A solubilized stable liquid preparation of turnera and a method of preparing the same

Abstract

The application discloses a solubilization-stable liquid preparation of Trollius chinensis Bunge and a preparation method thereof. Anionic surfactant is used as a micelle carrier. Through a stable micelle structure formed in the water phase, the poorly soluble active ingredients in Trollius chinensis Bunge extract are dissolved and uniformly dispersed in the water phase, so that the solubility and system stability are significantly improved, and the bacteriostatic effect is improved. Through screening of formula components and optimization and adjustment of the composition ratio, the application successfully prepares a clear liquid preparation with excellent bacteriostatic activity from the poorly soluble substances of Trollius chinensis Bunge. The inhibition rate of the preparation on Escherichia coli, Staphylococcus aureus and Candida albicans is more than 90% after 30s of contact. The preparation has good stability after being placed at room temperature for 24 months, and the inhibition rate on the three test bacteria is still more than 80%. The concentration of Trollius chinensis Bunge extract required to achieve the same or even better bacteriostatic effect is far lower than 1 / 16 of the Trollius chinensis Bunge liquid preparation assisted by dimethyl sulfoxide (DMSO) organic solvent.

Description

Technical Field

[0001] This invention relates to the technical field of personal care products, disinfection products, and pharmaceutical and health products, and in particular to a solubilized and stabilized nasturtium liquid preparation, its preparation method, and its application. Background Technology

[0002] As people's living standards improve, the incidence of oral problems also increases, seriously affecting their quality of life, social interactions, and mental health. Oral problems can be caused by a variety of pathogenic microorganisms, including bacteria and fungi. These pathogens can migrate downwards, leading to pneumonia, chronic gastritis, and other conditions. If local infections and inflammation in the mouth are neglected for a long time, bacteria, toxins, and inflammatory factors can spread throughout the body via the bloodstream and lymphatic system, inducing and aggravating various systemic diseases through mechanisms such as affecting nutrient absorption and triggering chronic inflammatory responses. Examples include atherosclerosis, coronary heart disease, and infective endocarditis. Therefore, oral health is an important component of overall health. Oral hygiene is not only key to preventing oral inflammation, toothache, and tooth loss, but also a crucial link in reducing the risk of systemic diseases and maintaining overall health. In recent years, mouthwash has become an increasingly popular and effective oral hygiene method. Currently, most oral care products on the market contain chemical ingredients, which, while having significant antibacterial effects, can have adverse effects on the human body. Some ingredients have poor solubility in water and require the addition of alcohol for dissolution, posing a risk of inadequate suitability for children and sensitive individuals. Natural plant extracts are gaining attention and popularity due to their low toxicity and multiple bioactivities. For example, Scutellaria baicalensis and honeysuckle have been used in oral care products such as mouthwash.

[0003] Golden lotus, a perennial herb belonging to the genus *Trollius* in the family Ranunculaceae. The *Compendium of Materia Medica Supplement* describes it as having the effects of reducing swelling, clearing heat and detoxifying, and improving eyesight. It is effective for colds with fever, sore throat, bleeding gums, and swollen and painful gums. It is particularly effective for upper respiratory tract infections caused by heat in the throat, mouth, eyes, and lungs. Golden lotus contains a large amount of flavonoids and small-molecule phenolic acids, which are the main substances responsible for its antibacterial and anti-inflammatory effects. However, these components have low solubility in water, often leading to the precipitation of large amounts of sediment in the extract, which limits the development of liquid preparations. Summary of the Invention

[0004] The purpose of this invention is to address the technical shortcomings of existing technologies where the extract of *Trollius chinensis* has low solubility and poor stability in aqueous phase due to its poorly soluble components, making it impossible to directly prepare a clear liquid preparation with antibacterial activity. This invention provides a solubilized and stabilized liquid preparation of *Trollius chinensis*, its preparation method, and its application. The preparation process is simple, the system is stable, and it has significant advantages such as safety, broad spectrum, and high efficiency in antibacterial effects.

[0005] To achieve the above objectives, the technical solution provided by this invention is as follows:

[0006] A solubilizing and stabilizing liquid formulation of *Trollius chinensis* comprises, by mass percentage: 0.02%-0.8% *Trollius chinensis* extract, 0.02%-0.8% micelle carrier, 4%-10% additives, and the remainder being water.

[0007] Preferably, the micelle carrier is constructed from a surfactant, which is an anionic surfactant, including at least one of sodium dodecyl sulfate, sodium dodecyl sulfonate, and sodium dodecylbenzene sulfonate.

[0008] Preferably, the additive includes at least one of flavoring agents, flavorings, and preservatives. The flavoring agents include xylitol, sorbitol, erythritol, and steviol glycosides. The flavorings include menthol, citronellol, and eugenol. The preservatives include parabens and their sodium salts, benzoic acid and its sodium salts, potassium sorbate, and chlorhexidine digluconate.

[0009] Preferably, the golden lotus extract is prepared by one of the following methods: water decoction, ethanol reflux extraction, and water extraction with alcohol precipitation. Its main active ingredients include flavonoids and phenolic acids, and it is rich in characteristic components such as styracin, vitexin, and veratril.

[0010] Preferably, the golden lotus extract is prepared by water decoction, and the specific preparation process is as follows:

[0011] Add 12 times the amount of purified water to dried golden lotus flowers and reflux extract 1-2 times at 100℃, each time for 0.5-2.5 hours. Combine the decoctions and concentrate under reduced pressure at 25℃ to a density of 1.2-1.3 g / mL. Dry the concentrate to obtain golden lotus flower water extract powder as golden lotus flower extract.

[0012] Preferably, the golden lotus extract is prepared by ethanol reflux extraction, and the specific preparation process is as follows:

[0013] Add 12 times the amount of 70% ethanol to dried golden lotus flowers and reflux extract 1-2 times at 80℃, each time for 0.5-2.5 hours. Combine the extracts and concentrate under reduced pressure at 25℃ to a density of 1.2-1.3 g / mL. Dry the concentrate to obtain golden lotus flower ethanol extract powder as golden lotus flower extract.

[0014] Preferably, the golden lotus extract is prepared by water extraction and alcohol precipitation, and the specific preparation process is as follows:

[0015] Add 8-12 times the amount of purified water to dried golden lotus flowers and decoct 1-2 times, 0.5-2.5 hours each time. Combine the decoctions and concentrate under reduced pressure at 25°C to a density of 1.2-1.3 g / mL. Then add ethanol while stirring, and precipitate the alcohol to an alcohol content of 65%-75%. Concentrate the supernatant under reduced pressure and dry to obtain golden lotus flower water-ethanol precipitate powder as golden lotus flower extract.

[0016] The above-mentioned method for preparing a solubilized and stabilized liquid formulation of *Trollius chinensis* involves the self-assembly of anionic surfactants in an aqueous phase to form stable micelles, which then interact with *Trollius chinensis* extract to generate a stable micelle-solute complex. This micelle-solute complex effectively disperses the poorly soluble components in the *Trollius chinensis* extract within the micelle system, ultimately forming a clear liquid formulation with antibacterial activity.

[0017] Preferably, the clear liquid preparation obtained by this method has an inhibitory effect on at least one of Escherichia coli, Staphylococcus aureus, and Candida albicans (inhibition rate >50%). The method for verifying the inhibitory effect is as follows: the clear liquid preparation is mixed with a suspension of the target bacteria (concentration 1×10⁻⁶). 5 ~1×10 7 After mixing the CFU / mL solution and contacting it for 30 seconds, take the mixture and place it in a sterile petri dish. Pour in nutrient agar medium (suitable for Escherichia coli and Staphylococcus aureus) or Sabouraud agar medium (suitable for Candida albicans). After incubating at 35-37°C for 48-72 hours, use the same volume of physiological saline as a blank control group to calculate the antibacterial rate.

[0018] The clear liquid preparation obtained above can be used in antibacterial cleaning products, oral care products, and health care products, specifically including antibacterial cleaning care for oral mucosa, pharyngeal mucosa, teeth, and gums, wherein the antibacterial cleaning care targets at least one of Escherichia coli, Staphylococcus aureus, and Candida albicans.

[0019] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0020] In a first aspect, the present invention provides an anionic surfactant micelle solubilization strategy for poorly soluble substances in nasturtium extract. By optimizing the specific formulation and preparation process for micelle solubilization of nasturtium extract, the solubilization of nasturtium extract is achieved, and it can be used for the construction of stable liquid systems and the application of clear micelle antibacterial agents or products.

[0021] Secondly, this invention exhibits significant advantages in antibacterial activity, greatly reducing the amount of raw materials required. The micelle solution of *Trollius chinensis* extract prepared by this invention effectively promotes the release and efficacy of active ingredients. Experimental results show that, using the technical solution of this invention, when the concentration of *Trollius chinensis* extract is as low as 0.2% (w / v), its antibacterial rate against *Escherichia coli*, *Staphylococcus aureus*, and *Candida albicans* can reach over 90% after 30 seconds of contact. In contrast, *Trollius chinensis* extract using dimethyl sulfoxide (DMSO) as a solvent, even at a concentration as high as 3.2% (w / v), still shows lower antibacterial effects against the above three test bacteria than the 0.2% concentration formulation of this invention. The amount of *Trollius chinensis* extract required to achieve the same or even better antibacterial effect is less than 1 / 16 of that in the comparative example. Therefore, this invention significantly improves efficacy by solubilizing and releasing the active ingredients of *Trollius chinensis* through micelles, greatly reducing the consumption of *Trollius chinensis* extract raw materials, and possesses outstanding economic efficiency, practicality, and real-world application value.

[0022] Thirdly, this invention utilizes anionic surfactants as carriers to form micelles, thereby solubilizing the poorly soluble substances in *Trollius chinensis* extract. This significantly improves the uniform dispersion and physical stability of the poorly soluble components in the aqueous system, achieving clarity, homogeneity, and long-term stability of the system. The *Trollius chinensis* extract micelle solution system provided by this invention maintains good stability in appearance after 24 months at room temperature, without precipitation, turbidity, or stratification. After 24 months of storage at room temperature, the *Trollius chinensis* extract micelle solution system provided by this invention still maintains good antibacterial activity, with an inhibition rate of over 90% against *Escherichia coli* and *Staphylococcus aureus*, and an inhibition rate of not less than 80% against *Candida albicans*. Compared to comparative studies using nonionic or cationic surfactants with added alkaline components for solubilization, this invention demonstrates superior technical effects in terms of antibacterial efficacy, clarity, and system stability.

[0023] Fourthly, this invention demonstrates significant advantages in environmental resistance. After three rounds of repeated freeze-thaw stability tests, the nasturtium extract micelle solution prepared using this invention remained stable, without precipitation, turbidity, or stratification, exhibiting excellent environmental stability and the ability to withstand low-temperature storage and transportation. Attached Figure Description

[0024] Figure 1 The stability appearance diagrams are for the newly formulated examples 1 and 1, 2 and 4.

[0025] Figure 2 The images show (a) an appearance diagram, (b) a particle size distribution, (c) high-performance liquid chromatograms of erythritol, vitexin and veratronic acid reference standards, and (d) a high-performance liquid chromatogram of the newly prepared liquid system in Example 2.

[0026] Figure 3 This is a schematic diagram illustrating the promoting effect of freshly prepared sample in culture plate for 30 seconds on the growth and reproduction of Candida albicans, which is a comparative example.

[0027] Figure 4 The images show the stability and appearance of Examples 1 and 3 after being placed at room temperature for 3, 6, 9, 12, and 24 months.

[0028] Figure 5 The image shows the stability of Example 1 after three rounds of freeze-thaw cycle testing.

[0029] Figure 6 This is a schematic diagram illustrating the antibacterial effect of the newly prepared composition of Example 1 after being placed in a culture plate for 30 seconds after 24 months of storage.

[0030] Figure 7 This is a schematic diagram illustrating the antibacterial effect of the newly prepared composition of Example 3 after 24 months of storage and contact with a culture plate for 30 seconds.

[0031] Figure 8 This is a schematic diagram showing the antibacterial rate of the newly prepared formulations in Examples 1 and 3 after contact with a culture plate for 30 seconds.

[0032] Figure 9 This is a schematic diagram showing the antibacterial rate of Examples 1 and 3 after 24 months of storage and contact with a culture plate for 30 seconds. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0034] Example 1

[0035] This embodiment discloses a solubilized and stabilized liquid formulation of *Trollius chinensis*, with the following specific formulation components and proportions: 0.2% *Trollius chinensis* extract, 0.2% sodium dodecyl sulfate, 4% xylitol, 0.02% menthol, 0.1% methylparaben, 0.02% propylparaben, and purified water added to 100 mL. The above raw materials are mixed and then dissolved by stirring and sonication to obtain a liquid formulation containing *Trollius chinensis* extract.

[0036] The above-mentioned golden lotus extract was prepared by water extraction and alcohol precipitation. The specific preparation process is as follows: dried golden lotus flowers were added to 12 times the amount of purified water and decocted twice, one hour each time. The decoctions were combined and concentrated under reduced pressure at 25°C to a density of 1.25 g / mL. Ethanol was then added while stirring, and the alcohol was precipitated until the alcohol content reached 70%. The supernatant was concentrated under reduced pressure and dried to obtain the golden lotus water-extracted alcohol-precipitated powder as the golden lotus extract.

[0037] Example 2

[0038] This embodiment discloses a solubilized and stabilized liquid formulation of *Trollius chinensis*, with the following specific formulation components and proportions: 0.2% *Trollius chinensis* extract, 0.2% sodium dodecyl sulfate, 6% sorbitol, 0.02% citronellol, and purified water added to 100 mL. The above raw materials are mixed and then dissolved by stirring and sonication to obtain a liquid formulation containing *Trollius chinensis* extract.

[0039] The above-mentioned golden lotus extract was prepared by water extraction and alcohol precipitation. The specific preparation process is as follows: dried golden lotus flowers were added to 10 times the amount of purified water and decocted once, each time for 2.5 hours. The decoctions were combined and concentrated under reduced pressure at 25°C to a density of 1.3 g / mL. Ethanol was then added while stirring, and the alcohol was precipitated until the alcohol content reached 65%. The supernatant was concentrated under reduced pressure and dried to obtain the golden lotus water-extracted alcohol-precipitated powder as the golden lotus extract.

[0040] Example 3

[0041] This embodiment discloses a solubilized and stabilized liquid formulation of *Trollius chinensis*, with the following specific formulation components and proportions: 0.2% *Trollius chinensis* extract, 0.1% sodium dodecyl sulfate, 4% xylitol, 0.02% menthol, 0.1% sodium methylparaben, 0.02% sodium propylparaben, and purified water added to 100 mL. The above raw materials are mixed and then dissolved by stirring and sonication to obtain a liquid formulation containing *Trollius chinensis* extract.

[0042] The above-mentioned golden lotus extract was prepared by water extraction and alcohol precipitation. The specific preparation process is as follows: dried golden lotus flowers were added to 8 times the amount of purified water and decocted once, each time for 2.5 hours. The solution was concentrated under reduced pressure at 25°C to a density of 1.3 g / mL, and then ethanol was added while stirring. The alcohol was precipitated until the alcohol content reached 75%. The supernatant was concentrated under reduced pressure and dried to obtain the golden lotus water-extracted alcohol-precipitated powder as the golden lotus extract.

[0043] Example 4

[0044] This embodiment discloses a solubilizing and stabilizing liquid formulation of *Trollius chinensis*, with the following specific formulation components and proportions: 0.02% *Trollius chinensis* extract, 0.02% sodium dodecyl sulfate, 8% erythritol, 0.02% eugenol, 0.6% sodium benzoate, and purified water added to 100 mL. The above raw materials are mixed and then dissolved by stirring and sonication to obtain a liquid formulation containing *Trollius chinensis* extract.

[0045] The above-mentioned golden lotus extract was prepared by water decoction method. The specific preparation process is as follows: dried golden lotus flowers were added to 12 times the amount of purified water and decocted twice, 0.5 hours each time. The decoctions were combined and concentrated under reduced pressure at 25°C to a density of 1.2 g / mL. The concentrate was dried to obtain golden lotus water decoction powder as golden lotus extract.

[0046] Example 5

[0047] This embodiment discloses a solubilizing and stabilizing liquid formulation of *Trollius chinensis*, with the following specific formulation components and proportions: 0.4% *Trollius chinensis* extract, 0.4% sodium dodecyl sulfate, 0.4% steviol glycosides, 0.02% citronellol, 0.2% potassium sorbate, and purified water added to 100 mL. The above raw materials are mixed and then dissolved by stirring and sonication to obtain a liquid formulation containing *Trollius chinensis* extract.

[0048] The above-mentioned golden lotus extract was prepared by water decoction method. The specific preparation process is as follows: dried golden lotus flowers were added to 8 times the amount of purified water and decocted once, each time for 2.5 hours. The decoctions were combined and concentrated under reduced pressure at 25°C to a density of 1.3 g / mL. The concentrate was dried to obtain golden lotus water decoction powder as golden lotus extract.

[0049] Example 6

[0050] This embodiment discloses a solubilized and stabilized liquid formulation of *Trollius chinensis*, with the following specific formulation components and proportions: 0.8% *Trollius chinensis* extract, 0.8% sodium dodecylbenzenesulfonate, 0.4% steviol glycosides, 0.02% citronellol, 0.1% methylparaben, 0.02% propylparaben, and purified water added to 100 mL. The above raw materials are mixed and then dissolved by stirring and sonication to obtain a liquid formulation containing *Trollius chinensis* extract.

[0051] The above-mentioned nasturtium extract was prepared by ethanol reflux extraction. The specific preparation process is as follows: dried nasturtium flowers were added to 8 times the amount of 70% ethanol and decocted once, for 2 hours each time. The solution was concentrated under reduced pressure at 25°C to a density of 1.3 g / mL. The concentrate was dried to obtain nasturtium ethanol powder as the nasturtium extract.

[0052] Example 7

[0053] This embodiment discloses a solubilizing and stabilizing liquid formulation of *Trollius chinensis*, with the following specific formulation components and proportions: 0.2% *Trollius chinensis* extract, 0.1% sodium dodecylbenzenesulfonate, 4% xylitol, 0.02% menthol, 0.1% sodium methylparaben, 0.02% sodium propylparaben, and purified water added to 100 mL. The above raw materials are mixed and then dissolved by stirring and sonication to obtain a liquid formulation containing *Trollius chinensis* extract.

[0054] The above-mentioned nasturtium extract was prepared by ethanol reflux extraction. The specific preparation process is as follows: dried nasturtium was added to 12 times the amount of 70% ethanol and decocted twice, 1 hour each time. The decoctions were combined and concentrated under reduced pressure at 25°C to a density of 1.3 g / mL. The concentrate was dried to obtain nasturtium ethanol powder as the nasturtium extract.

[0055] Comparative Example 1

[0056] This comparative example discloses an alkaline-based solubilizing liquid formulation of *Trollius chinensis*, with the following specific formulation components and proportions: 0.2% *Trollius chinensis* extract, 0.2% sodium bicarbonate, 4% xylitol, 0.02% menthol, 0.2% chlorhexidine digluconate, and purified water added to 100 mL. The above raw materials are mixed, stirred, and ultrasonically dissolved to obtain a liquid formulation containing *Trollius chinensis* extract.

[0057] The above-mentioned golden lotus extract was prepared by water extraction and alcohol precipitation. The specific preparation process is as follows: dried golden lotus flowers were added to 12 times the amount of purified water and decocted twice, 2 hours each time. The decoctions were combined and concentrated under reduced pressure at 25°C to a density of 1.25 g / mL. Ethanol was then added while stirring, and the alcohol was precipitated until the alcohol content reached 75%. The supernatant was concentrated under reduced pressure and dried to obtain the golden lotus water-extracted alcohol-precipitated powder as the golden lotus extract.

[0058] Comparative Example 2

[0059] This comparative example discloses a liquid formulation of *Trollius chinensis* containing a nonionic surfactant and an alkaline component for solubilization. The specific formulation components and proportions are as follows: 0.2% *Trollius chinensis* extract, 0.2% sodium bicarbonate, 0.1% polysorbate-20, 6% sorbitol, 0.02% citronellol, 0.1% sodium methylparaben, 0.02% sodium propylparaben, and purified water to 100 mL. After mixing the above raw materials, the mixture is stirred, ultrasonically dissolved, and filtered to obtain a liquid formulation containing *Trollius chinensis* extract.

[0060] The above-mentioned golden lotus extract was prepared by water extraction and alcohol precipitation. The specific preparation process is as follows: dried golden lotus flowers were added to 8 times the amount of purified water and decocted once, each time for 2.5 hours. The solution was concentrated under reduced pressure at 25°C to a density of 1.3 g / mL, and then ethanol was added while stirring. The alcohol was precipitated until the alcohol content reached 75%. The supernatant was concentrated under reduced pressure and dried to obtain the golden lotus water-extracted alcohol-precipitated powder as the golden lotus extract.

[0061] Comparative Example 3

[0062] This comparative example discloses a liquid formulation of *Trollius chinensis* containing a nonionic surfactant and 1,2-propanediol as a solubilizer. The specific formulation components and proportions are as follows: 0.2% *Trollius chinensis* extract, 0.1% polysorbate-20, 8% erythritol, 0.02% eugenol, 0.2% methylparaben, 0.14% propylparaben, 4% 1,2-propanediol, and purified water to 100 mL. After mixing the above raw materials, the mixture is stirred, ultrasonically dissolved, and filtered to obtain a liquid formulation containing *Trollius chinensis* extract.

[0063] The above-mentioned golden lotus extract was prepared by water decoction. The specific preparation process is as follows: dried golden lotus flowers were added to 12 times the amount of purified water and decocted twice, 1 hour each time. The decoctions were combined and concentrated under reduced pressure at 25°C to a density of 1.3 g / mL. The concentrate was dried to obtain golden lotus water decoction powder as the golden lotus extract.

[0064] Comparative Example 4

[0065] This comparative example discloses a liquid formulation of *Trollius chinensis* with added cationic surfactant for solubilization. The specific formulation components and proportions are as follows: 0.2% *Trollius chinensis* extract, 0.1% cetyltrimethylammonium bromide, 4% xylitol, 0.02% menthol, 0.1% methylparaben, 0.02% propylparaben, and purified water to 100 mL. After mixing the above raw materials, the mixture is stirred, ultrasonically dissolved, and filtered to obtain a liquid formulation containing *Trollius chinensis* extract.

[0066] The above-mentioned nasturtium extract was prepared by ethanol reflux extraction. The specific preparation process is as follows: dried nasturtium flowers were added to 10 times the amount of 70% ethanol and decocted once, each time for 2.5 hours. The solution was concentrated under reduced pressure at 25°C to a density of 1.2 g / mL, and the concentrate was dried to obtain nasturtium ethanol powder as the nasturtium extract.

[0067] Comparative Example 5

[0068] This comparative example discloses a liquid formulation of *Trollius chinensis* with the aid of an organic solvent. The formulation contains 3.2% *Trollius chinensis* extract, with 20% dimethyl sulfoxide (DMSO) added to 100 mL. The above raw materials are mixed, stirred, ultrasonically dissolved, and filtered to obtain a liquid formulation containing *Trollius chinensis* extract.

[0069] The above-mentioned golden lotus extract was prepared by water extraction and alcohol precipitation. The specific preparation process is as follows: dried golden lotus flowers were added to 10 times the amount of purified water and decocted twice, 2.5 hours each time. The decoctions were combined and concentrated under reduced pressure at 25°C to a density of 1.2 g / mL. Ethanol was then added while stirring, and the alcohol was precipitated until the alcohol content reached 70%. The supernatant was concentrated under reduced pressure and dried to obtain the golden lotus water-extracted alcohol-precipitated powder as the golden lotus extract.

[0070] The physical appearance diagrams of Embodiment 1 and Comparative Examples 1, 2, and 4 are attached. Figure 1 .

[0071] The appearance diagram, particle size distribution, and high-performance liquid chromatography (HPLC) chromatogram of the newly formulated product in Example 2 above, as well as the HPLC chromatograms of the standards for scutellarin, vitexin, and veratril, are attached. Figure 2 .

[0072] From the appendix Figure 2 As shown in (a), the newly formulated solubilized and stabilized nasturtium liquid preparation is clear and transparent in appearance, with no obvious precipitation. (From the appendix...) Figure 2As shown in (b), the particle size curve exhibits a narrow, single-peak distribution with no obvious secondary peaks, indicating that the particles in the system are uniform in size and well-dispersed. The peak particle size is 197 nm, and the overall particle size range is concentrated between approximately 100-300 nm, belonging to a typical nanoscale colloidal dispersion system. This indicates that the effective components of *Trollius chinensis* are uniformly encapsulated by anionic surfactants to form micelles or nano-aggregates, with no large particle residue, and high and uniform solubility. (See attached image.) Figure 2 (c) and by appendix Figure 2 As shown in the comparison (d), the newly prepared solubilized and stabilized nasturtium liquid system contains characteristic components of flavonoids and phenolic acids, such as rutin, vitexin, and veratric acid.

[0073] The compositions of Examples 1-7 and Comparative Examples 1-5 were subjected to the following performance tests. The test methods and results are as follows:

[0074] Experiment 1: Determination of antibacterial properties

[0075] This invention employs the People's Republic of China Health Industry Standard WS / T 650-2019 "Quantitative Suspension Antibacterial Test" method to examine the antibacterial performance of each embodiment and comparative example against Escherichia coli, Staphylococcus aureus, and Candida albicans. An inhibition rate >50% indicates antibacterial activity, and an inhibition rate >90% indicates bactericidal activity (culture media: nutrient agar was used for Staphylococcus aureus and Escherichia coli, and Sabouraud agar was used for Candida albicans).

[0076] The antibacterial effect test includes the following steps:

[0077] 1) Activation of the strain: Pick a single colony from a well-preserved solid culture medium and place it in a liquid culture medium. Amplify and culture it in a constant temperature shaker at 37℃ for 24 h at a speed of 80 rpm.

[0078] 2) Preparation of bacterial suspension: Take 6 mL of bacterial suspension into each centrifuge tube and centrifuge for 2 min at 4000 rpm. Remove the supernatant, wash the bacterial precipitate with physiological saline, centrifuge, and wash a total of 3 times. Discard the supernatant, add 4 mL of physiological saline, and mix thoroughly using a vortex mixer. Take 2 mL of bacterial suspension and measure the absorbance at 600 nm to calculate the bacterial concentration.

[0079] 3) Antibacterial test of liquid preparation of golden lotus extract: Dilute the above activated bacterial solution with physiological saline (concentration 1×10⁻⁶). 5 ~1×10 7Prepare a bacterial suspension (CFU / mL) for later use. Take a sterile test tube, add 5.0 mL of the sample, and incubate in a 20℃±1℃ water bath for 5 min. Then add 0.1 mL of the test bacterial suspension, mix quickly, and start timing immediately. After 30 s of contact between the test bacteria and the sample, inoculate 1 mL of the test bacteria and sample mixture into two petri dishes and pour the culture medium. If the bacterial count is uncountable, perform a 10-fold serial dilution with physiological saline, select an appropriate dilution, and inoculate 1.0 mL into two petri dishes for viable cell counting. Use the same volume of physiological saline as a blank control group. All test and control samples were incubated at 36℃±1℃. Bacterial vegetative cells were cultured for 48 h to observe the final results; Candida albicans was cultured for 72 h to observe the final results. The experiment was repeated three times, and the inhibition rate (%) of each liquid preparation after 30 s of contact was calculated.

[0080] Antibacterial rate % = (Number of colonies in the saline blank control group - Number of colonies in the experimental group) / Number of colonies in the saline blank control group × 100%;

[0081] The liquid formulations in Examples 1-7 and Comparative Examples 1-5 were tested according to the above method, and the results are shown in Table 1.

[0082] Table 1 Antibacterial effect test

[0083]

[0084] Note: ++ indicates bactericidal effect (bacteriostatic rate > 90%), + indicates bacteriostatic effect (50% < bacteriostatic rate < 90%), - indicates no bacteriostatic effect (bacteriostatic rate < 50%).

[0085] The following information can be obtained from Table 1:

[0086] 1) Examples 1-7 show that the inhibition rate against the three bacteria is >50%, indicating that it has an inhibitory effect on the growth of the three test bacteria.

[0087] 2) In Comparative Example 1, chlorhexidine digluconate was introduced as a preservative, which showed a certain inhibitory effect on the growth of all three test bacteria.

[0088] 3) In Comparative Example 2, the nonionic surfactant polysorbate was used, and sodium bicarbonate was added to aid dissolution. It was able to inhibit Escherichia coli and Staphylococcus aureus, but had no inhibitory effect on Candida albicans.

[0089] 4) Although the addition of 1,2-propanediol to Comparative Example 3 promoted the dissolution of parabens in the system, the system not only exhibited reduced antibacterial activity but also promoted the growth of Candida albicans, resulting in a denser increase in colony count. This indicates that the formulation is unsuitable as an antibacterial product. See the appendix for detailed results. Figure 3 .

[0090] 5) In Comparative Example 4, the hexadecyltrimethylammonium bromide cationic surfactant was used for solubilization. The formulation had no antibacterial effect on the three test bacteria, indicating that compared with the use of anionic surfactants, the cationic surfactant caused a significant decrease in antibacterial activity.

[0091] 6) In Comparative Example 5, with the addition of 20% DMSO organic solvent to aid dissolution, the high concentration group of golden lotus extract (3.2%) was able to inhibit the three test bacteria to a certain extent.

[0092] The inhibition rates of the liquid formulations in Examples 1-7 against Escherichia coli, Staphylococcus aureus, and Candida albicans are shown in Table 2.

[0093] Table 2. Inhibition rates (%) of the liquid formulations of Examples 1-7 and Comparative Example 5 against the three test bacteria.

[0094]

[0095] As can be seen from the data in Tables 1 and 2, Examples 1-7 can inhibit the growth of the three test bacteria, while Examples 1 and 3 can inhibit the growth of the three test bacteria by more than 90%. Therefore, we selected Examples 1 and 3 as the preferred formulations.

[0096] Experiment 2: Physical Stability Assessment

[0097] During long-term storage or transportation, the stability of liquid formulations may decrease due to environmental factors, manifesting as sedimentation of components or degradation of active ingredients. Long-term stability tests were conducted on the liquid formulations of Examples 1-7 and Comparative Examples 1-5.

[0098] Long-term stability test: The liquid formulation was stored at room temperature (25°C) in the dark for 24 months, and its appearance and clarity were checked to evaluate the long-term stability of the composition.

[0099] Examples 1-7 and Comparative Examples 1-5 were tested according to the above method, and the results are shown in Table 3. The physical appearance of Examples 1 and 3 after 3, 6, 9, 12, and 24 months of storage is attached. Figure 4 As shown.

[0100] Table 3 Physical stability assessment

[0101]

[0102] Note: "↓" indicates the formation of precipitate.

[0103] As shown in Table 3:

[0104] 1) The results of Examples 1-7 show that the micelle dispersion system of the present invention exhibits excellent physical stability, wherein the anionic surfactant has a significant solubilizing effect on the extract of Napier lotus, and the dispersion system can maintain good system stability during long-term storage.

[0105] 2) In Comparative Example 1, the system color darkened and white precipitate appeared during the preparation process. This was because chlorhexidine digluconate reacted with sodium bicarbonate to generate chlorhexidine free base, which has poor water solubility, resulting in turbidity of the system. Furthermore, the precipitation occurred after long-term standing, indicating that the physical stability of the formula was poor.

[0106] 3) In Comparative Example 2, the extract of *Trollius chinensis* was solubilized using the nonionic surfactant polysorbate-20, and sodium bicarbonate was added to aid in its dissolution. However, this formulation exhibited poor stability, with precipitation observed after 24 months of storage at room temperature.

[0107] 4) In Comparative Example 3, polysorbate-20 and 1,2-propanediol were used as solubilizer and co-solvent, respectively. Although the system showed good stability at room temperature, it promoted the growth of Candida albicans, resulting in a dense increase in colony count. This indicates that the formulation is not suitable as an antibacterial product.

[0108] 5) Comparative Example 4 used the cationic surfactant hexadecyltrimethylammonium bromide to solubilize the extract of *Trollius chinensis*. The system darkened in color during preparation, but the solubilization effect was achieved. After 24 months at room temperature, a precipitate formed, indicating poor long-term stability. Compared to the system constructed using anionic surfactants, the system's stability was significantly insufficient.

[0109] 6) In Comparative Example 5, the concentration of golden lotus extract was relatively high. 20% DMSO organic solvent was added to aid dissolution, but precipitation still occurred in the system after standing.

[0110] Experiment 3: Freeze-thaw stability test

[0111] Freeze-thaw stability test: The liquid formulation was placed at a low temperature (-5℃) for 24 hours, and then placed at 40℃ for 24 hours. The freeze-thaw cycle was repeated three times. After each cycle, the appearance (layering, crystallization, precipitation) and clarity of the sample were checked to evaluate the freeze-thaw stability of the sample.

[0112] Examples 1-7 and Comparative Examples 1-5 were tested according to the above method, and the results are shown in Table 4. The stability appearance diagram of Example 1 after three rounds of freeze-thaw cycle testing is attached. Figure 5 As shown.

[0113] Table 4. Stability assessment of samples after 3 freeze-thaw cycles

[0114]

[0115] Note: "↓" indicates the formation of precipitate.

[0116] As shown in Table 4:

[0117] 1) Examples 1-7 demonstrate good physical stability. The anionic surfactants achieved a significant solubilizing effect on the extract of Napier lotus and maintained good physical stability in the freeze-thaw stability test, which reflects the excellent temperature adaptability of the system.

[0118] 2) Comparative Example 1 showed poor stability under low temperature conditions and precipitation occurred during freeze-thaw stability testing.

[0119] 3) Comparative Example 2 used nonionic surfactant polysorbate-20 for solubilization and added sodium bicarbonate to assist in the dissolution of nasturtium extract, but the system stability was poor and precipitation occurred after freeze-thaw test.

[0120] 4) Comparative Example 3 was solubilized using the nonionic surfactant polysorbate-20, and 1,2-propanediol was added as a co-solvent. This system still showed precipitation at low temperatures, indicating insufficient low-temperature stability and inability to meet the storage requirements for refrigerated transport.

[0121] 5) In Comparative Example 4, the cationic surfactant cetyltrimethylammonium bromide was used for solubilization. The system darkened in color during preparation, and precipitation occurred during freeze-thaw stability testing, indicating poor physical stability against temperature changes. Compared to anionic surfactants, this system exhibits poor stability, with the insoluble components more prone to precipitation under fluctuating temperatures.

[0122] 6) In Comparative Example 5, the concentration of golden lotus extract was relatively high, and most of it precipitated out at low temperature, indicating poor system stability.

[0123] Based on the combined results of antibacterial activity tests and physical stability studies, Examples 1 and 3 demonstrated excellent technical effects. Further investigation of their antibacterial activity after 24 months at room temperature showed no significant decrease in antibacterial performance. The inhibition rates of Examples 1 and 3 against the three test bacteria after 24 months are shown in Table 5. The antibacterial activity graphs of Examples 1 and 3 after fresh preparation and after 24 months of storage are attached. Figure 6 , 7 Antibacterial rate is shown in the appendix. Figure 8 , 9 .

[0124] Table 5. Antibacterial rate (%) of Examples 1 and 3 after 24 months of storage.

[0125]

[0126] Table 5 shows that after 24 months of storage at room temperature, the inhibition rates of Examples 1 and 3 against the three test bacteria did not decrease significantly, and the inhibition rates against the three test bacteria could still reach more than 80%, indicating that they have strong chemical stability.

[0127] Examples 1-7 above are preferred embodiments of the present invention. Examples 1 and 3 show good performance in antibacterial properties and stability, and are therefore suitable as the best formulations.

[0128] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A solubilizing and stabilizing liquid formulation of *Trollius chinensis*, characterized in that, By weight percentage, it includes: 0.02%-0.8% golden lotus extract, 0.02%-0.8% micelle carrier, 4%-10% additives, and the remainder is water.

2. The solubilized and stabilized nasturtium liquid formulation according to claim 1, characterized in that, The micelle carrier is constructed from a surfactant, which is an anionic surfactant, including at least one of sodium dodecyl sulfate, sodium dodecyl sulfonate, and sodium dodecylbenzene sulfonate.

3. The solubilized and stabilized nasturtium liquid formulation according to claim 1, characterized in that, The additives include at least one of flavoring agents, flavorings, and preservatives. The flavoring agents include xylitol, sorbitol, erythritol, and steviol glycosides. The flavorings include menthol, citronellol, and eugenol. The preservatives include parabens and their sodium salts, benzoic acid and its sodium salts, potassium sorbate, and chlorhexidine digluconate.

4. The solubilized and stabilized nasturtium liquid formulation according to claim 1, characterized in that, The golden lotus extract is prepared by one of the following methods: water decoction, ethanol reflux extraction, and water extraction with alcohol precipitation. Its main active ingredients include flavonoids and phenolic acids, and it is rich in characteristic components such as strychnine, vitexin, and veratrolic acid.

5. The solubilized and stabilized nasturtium liquid formulation according to claim 4, characterized in that, The golden lotus extract was prepared by water decoction, and the specific preparation process is as follows: Add 12 times the amount of purified water to dried golden lotus flowers and reflux extract 1-2 times at 100℃, each time for 0.5-2.5 hours. Combine the decoctions and concentrate under reduced pressure at 25℃ to a density of 1.2-1.3 g / mL. Dry the concentrate to obtain golden lotus flower water extract powder as golden lotus flower extract.

6. The solubilized and stabilized nasturtium liquid formulation according to claim 4, characterized in that, The golden lotus extract was prepared by ethanol reflux extraction, and the specific preparation process is as follows: Add 12 times the amount of 70% ethanol to dried golden lotus flowers and reflux extract 1-2 times at 80℃, each time for 0.5-2.5 hours. Combine the extracts and concentrate under reduced pressure at 25℃ to a density of 1.2-1.3 g / mL. Dry the concentrate to obtain golden lotus flower ethanol extract powder as golden lotus flower extract.

7. The solubilized and stabilized nasturtium liquid formulation according to claim 4, characterized in that, The golden lotus extract was prepared by water extraction and alcohol precipitation, and the specific preparation process is as follows: Add 8-12 times the amount of purified water to dried golden lotus flowers and decoct 1-2 times, 0.5-2.5 hours each time. Combine the decoctions and concentrate under reduced pressure at 25°C to a density of 1.2-1.3 g / mL. Then add ethanol while stirring, and precipitate the alcohol to an alcohol content of 65%-75%. Concentrate the supernatant under reduced pressure and dry to obtain golden lotus flower water-ethanol precipitate powder as golden lotus flower extract.

8. The method for preparing the solubilized and stabilized nasturtium liquid formulation according to any one of claims 1-7, characterized in that, Anionic surfactants self-assemble in the aqueous phase to form stable micelles, which then interact with the extract of *Trollius chinensis* to generate a stable micelle-solute complex. This micelle-solute complex effectively disperses the poorly soluble components of the *Trollius chinensis* extract within the micelle system, ultimately forming a clear liquid formulation with antibacterial activity.

9. The method for preparing a solubilized and stabilized nasturtium liquid formulation according to claim 8, characterized in that, The clear liquid preparation obtained by this method exhibits inhibitory activity against at least one of Escherichia coli, Staphylococcus aureus, and Candida albicans, with an inhibition rate >50%. The method for verifying this inhibitory activity is as follows: the clear liquid preparation is mixed with a solution of 1×10⁻⁶... 5 ~1×10 7 After mixing the target bacterial suspension (CFU / mL) for 30 seconds, place the mixture in a sterile petri dish and pour in nutrient agar, Sabouraud agar, or Sabouraud weak agar. After incubation at 35-37°C for 48-72 hours, use the same volume of physiological saline as a blank control group to calculate the inhibition rate.

10. The application of the clear liquid formulation prepared by the method of claim 9, characterized in that, It is used in antibacterial cleaning products, oral care products, and health care products, specifically including antibacterial cleaning care for oral mucosa, pharyngeal mucosa, teeth, and gums, wherein the antibacterial cleaning care targets at least one of Escherichia coli, Staphylococcus aureus, and Candida albicans.

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