Camellia sinensis flower whitening extract, preparation method and application thereof

Abstract

This invention relates to a whitening extract from Camellia yunnanensis, its preparation method, and its applications, belonging to the field of plant extract preparation and cosmetic technology. The extraction method includes: preparing a eutectic solvent by mixing choline chloride and citric acid in a molar ratio of 1:1 to 1:5, and adjusting the water content to 40%–80%; mixing Camellia yunnanensis raw material powder with the eutectic solvent at a material-to-liquid ratio of 1:10–30 (g / mL), and performing ultrasound-assisted extraction. This extract can be used to prepare cosmetics or topical skin preparations with whitening, brightening, antioxidant, or pigmentation-improving effects, as well as tyrosinase activity inhibitors and α-amylase activity inhibitors. This invention provides a green and efficient method for extracting the whitening active ingredients from Camellia yunnanensis.

Description

Technical Field

[0001] This invention belongs to the field of plant extract preparation and cosmetic technology, specifically relating to a whitening extract of Camellia yunnanensis, its preparation method and application. Background Technology

[0002] With the increasing demand for skin whitening products, research on natural plant extracts as whitening ingredients is receiving more and more attention. Currently, common whitening ingredients on the market mainly include hydroquinone, arbutin, vitamin C and its derivatives, but these ingredients may cause adverse reactions such as skin irritation and rebound pigmentation during long-term use, and the production process of some chemically synthesized ingredients may cause environmental pollution.

[0003] Dianshan Tea ( Camellia reticulata Camellia yunnanensis is a medicinal plant unique to Yunnan Province. Its flowers are rich in flavonoids, triterpenoids, organic acids, and other active ingredients, possessing antioxidant, anti-inflammatory, and tyrosinase-inhibiting effects. Traditional plant extraction methods mainly include water extraction, organic solvent extraction, and supercritical CO2 extraction. However, existing methods for preparing whitening extracts from Camellia yunnanensis still have some problems: First, traditional extraction methods are inefficient and cannot fully extract the whitening active ingredients from Camellia yunnanensis; second, conventional extraction processes often require large amounts of organic solvents, which are not only costly but may also pollute the environment; third, active ingredients are prone to oxidation and degradation during extraction, leading to unstable whitening effects of the extract. Therefore, developing an efficient, green, and stable method for preparing whitening extracts from Camellia yunnanensis is of great significance for meeting the cosmetics industry's demand for natural, safe, and highly effective whitening ingredients. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing a whitening extract of Camellia yunnanensis. This method uses a green eutectic solvent combined with ultrasound-assisted extraction technology, which is simple and environmentally friendly.

[0005] Another object of the present invention is to provide a whitening extract of Camellia yunnanensis obtained by the above method, which has excellent tyrosinase inhibitory activity and whitening effect.

[0006] Another object of the present invention is to provide the application of the above-mentioned Camellia yunnanensis whitening extract in the preparation of whitening, brightening, antioxidant or pigmentation-improving cosmetics and topical skin preparations.

[0007] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for preparing a whitening extract of Camellia yunnanensis, comprising the following steps: (1) Preparation of eutectic solvent: Mix choline chloride and citric acid in a molar ratio of 1:1 to 1:5, heat and stir until a homogeneous and transparent liquid is formed, and add water to adjust the water content to 40% to 80%; (2) Mix the Yunnan camellia flower raw material with the eutectic solvent of step (1) and perform ultrasonic-assisted extraction to obtain the extract; (3) The extract from step (2) is concentrated and dried to obtain Camellia yunnanensis extract.

[0008] Furthermore, the Yunnan camellia flower raw material undergoes drying, pulverization, and sieving pretreatment before being mixed with the eutectic solvent in step (1). Preferably, the sieving is performed through a 50-150 mesh sieve. This pretreatment can increase the contact area between the raw material and the extraction solvent, thereby improving the extraction efficiency.

[0009] Preferably, the heating and stirring temperature is 30–80°C, and more preferably 80°C.

[0010] Preferably, the molar ratio of choline chloride to citric acid in step (1) is 1:3. The water content is 40% to 60%, more preferably 48% to 50%.

[0011] Furthermore, the material-to-liquid ratio for ultrasonic-assisted extraction in step (2) is 1:15 to 30, in g / mL, and more preferably 1:25.

[0012] Furthermore, the ultrasound-assisted extraction time in step (2) is 15 to 75 minutes, and more preferably 30 minutes.

[0013] Furthermore, in step (2), the ultrasonic-assisted extraction temperature is 50–80°C, and the extraction is performed 2–3 times. Under these parameters, the active ingredients in Camellia yunnanensis can be fully extracted while avoiding degradation of the active ingredients caused by prolonged high-temperature treatment.

[0014] Furthermore, the concentration in step (3) is vacuum concentration at a temperature of 50–65°C; the drying is freeze drying at a temperature of -60–-70°C. These conditions can effectively protect the heat-sensitive active ingredients in the extract.

[0015] Secondly, the present invention also provides a whitening extract of Camellia yunnanensis prepared by the above method.

[0016] Thirdly, the present invention also provides the application of the above-mentioned Camellia yunnanensis whitening extract in the preparation of cosmetics or topical skin preparations with whitening, brightening, antioxidant or pigmentation-improving effects.

[0017] Preferably, the cosmetic is an aqueous solution, essence, mask, oil, cream, emulsion, ointment, gel, aerosol, or patch.

[0018] Fourthly, the present invention also provides the application of the above-mentioned Camellia yunnanensis whitening extract in the preparation of tyrosinase activity inhibitors, α-amylase activity inhibitors, or adjuvant hypoglycemic products.

[0019] Fifthly, the present invention also provides a cosmetic or topical skin preparation having whitening, brightening, antioxidant, or pigmentation-improving effects, comprising the above-mentioned Yunnan camellia whitening extract.

[0020] Preferably, the content of the Yunnan camellia whitening extract in cosmetics or topical skin preparations is 0.2% to 5%.

[0021] Compared with the prior art, the present invention has the following advantages and beneficial effects: Green and environmentally friendly: This invention uses choline chloride-citric acid eutectic solvent as the extraction medium. This solvent is biodegradable, non-toxic and harmless, and can replace traditional organic solvents. It conforms to the concept of green chemistry and avoids the safety hazards caused by organic solvent residues.

[0022] High extraction efficiency: The eutectic solvent has good solubility and extraction selectivity for plant active ingredients such as flavonoids and polyphenols. Combined with the mechanical and cavitation effects of ultrasound-assisted extraction, the extraction efficiency can be significantly improved, the extraction time can be shortened, and energy consumption can be reduced.

[0023] Significant whitening effect: The extract of this invention showed significant tyrosinase inhibition activity and melanin synthesis inhibition in in vitro tyrosinase inhibition tests, B16 cell models and zebrafish models, which can effectively improve skin pigmentation and achieve whitening and brightening effects.

[0024] Additional activity: The extract of this invention also has good α-amylase inhibitory activity and has the potential to be used as an adjunct to lower blood sugar.

[0025] Good safety profile: Cytotoxicity tests have verified that the extract of this invention has no significant toxicity to cells within the effective concentration range, demonstrating good safety profile and making it suitable for cosmetics and topical skin preparations. Attached Figure Description

[0026] Figure 1 The DPPH free radical scavenging capacity curve of Camellia yunnanensis flower extract; Figure 2 ABTS for Camellia yunnanensis flower extract + Free radical scavenging capacity curve; Figure 3 The α-amylase inhibitory activity curve of Camellia yunnanensis flower extract; Figure 4 The results of the B16 cell safety evaluation of the extract of Camellia yunnanensis; Figure 5The results of the safety evaluation of the Yunnan camellia flower extract in A375 cells; Figure 6 The effect of Camellia yunnanensis flower extract on melanin content in B16 cells; Figure 7 The effect of Camellia yunnanensis flower extract on tyrosinase activity in B16 cells; Figure 8 The effect of Camellia yunnanensis flower extract on melanin content in A375 cells; Figure 9 The effect of Camellia yunnanensis flower extract on tyrosinase activity in A375 cells; Figure 10 The effect of Camellia yunnanensis extract on melanin content in zebrafish; Figure 11 The effect of Camellia yunnanensis flower extract on zebrafish tyrosinase activity; Figure 12 Photographs showing the effect of Camellia yunnanensis extract on melanin distribution in zebrafish. Detailed Implementation

[0027] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0028] The processes, conditions, reagents, and experimental methods used in implementing this invention, except as specifically mentioned below, are all common knowledge and general knowledge in the field, and this invention does not have any particular limitations. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0029] Unless otherwise stated, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. However, in the event of any conflict, the specification containing the definitions shall prevail.

[0030] The following are the main raw material sources and pretreatment methods in the examples: Yunnan Camellia ( Camellia reticulata Collected from the Chinese Herbal Medicine Garden of Yunnan University of Traditional Chinese Medicine.

[0031] Pretreatment method: Freeze-dry and pulverize Yunnan camellia flowers, and pass them through a 100-mesh sieve to obtain Yunnan camellia flower powder.

[0032] Example 1 A method for preparing a skin-whitening extract from Camellia yunnanensis includes the following steps: (1) Preparation of eutectic solvent: Choline chloride and citric acid are mixed at a molar ratio of 1:3.03 and heated and stirred at 80°C until a homogeneous transparent liquid is formed. Water is added to adjust the water content to 48.01%. During the heating and stirring process, the temperature is controlled at around 80°C and stirring is continued for about 30 minutes until the liquid is completely dissolved and a transparent liquid is formed.

[0033] (2) Ultrasonic-assisted extraction: 1 g of pretreated Camellia yunnanensis powder was mixed with the eutectic solvent prepared in step (1) at a material-to-liquid ratio of 1:25.2 (g / mL), and placed in an ultrasonic extractor. Ultrasonic extraction was performed at 60℃ for 30.4 min, with the ultrasonic frequency set to 40 kHz and the power to 500 W. After extraction, the mixture was centrifuged, and the supernatant was collected. The residue was then treated with the same volume of eutectic solvent, and the extraction was repeated twice. All supernatants were then combined.

[0034] (3) Concentration and drying: The extract obtained in step (2) is concentrated under reduced pressure at a temperature of 60°C to 1 / 5 of its original volume. The concentrate is then freeze-dried at a temperature of -65°C and a vacuum of 30 Pa to obtain the whitening extract of Camellia yunnanensis.

[0035] Example 2 A method for preparing a skin-whitening extract from Camellia yunnanensis includes the following steps: (1) Preparation of eutectic solvent: Mix choline chloride and citric acid in a molar ratio of 1:3, heat and stir at 80°C until a homogeneous transparent liquid is formed, and add water to adjust the water content to 50%. During the heating and stirring process, the temperature is controlled at about 80°C, and stirring is continued for about 30 minutes until it is completely dissolved and forms a transparent liquid.

[0036] (2) Ultrasonic-assisted extraction: 1 g of pretreated Camellia yunnanensis powder was mixed with the eutectic solvent prepared in step (1) at a material-to-liquid ratio of 1:25 (g / mL), and placed in an ultrasonic extractor. Ultrasonic extraction was performed at 60℃ for 30 min, with the ultrasonic frequency set to 40 kHz and the power to 500 W. After extraction, the mixture was centrifuged, and the supernatant was collected. The residue was then treated with the same volume of eutectic solvent, and the extraction was repeated twice. All supernatants were then combined.

[0037] (3) Concentration and drying: The extract obtained in step (2) is concentrated under reduced pressure at a temperature of 60°C to 1 / 5 of its original volume. The concentrate is then freeze-dried at a temperature of -65°C and a vacuum of 30 Pa to obtain the whitening extract of Camellia yunnanensis.

[0038] Example 3 The difference from Example 2 is that the molar ratio of choline chloride to citric acid is 1:1, while the other conditions are the same as in Example 2.

[0039] Example 4 The difference from Example 2 is that the molar ratio of choline chloride to citric acid is 1:2, while the other conditions are the same as in Example 2.

[0040] Example 5 The difference from Example 2 is that the molar ratio of choline chloride to citric acid is 1:4, while the other conditions are the same as in Example 2.

[0041] Example 6 The difference from Example 2 is that the molar ratio of choline chloride to citric acid is 1:5, while the other conditions are the same as in Example 2.

[0042] Example 7 The difference from Example 2 is that the water content of the eutectic solvent is 40%, while the other conditions are the same as in Example 2.

[0043] Example 8 The difference from Example 2 is that the water content of the eutectic solvent is 60%, while the other conditions are the same as in Example 2.

[0044] Example 9 The difference from Example 2 is that the water content of the eutectic solvent is 70%, while the other conditions are the same as in Example 2.

[0045] Example 10 The difference from Example 2 is that the water content of the eutectic solvent is 80%, while the other conditions are the same as in Example 2.

[0046] Example 11 The difference from Example 2 is that the extraction time is 15 min, while the other conditions are the same as in Example 2.

[0047] Example 12 The difference from Example 2 is that the extraction time is 45 minutes, while the other conditions are the same as in Example 2.

[0048] Example 13 The difference from Example 2 is that the extraction time is 60 minutes, while the other conditions are the same as in Example 2.

[0049] Example 14 The difference from Example 2 is that the extraction time is 75 minutes, while the other conditions are the same as in Example 2.

[0050] Example 15 The difference from Example 2 is that the material-to-liquid ratio is 1:10, while the other conditions are the same as in Example 2.

[0051] Example 16 The difference from Example 2 is that the material-to-liquid ratio is 1:15, while the other conditions are the same as in Example 2.

[0052] Example 17 The difference from Example 2 is that the material-to-liquid ratio is 1:20, while the other conditions are the same as in Example 2.

[0053] Example 18 The difference from Example 2 is that the material-to-liquid ratio is 1:30, while the other conditions are the same as in Example 2.

[0054] Comparative Example 1 (Ultrasound-assisted ethanol extraction) Accurately weigh 1g of Yunnan camellia flower powder and add 70% ethanol solution at a material-to-liquid ratio of 1:25. Extract by ultrasonication at 60℃ for 30 min, repeating the extraction process twice. Combine all supernatants. Concentrate under reduced pressure at 60℃ to 1 / 5 of the original volume. Then freeze-dry the concentrate at -65℃ under a vacuum of 30 Pa to obtain the Yunnan camellia flower extract.

[0055] Comparative Example 2 (Enzymatic hydrolysis and ultrasonic extraction) Accurately weigh 1g of Yunnan camellia flower powder, add pure water at a material-to-liquid ratio of 1:25, and add 1.5% of a compound enzyme consisting of cellulose and pectinase (1:1). Extract at 60℃ for 30 min, repeating the extraction process twice, and combine all supernatants. Concentrate under reduced pressure at 60℃ to 1 / 5 of the original volume. Then freeze-dry the concentrate at -65℃ under a vacuum of 30 Pa to obtain the Yunnan camellia flower extract.

[0056] Comparative Example 3 (choline chloride-urea DES) The difference from Example 1 is that the eutectic solvent of choline chloride and citric acid is replaced with a eutectic solvent prepared by choline chloride and urea at a molar ratio of 1:2, while the other conditions are the same as in Example 1.

[0057] Comparative Example 4 (choline chloride-oxalate DES) The difference from Example 1 is that the eutectic solvent of choline chloride and citric acid is replaced with a eutectic solvent prepared by choline chloride and oxalic acid in a molar ratio of 1:2, while the other conditions are the same as in Example 1.

[0058] Experimental Example 1: Determination of Tyrosinase Inhibition Rate The camellia flower extracts prepared in the above examples and comparative examples were subjected to in vitro tyrosinase inhibition rate tests.

[0059] Sample preparation: Take samples from Examples 1-18 and Comparative Examples 1-4 respectively, and prepare sample solutions of appropriate concentrations using sodium phosphate buffer solvent at pH 6.8.

[0060] Experimental Method: 75 μL of sample was mixed with 25 μL of tyrosinase solution and incubated at 37℃ for 10 min. Then, 100 μL of 1 mol / L levodopa solution was added to initiate the reaction. The absorbance was measured at 475 nm, and the tyrosinase inhibition rate was calculated using the following formula:

[0061] In the formula, Ab is the absorbance of the tyrosinase solution to the sample solution, A is the absorbance of the sodium phosphate buffer (pH 6.8) to the sample solution; Cb is the absorbance of the sodium phosphate buffer (pH 6.8) to the tyrosinase solution, and C is the absorbance of the sodium phosphate buffer (pH 6.8).

[0062] The results are shown in Table 1.

[0063] Table 1. Results of tyrosinase inhibition rate detection in Examples 1-18 and Comparative Examples 1-4

[0064] The results in the table show that the Camellia yunnanensis extracts prepared in Examples 1-18 of this invention all exhibited high tyrosinase inhibition rates, ranging from 77.97% to 95.39%, which were significantly better than the comparative examples (30.46% to 68.3%). This indicates that using a specific ratio of choline chloride-citric acid eutectic solvent combined with ultrasound-assisted extraction can effectively extract the whitening components with tyrosinase inhibitory activity from Camellia yunnanensis.

[0065] Comparative analysis of the results from Examples 2-6 shows that when the molar ratio is 1:1 to 1:5 (Examples 2, 3, 4, 5, and 6), the tyrosinase inhibition rate reaches over 78%, with the highest inhibition rate (94.21%) at a molar ratio of 1:3, followed by 90.44% at a molar ratio of 1:2. When the molar ratio is too low (1:1) or too high (1:5), the inhibition rate decreases significantly. This indicates that a suitable ratio of hydrogen bond donor to acceptor helps form a high-quality eutectic solvent, enhancing the selective extraction of the active ingredient.

[0066] Comparative analysis of the results in Examples 2 and 7-10 shows that the inhibition rate remains above 77% when the water content is 40% to 80%, with the highest inhibition rate at a water content of 50%. When the water content increases to 60%, the inhibition rate decreases to 86.19%, indicating that excessively high water content weakens the hydrogen bond network structure of the eutectic solvent and reduces its solubility for the target component. A water content of 40% to 60% is the preferred range.

[0067] Comparative analysis of the results in Examples 2 and 11-14 showed that when the extraction time was extended from 15 min to 30 min, the inhibition rate significantly increased from 90.93% to 94.21%; when the time reached 45 min, the inhibition rate was 90.14%; and when it was further extended to 75 min, the inhibition rate decreased to 86.4%. This indicates that an ultrasonic time of about 30 min is sufficient to fully dissolve the active ingredients, and an excessively long ultrasonic time will destroy the active ingredients. The preferred ultrasonic time is 30 min.

[0068] Comparative analysis of the results in Examples 2 and 15-18 shows that the inhibition rate was highest (94.21%) when the material-to-liquid ratio was 1:25; the inhibition rates were 88.28% and 90.62% when the material-to-liquid ratios were 1:20 and 1:30, respectively; and the inhibition rate was only 82.15% when the material-to-liquid ratio was 1:10. This indicates that a material-to-liquid ratio of 1:25 is the optimal range.

[0069] The inhibition rates of Comparative Example 1 (ultrasound-assisted ethanol extraction) and Comparative Example 2 (ultrasound-assisted enzymatic hydrolysis extraction) were 54.62% and 30.46%, respectively, which were significantly lower than those of the Example, indicating that the low eutectic solvent extraction efficiency of the specific composition ratio of the present invention is better than that of traditional solvent extraction.

[0070] Comparative Examples 3 and 4 used choline chloride-urea and choline chloride-oxalic acid eutectic solvent systems, respectively, and their inhibition rates were 68.30% and 66.91%, respectively, both lower than that of Example 1 of the present invention (95.39%). This indicates that the choline chloride-citric acid system has a specific advantage in the extraction of whitening active ingredients from Camellia yunnanensis flowers, which may be related to the carboxyl structure of citric acid and the appropriate strength of hydrogen bonding.

[0071] The above results demonstrate that the Camellia yunnanensis extract prepared by this invention, using choline chloride-citric acid eutectic solvent as the extraction medium and combined with ultrasound-assisted extraction, exhibits a significant inhibitory effect on tyrosinase, with an inhibition rate exceeding 95%, which is significantly superior to traditional solvent extraction and other types of eutectic solvent extraction methods. This extraction process is green, environmentally friendly, and simple to operate, showing promising application prospects.

[0072] Experimental Example 2: Evaluation of Antioxidant Activity 1. Determination of DPPH free radical scavenging ability The extract obtained in Example 1 was prepared into concentration gradient solutions (0, 0.125, 0.25, 0.5, 1.0, 2.0 mg / mL) with methanol. 2.0 mL of each concentration sample solution was added, and 2.0 mL of 0.1 mmol / L DPPH methanol solution was added to each solution. After mixing, the solution was allowed to stand in the dark for 30 min, and the absorbance was measured at 517 nm (sample A). Methanol was used as a blank control (blank A), and vitamin C (with the same concentration gradient) was used as a positive control. The clearance rate was calculated using the following formula: Clearance rate (%) = [1 (A sample [Sample background (A) / Blank (A)] × 100% Among them, the background of sample A is the absorbance of the sample solution after mixing with methanol (to eliminate the interference of the sample's own color).

[0073] Regression analysis was used to calculate the half-maximal inhibitory concentration (IC50).

[0074] 2. ABTS + Free radical scavenging capacity determination ABTS was prepared by mixing an equal volume of 7 mmol / L ABTS solution with 2.45 mmol / L potassium persulfate solution and reacting in the dark for 12-16 hours. + The stock solution was diluted with phosphate-buffered saline (PBS, pH 7.4) to an absorbance of 0.70 ± 0.02 at 734 nm before use.

[0075] The extract obtained in Example 1 was used to prepare concentration gradient solutions (0, 0.125, 0.25, 0.5, 1.0, 2.0 mg / mL) with PBS. 0.1 mL of each concentration sample solution was taken and ABTS was added. + 3.9 mL of working solution was mixed thoroughly and reacted in the dark for 30 min. The absorbance was measured at 734 nm (sample A). PBS was used as a blank control (blank A) instead of the sample, and vitamin C (at the same concentration gradient) was used as a positive control. The clearance rate was calculated using the same formula as above.

[0076] See results Figure 1-2 The results showed that the IC50 of the extract for scavenging DPPH free radicals was [missing information]. 50 The concentration was 350 μg / mL, and the clearance rate reached 87.4% at 2.0 mg / mL; for ABTS + IC50 of free radical scavenging rate 50 The concentration was 410 ug / mL, and the scavenging rate reached 93.3% at 2.0 mg / mL. This indicates that the extract has good antioxidant activity.

[0077] Experimental Example 3: Evaluation of α-amylase inhibitory activity 1. Solution preparation α-Amylase solution: Prepare an enzyme solution of 0.5 mg / mL by dissolving α-amylase (derived from porcine pancreas, with an activity ≥10 U / mg) in 20 mmol / L PBS (pH 6.9, containing 6.7 mmol / L NaCl). Prepare fresh before use.

[0078] Substrate solution: Prepare a 1% (w / v) starch solution from soluble starch using the PBS described above, and preheat it to 37°C for later use.

[0079] Sample solutions: Take the extract obtained in Example 1 and prepare concentration gradient solutions (0, 0.625, 1.25, 2.5, 5.0, 10.0 mg / mL) with PBS.

[0080] Positive control: Acarbose was prepared into a gradient solution of the same concentration using PBS.

[0081] 2. Measurement Method The Bernfeld method was used with slight modifications. 0.2 mL of each concentration sample solution was taken, and 0.2 mL of α-amylase solution was added. The mixture was pre-incubated at 37°C for 10 min. Then, 0.2 mL of 1% starch solution was added, and the reaction was carried out at 37°C for 10 min. Finally, 0.5 mL of DNS chromogenic reagent (3,5-dinitrosalicylic acid) was added, and the reaction was terminated by boiling in a water bath for 5 min. After cooling, the absorbance was measured at 540 nm (sample A).

[0082] Simultaneously set the following controls: Blank control: PBS was used instead of the sample solution. Sample background control: PBS was used instead of starch solution (to eliminate interference from the sample's own absorbance). Positive control: The inhibition rate of acarbose at various concentrations was determined using the same method. 3. Calculation Formula Inhibition rate (%) = [1 (A sample [Sample background (A) / Blank (A)] × 100%; See results Figure 3 The results showed that the inhibitory rate of the extract on α-amylase was dose-dependent, with an IC50 value of [missing value]. 50 The concentration was 1.25 mg / mL, indicating that the extract has potential adjuvant hypoglycemic activity.

[0083] Test Example 4 Safety Evaluation Logarithmic growth phase B16 cells and A375 cells were collected and the cell concentration was adjusted to 1×10⁻⁶. 5 Cells were seeded at a density of 1 / mL in 96-well plates and cultured for 24 h. Different concentrations (20, 40, 60, 80, 100 μg / mL) of Camellia yunnanensis extract (samples prepared in Example 1) were added, and the plates were cultured for another 48 h. 10 μL of CCK-8 solution was added to each well, and the plates were incubated for 4 h. The absorbance was measured at 450 nm, and cell viability was calculated.

[0084] See results Figure 4 (B16 cell safety evaluation) and Figure 5(A375 cell safety evaluation) As shown in the figure, when the extract concentration is below 100 μg / mL, the cell survival rate is greater than 90%, indicating that it has good safety within this concentration range.

[0085] Experimental Example 5: Effects of Camellia yunnanensis flower extract on melanin synthesis and tyrosinase activity in B16 cells 1. Effects on melanin content B16 cells were seeded at 1.5 mL / well in 6-well plates and cultured for 24 h. Different concentrations (0.25 mg / mL, 0.5 mg / mL) of Camellia yunnanensis extract (samples prepared in Example 1) were added to the cells, and the cells were cultured for another 48 h. Cells were collected, and 200 μL of NaOH solution containing 10% DMSO was added. The cells were heated in an 80°C water bath until the melanin was completely dissolved. The absorbance was measured at 405 nm, and the relative melanin content was calculated. Arbutin (600 μM) was used as a positive control.

[0086] See results Figure 6 The results showed that, compared with the model group, the extract significantly reduced melanin content in a dose-dependent manner.

[0087] 2. Effects on tyrosinase activity Cell culture and drug administration were performed as described above. Cells were collected and lysed by repeated freeze-thaw cycles in 150 μL of PBS solution containing 1% Triton X-100. 100 μL of the supernatant was taken and 100 μL of 1 mg / mL L-DOPA solution was added. The cells were incubated at 37°C for 30 min, and the absorbance was measured at 475 nm to calculate the relative tyrosinase activity.

[0088] See results Figure 7 The results showed that, compared with the model group, the extract prepared in this invention could significantly inhibit tyrosinase activity in a dose-dependent manner.

[0089] Experimental Example 6: Effects of Camellia yunnanensis flower extract on melanin synthesis and tyrosinase activity in A375 cells Following the method in Example 5, the effects of Camellia yunnanensis flower extract on melanin content and tyrosinase activity in A375 cells were determined. The results are shown in [Figure 5]. Figure 8-9 The extract also showed a significant inhibitory effect.

[0090] Experimental Example 7: Effect of Camellia yunnanensis flower extract on melanin synthesis in zebrafish 1. Effects on melanin content and distribution Zebrafish embryos aged 7-9 hpf were randomly divided into a blank control group, a model group (α-MSH induced), a positive control group (arbutin, 600 μM), and a sample group (extract samples prepared in Example 1 at different concentrations (0.125, 0.25, 0.5 mg / mL), with 50 embryos in each group. The embryos were induced daily and cultured to 72 hpf. A portion of the juveniles from each group were observed and photographed under a microscope to record the distribution of melanin (results are shown in [see details]). Figure 12 The remaining juvenile fish were collected, homogenized with RIPA lysis buffer, centrifuged to collect the precipitate, and then dissolved in NaOH solution containing 10% DMSO. The absorbance was measured at 405 nm, and the relative melanin content was calculated.

[0091] See results Figure 10 Compared with the model group, the extract significantly reduced melanin content and improved melanin distribution in zebrafish.

[0092] 2. Effects on tyrosinase activity Zebrafish embryo culture and drug administration were performed as described above. Juvenile fish were collected, and the pellet was lysed with PBS solution containing 1% Triton X-100, followed by repeated freeze-thaw cycles and centrifugation. 100 μL of the supernatant was taken, and 100 μL of 1 mg / mL L-DOPA solution was added. The mixture was incubated at 37°C for 30 min, and the absorbance was measured at 475 nm to calculate the relative tyrosinase activity.

[0093] See results Figure 11 Compared with the model group, the extract prepared in this invention can significantly inhibit the activity of tyrosinase in zebrafish.

Claims

1. A method for preparing a Camellia retusa flower whitening extract, characterized in that, Includes the following steps: (1) Preparation of eutectic solvent: Mix choline chloride and citric acid in a molar ratio of 1:1 to 1:5, heat and stir until a homogeneous and transparent liquid is formed, and add water to adjust the water content to 40% to 80%; (2) Mix the Yunnan camellia flower raw material with the eutectic solvent of step (1) and perform ultrasonic-assisted extraction to obtain the extract; (3) The extract from step (2) is concentrated and dried to obtain Camellia yunnanensis extract.

2. The preparation method according to claim 1, characterized in that, The Yunnan camellia raw material is pretreated by drying, crushing and sieving before being mixed with the eutectic solvent in step (1).

3. The preparation method according to claim 1, characterized in that, The material-to-liquid ratio for ultrasonic-assisted extraction in step (2) is 1:10 to 30, with units of g / mL.

4. The preparation method according to claim 1, characterized in that, The ultrasound-assisted extraction time in step (2) is 15 to 75 minutes.

5. The preparation method according to claim 1, characterized in that, The concentration in step (3) is vacuum concentration; the drying is freeze drying.

6. The whitening extract of Camellia yunnanensis prepared by the preparation method according to any one of claims 1-5.

7. The use of the Camellia yunnanensis whitening extract according to claim 6 in the preparation of cosmetics or topical skin preparations with whitening, brightening, antioxidant or pigmentation-improving effects.

8. The application according to claim 7, characterized in that, The cosmetics mentioned are liquids, serums, masks, oils, creams, emulsions, ointments, gels, aerosols, or patches.

9. The use of the Camellia yunnanensis whitening extract according to claim 6 in the preparation of tyrosinase activity inhibitors, α-amylase activity inhibitors, or adjunctive hypoglycemic products.

10. A cosmetic or topical skin preparation having whitening, brightening, antioxidant, or pigmentation-improving effects, characterized in that, It contains the Yunnan camellia whitening extract as described in claim 6.

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