsing, and its excessive cleaning power may irritate the oral mucosa. Groups 2 and 3 do not produce a noticeable bitter or astringent feeling after rinsing, and their cleaning power is within an acceptable range.

[0104] ">>" indicates much greater than.

[0105] Therefore, cocamidopropyl betaine, as a surfactant, provides mild foaming, moderate cleaning power without producing astringency, and does not significantly alter the overall taste of the formulation.

[0106] Comparative Experiment 2 Unlike Application Example 1, the amount of cocamidopropyl betaine used is different.

[0107] Five parallel experiments were conducted with concentrations of cocamidopropyl betaine of 0%, 0.1%, 0.3%, 0.5%, and 1%, respectively.

[0108] Take 10g of sample and rinse your mouth for 3-5 minutes. Compare the differences in taste between mouthwash formulas, i.e., taste and foaming properties. Ten testers evaluated the different mouthwashes.

[0109] The results show that Taste: There were no significant changes in taste in the five groups.

[0110] Foaming properties: The foaming power of the 0.1% concentration group is almost similar to that of the 0% group. The foaming power of the 1% concentration group is too strong. The foam fills the mouth with a little rinsing and may even overflow. The foaming power of the 0.3% and 0.5% groups is appropriate and does not produce any adverse reactions. There is no significant difference in the amount of foam produced between the two groups.

[0111] Considering both foaming power and economic efficiency, 0.3% cocamidopropyl betaine is the most suitable, offering appropriate foaming power and cleaning ability.

[0112] Comparative Experiment 3 Unlike Application Example 1, the amount of golden chrysanthemum tea extract used is different.

[0113] Golden camellia tea extract was used as a plant antibacterial agent. It is rich in triterpenoid saponins and terpenoid compounds that alter the cell membrane permeability of acid-producing bacteria. 2 mg / mL of golden camellia tea extract has an effect on acid-producing bacteria.

[0114] ① Five parallel experiments were conducted using extracts of golden chrysanthemum tea leaves as antibacterial agents, with concentrations of 0%, 0.2%, 0.5%, 1%, and 2%, respectively.

[0115] ② Take 10g of sample and rinse your mouth for 3-5 minutes to compare the differences in taste between the different formulas, namely, the irritation and the flavor.

[0116] Ten testers evaluated different mouthwashes.

[0117] The results show that: Irritation: No obvious oral irritation was observed in any of the five groups.

[0118] Taste: At concentrations of 0.2-0.5%, there is almost no significant difference from the 0% group. The 0.5% group has a slight tea flavor, the 1% group has a slightly bitter taste, and the 2% group has a stronger bitter taste, which directly affects the overall taste of the mouthwash.

[0119] Comparative Experiment 4 Unlike Application Example 1, the buffer is different. The normal oral pH is 6.8-7.8. Saliva acts as the main buffer, primarily composed of dihydrophosphate / hydrophosphate and carbonate / bicarbonate. This formula uses a buffer to mimic the pH of oral saliva, thus mitigating the problem of decreased oral pH and maintaining a healthy oral pH level.

[0120] The pH of the mouthwash was adjusted to between 7.2 and 7.4 using appropriate amounts of citric acid / sodium citrate, citric acid / zinc citrate / potassium dihydrogen phosphate, and 10% HCl / sodium citrate, respectively, as detailed in Table 9.

[0121] ① Take 10g of sample and rinse your mouth for 3-5 minutes to compare the differences in taste between the formulas.

[0122] ②Measure the oral pH value after 0h, 2h and 6h for unused products and single-use samples. The specific values ​​are shown in Table 10.

[0123] Table 9 Buffers

[0124] Ten testers evaluated different mouthwashes.

[0125] result: Irritation: No. 1 and 2 have no obvious oral irritation, while No. 3 has a slightly stinging sensation; Taste: No obvious astringency or bitterness was observed in any of the three experimental groups.

[0126] Table 10 Changes in oral pH before and after using mouthwash

[0127] result: All three groups of experiments showed that a single application for 0-2 hours effectively reduced oral pH levels and maintained a healthy pH range of 6.8-7.8 compared to no application.

[0128] After 6 hours, the oral pH of experimental groups 1 and 3 decreased significantly, but the oral pH of experimental group 2 remained within the healthy range. Therefore, buffer reagent citric acid / zinc citrate / potassium dihydrogen phosphate (application example 1) is better.

[0129] Comparative Experiment 5 Unlike Application Example 1, the sweetener is different.

[0130] Sweeteners are mainly used to neutralize the bitterness of the cooling agent itself and improve the taste of mouthwash.

[0131] Group 1 (Application Example 1): A compound of 0.03% sodium saccharin, 5% xylitol, and 0.02% trichlorogalactose; Group 2: Single ingredient 5.5% sodium saccharin; Group 3: Single ingredient 5.5% xylitol; Group 4: Single ingredient 5.5% trichlorogalactose.

[0132] Ten testers evaluated different mouthwashes.

[0133] Single sweeteners have a monotonous and cloying taste; sodium saccharin has insufficient aftertaste and a slightly metallic taste; xylitol is not sweet enough overall; trichlorogalactose has a slow onset of sweetness; the combination of sodium saccharin, xylitol and trichlorogalactose has a moderate sweetness and reduces the cloying sweetness of using a single sweetener.

[0134] Comparative Experiment 6 Unlike Application Example 1, the preservative is different.

[0135] Preservatives ensure that products do not deteriorate. Common preservatives include phenoxyethanol, triclosan, and sodium benzoate.

[0136] Modify the preservative composition in Application Example 1: Group 1: 0.2% phenoxyethanol; Group 2: 0.2% triclosan; Group 3: 0.2% sodium benzoate (application is in Example 1).

[0137] Take 10g of the sample and rinse your mouth for 3-5 minutes.

[0138] Ten testers evaluated different mouthwashes.

[0139] Both Group 1 and Group 2 produce a slightly astringent taste. The 0.2% sodium benzoate group has no obvious astringency and has little impact on the taste. Therefore, odorless sodium benzoate is used as the preservative, and glycerin humectant and flavoring can be added appropriately.

[0140] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing an extract of *Tea japonica* with oral care and antibacterial effects, characterized in that, Includes the following steps: (1) The golden flower tea leaves are crushed to obtain golden flower tea powder; (2) Mix the golden flower tea powder obtained in step (1) with an ethanol solution with a volume fraction of 75%-95%, extract at 70-100℃, filter, and collect the extract; (3) Purification: The extract obtained in step (2) is concentrated and purified by elution with 60%-80% ethanol solution and dried to obtain the extract of golden flower tea leaves.

2. The preparation method according to claim 1, characterized in that, In step (1), the particle size of the powder is 80-120 mesh.

3. The preparation method according to claim 1, characterized in that, In step (2), the volume fraction of the ethanol solution is 75%.

4. The preparation method according to claim 1, characterized in that, In step (2), the extraction temperature is 70-80℃.

5. The preparation method according to claim 4, characterized in that, In step (2), the extraction temperature is 80°C.

6. The preparation method according to claim 1, characterized in that, In step (2), the extraction is performed 3-5 times; the extraction time is 2-4 hours each time; the ratio of the golden flower tea powder to the ethanol solution is 1g:20-30mL.

7. The preparation method according to claim 1, characterized in that, In step (3), the concentration specifically refers to: concentrating the extract until the solid content is 1-3g:1mL to obtain a concentrated solution; In step (3), the purification process specifically involves the following steps: packing the pretreated resin into a column, with a weight ratio of concentrate to resin of 1:5-20; loading the sample at a rate of 2-4 mL / min, allowing it to stand for 2-3 hours after adsorption; rinsing to remove impurities at a rate of 2-4 mL / min, and then eluting with 2-5 column volumes of 60%-80% ethanol at a rate of 2-4 mL / min and an elution volume of 2-3 BV; collecting the ethanol wash solution and concentrating it to 8%-15% of the total volume; the resin is AB-8 resin.

8. The golden flower tea extract prepared by the preparation method according to any one of claims 1-7.

9. The application of the golden chrysanthemum tea extract according to claim 8 in the preparation of oral care products.

10. An oral care product containing extract of golden chrysanthemum tea leaves, characterized in that, The product includes the following components: the golden flower tea extract prepared by the preparation method according to any one of claims 1-7.

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