Extraction method and application of fresh acer truncatum leaf essential oil

Abstract

The invention relates to the technical field of natural product extraction, in particular to an extraction method and application of fresh acer truncatum leaf essential oil, and the method comprises the following steps: (1) crushing, (2) mixing and (3) extracting, the method adopts a salt-assisted steam distillation method to extract the fresh acer truncatum leaf essential oil as a Yao medicine. Auxiliary extraction has the advantages of less pollution, high extraction rate, low cost, high efficiency, energy conservation and the like. In addition, the fresh acer truncatum leaf essential oil can be applied to preparation of skin care products, and the skin care products prepared by taking the fresh acer truncatum leaf essential oil as the raw material have the effects of inhibiting bacteria, resisting oxidation and whitening.

Description

[Technical Field]

[0001] This invention belongs to the field of natural product extraction technology, and specifically relates to a method for extracting essential oil from fresh Acer palmatum leaves and its application. [Background Technology]

[0002] *Callicarpa dolichophylla*, a shrub or small tree belonging to the genus *Callicarpa* in the Lamiaceae family, is used medicinally in its entirety. It is one of the "Seventy-Two Winds" in the traditional Yao medicine formula "Tiger and Ox Drilling Wind," possessing hemostatic, analgesic, blood-stasis-dispersing, swelling-reducing, and rheumatic effects. It is also used to treat external bleeding, hemoptysis, postpartum wind-dampness, limb paralysis, and rheumatic pain. It is currently included in the *Dictionary of Traditional Chinese Medicine*, *Seeking the Origins of Materia Medica*, the *Handbook of Commonly Used Chinese Herbal Medicines* (Guangzhou Military Region), and *Chinese Materia Medica*. In folk medicine, this herb is commonly used for medicinal baths; Yao medicinal baths are believed to have therapeutic, health-preserving, detoxifying, beautifying, antibacterial, antipruritic, and anti-inflammatory effects.

[0003] Essential oils, also known as volatile oils, are extracted from the flowers, leaves, roots, bark, fruits, seeds, and resins of herbaceous plants through distillation and compression. Plant essential oils generally contain chemical components such as alcohols, aldehydes, acids, acetones, and terpenes, exhibiting antibacterial, antioxidant, anti-inflammatory, and insecticidal biological activities. Therefore, plant essential oils are widely used in the food, medical, and skincare industries.

[0004] The concept of "green" aims to follow the sustainable development strategy, achieve resource reuse, and reduce carbon emissions during processing. Existing methods for extracting plant essential oils mainly include organic solvent extraction, steam distillation, supercritical CO2 extraction, and pressing. Commonly used traditional extraction methods suffer from drawbacks such as high energy consumption, large solvent usage, significant environmental pollution, and low extraction rates.

[0005] Furthermore, plant resources have not been well developed and utilized. Therefore, developing new applications for the essential oil of fresh Acer palmatum leaves under specific extraction operations is the subject of further research in this application. [Summary of the invention]

[0006] In view of the above, it is necessary to provide a method for extracting essential oil from fresh leaves of Acer buergerianum and its application. This method uses salt-assisted steam distillation to extract essential oil from fresh leaves of Acer buergerianum, a traditional Chinese medicine. Compared with traditional extraction methods, assisted extraction has the advantages of less pollution, higher extraction rate, lower cost, and high efficiency and energy saving.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A method for extracting essential oil from fresh Acer palmatum leaves and its application, the method comprising the following steps:

[0009] (1) Crushing: Collect fresh, disease-free leaves of the Acer truncatum and crush them with a crusher to obtain fresh leaf powder. Then seal and store it for later use.

[0010] (2) Mixing: The fresh leaf powder obtained in step (1) and NaCl are mixed at a liquid-to-solid ratio of 5-7 mL / g to obtain a mixed liquid.

[0011] (3) Extraction: The mixture was extracted by water distillation to obtain the essential oil of fresh maple leaves.

[0012] In this invention, further, step (1) specifically involves pulverizing the material to a particle size of 0.6 mm.

[0013] In this invention, further, the concentration of the NaCl solution in step (2) is 5%.

[0014] In this invention, further, in step (2), the liquid-to-solid ratio of fresh leaf powder to NaCl is 6 mL / g.

[0015] In this invention, the water distillation extraction time in step (3) is 5 hours.

[0016] In the extraction process described in this invention, the three single factors with the greatest influence on the oil yield are selected from four single factors: particle size, liquid-to-solid ratio, distillation time, and NaCl concentration. The parameter selection is carried out by designing a three-factor, three-level experiment using the Box-Behnken response surface design method, with the oil yield (Y) of the essential oil as the response value. The extraction conditions of fresh Acer palmatum leaf essential oil are determined by combining the response surface optimization results.

[0017] Furthermore, this invention also proposes the application of the essential oil from fresh maple leaves extracted using the above method in skincare products.

[0018] In this invention, the application specifically involves using fresh maple leaf essential oil as a raw material to prepare skincare products.

[0019] The present invention has the following beneficial effects:

[0020] 1. The present invention relates to a method for extracting essential oil from fresh leaves of *Acer buergerianum*, using fresh leaves of this plant as material. Based on steam distillation, NaCl salt is added to assist extraction. Using the oil yield as an indicator, the extraction process of *Acer buergerianum* fresh leaf essential oil is optimized through response surface methodology. Single-factor analysis was conducted on process parameters such as extraction time, particle size, liquid-to-solid ratio, and NaCl concentration. The optimal extraction parameters were thus achieved by optimizing the extraction method. Finally, it was determined that a higher oil yield was obtained under the following conditions: NaCl concentration of 5%, liquid-to-solid ratio of 6 mL / g, distillation time of 5 h, and particle size of 0.6 mm.

[0021] 2. The extraction method of this invention is simple, has a high extraction rate, reduces the use of organic reagents, preserves the original state of fresh maple leaf essential oil, and is not contaminated by reagents; it also reduces reagent consumption and saves energy; and reduces the generation and emission of pollutants during the production process, which is more friendly to the environment and human health.

[0022] 3. This invention also proposes using the *Acer buergerianum* leaf essential oil extracted by the method of this invention as a raw material for preparing skincare products. Firstly, the *Acer buergerianum* leaf essential oil extracted by steam distillation exhibits good antibacterial activity, with a MIC of 0.31% against *Staphylococcus epidermidis*, 5% against *Staphylococcus aureus*, and 10% against *Micrococcus luteus*, providing a new additive for the development of antibacterial and anti-inflammatory skincare products. Secondly, the *Acer buergerianum* leaf essential oil exhibits good antioxidant and tyrosinase inhibitory activity. The antioxidant capacity of the *Acer buergerianum* leaf essential oil obtained by this method was measured using the FRAP method. 50 =0.30 mg / mL, IC50 of antioxidant capacity in the total reducing power method 50 =2.31 mg / mL. Furthermore, at a concentration of 12.5 mg / mL, the essential oil of fresh Acer truncatum leaves exhibited a 52% inhibition rate against mushroom tyrosinase, with an IC50 value of 2.31 mg / mL. 50 =0.48mg / mL, exhibiting a certain whitening effect. In summary, the essential oil of fresh Acer palmatum leaves prepared using the method of this application possesses antibacterial, antioxidant, and whitening effects, and has significant potential for application in skincare products. [Attached Image Description]

[0023] Figure 1 The effect of NaCl mass fraction on the oil yield of fresh Acer palmatum leaves;

[0024] Figure 2 The effect of particle size on the oil yield of fresh Acer truncatum leaves;

[0025] Figure 3 The effect of liquid-to-solid ratio on the oil yield of fresh Acer truncatum leaves;

[0026] Figure 4 The effect of distillation time on the oil yield of fresh Acer palmatum leaves;

[0027] Figure 5 The effect of the interaction between distillation time and liquid-to-solid ratio on the oil yield of fresh Acer truncatum leaves;

[0028] Figure 6 The effect of the interaction between distillation time and particle size on the oil yield of fresh Acer truncatum leaves;

[0029] Figure 7 The effect of the interaction between particle size and liquid-to-solid ratio on the oil yield of fresh Acer truncatum leaves;

[0030] Figure 8 Chromatogram of essential oil from fresh leaves of *Acer buergerianum*;

[0031] Figure 9 The graph shows the test results of the iron ion reduction capacity of fresh maple leaf essential oil.

[0032] Figure 10 The graph shows the test results of the total reducing power of fresh maple leaf essential oil.

[0033] Figure 11 This is a comparison chart showing the inhibitory effects of fresh Acer palmatum leaf essential oil and kojic acid on tyrosinase activity. [Specific implementation method]

[0034] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0035] Example 1:

[0036] This embodiment provides a method for extracting essential oil from fresh Acer palmatum leaves and its application. The method includes the following steps:

[0037] (1) Crushing: Collect fresh, disease-free leaves of the maple tree and crush them with a crusher to a particle size of 0.6 mm, so that they can pass through a 30-mesh standard sieve to obtain fresh leaf powder. Then seal and store for later use.

[0038] (2) Mixing: The fresh leaf powder obtained in step (1) and NaCl are mixed at a liquid-to-solid ratio of 6 mL / g to obtain a mixed liquid.

[0039] (3) Extraction: The mixture was extracted by water distillation for 5 hours to obtain the essential oil of fresh maple leaves.

[0040] Example 2:

[0041] This embodiment provides an experimental process for designing a three-factor, three-level experiment using the Box-Behnken response surface methodology, with the oil yield (Y) of the essential oil as the response value, and determining the extraction conditions of fresh Acer palmatum leaf essential oil based on the response surface optimization results.

[0042] I. Experimental Procedure:

[0043] Experiment 1: Optimization of Extraction of Essential Oil from Fresh Maple Leaves Using Response Surface Methodology

[0044] a. Single-factor experiments on the extraction of essential oil from fresh leaves of *Acer buergerianum*, including the following:

[0045] (1) Effect of NaCl concentration on the oil yield of fresh Acer palmatum leaves

[0046] This experiment explored the effect of different NaCl mass fractions on the oil yield of fresh Acer buergerianum leaves. 100g of crushed fresh Acer buergerianum leaves were weighed and placed in a round-bottom flask. 600mL of NaCl solutions with mass fractions of 0%, 2%, 5%, 10%, and 12% were added, respectively. The mixture was then subjected to electric heating distillation. The oil yield was recorded every hour until no significant increase was observed, at which point the oil yield was recorded again. The oil yields of the fresh Acer buergerianum leaves were then compared.

[0047] (2) Effect of particle size on the oil yield of fresh Acer palmatum leaves

[0048] Steam distillation was used to extract essential oil from *Acer buergerianum*. 100g of fresh *Acer buergerianum* leaves with particle sizes of 0.4mm, 0.6mm, 0.8mm, 2.0mm, and 4.0mm (i.e., 40 mesh, 30 mesh, 20 mesh, 10 mesh, and 5 mesh) were weighed out and placed in a round-bottom beaker with zeolite. 600mL of distilled water was added, and the oil yield was recorded every hour until there was no significant increase. The oil yield of the essential oil was then compared.

[0049] (3) Effect of liquid-to-solid ratio on oil yield of fresh Acer palmatum leaves

[0050] This experiment used 100g of fresh, pulverized maple leaves as the experimental material. The material-to-liquid ratio was 1:4, 1:5, 1:6, 1:7, and 1:8. The oil yield was recorded every hour until there was no significant increase. The oil yield of the essential oils was then compared.

[0051] (4) Effect of distillation time on the yield of essential oil from fresh Acer palmatum leaves

[0052] This experiment used 100g of fresh, pulverized maple leaves as experimental material. Distillation time was used as the single factor in the experiment, and the oil yield was recorded at 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, and 9h.

[0053] b. Response surface optimization analysis of Maple Leaf Essential Oil

[0054] The response surface methodology optimization experiment for extracting essential oil from fresh leaves of *Acer buergerianum* is as follows:

[0055] Based on the above single-factor experiments, the results showed that particle size, distillation time, and liquid-to-solid ratio had the greatest impact on the oil yield of fresh Acer truncatum leaves. A Box-Behnken orthogonal experimental design was used, with particle size (X1), distillation time (X2), and liquid-to-solid ratio (X3) as variables, each with three levels, to find the optimal process parameters for extracting essential oil from fresh Acer truncatum leaves.

[0056] The experimental design of factor levels for the extraction of essential oil from fresh leaves of *Acer buergerianum* is shown in Table 1.

[0057] Table 1. Experimental factor level design for extracting essential oil from fresh leaves of *Acer truncatum*.

[0058]

[0059] Experiment 2: GC-MS Analysis of Fresh Maple Leaf Essential Oil

[0060] The optimal extraction process for *Acer buergerianum* leaf essential oil was employed using response surface methodology. The extracted essential oil was dried using anhydrous sodium sulfate, and then diluted to a suitable concentration using n-hexane as a solvent. Chemical composition analysis was performed using gas chromatography-mass spectrometry (GC-MS).

[0061] GC-MS injection conditions

[0062] 1. Chromatographic conditions: Agilent 8890 gas chromatograph, column model: HP-5MS (30m×25mm×25μm), carrier gas: He, injection volume: 1μL, split injection, split ratio: 20:1, injection port temperature: 260℃. Initial column temperature: 80℃, hold for 1 min, then increase to 220℃ at a rate of 5℃ / min, hold for 5 min.

[0063] 2. Mass spectrometry conditions: Agilent 7250MSD mass spectrometer, ion source: EI source, ion source temperature: 230℃, scan range: 30-500, scan time: 5 times / s, transfer line temperature: 250℃.

[0064] II. Experimental Results and Analysis:

[0065] Experiment 1 Results a: Single-factor experiment on essential oil extraction from fresh Acer palmatum leaves

[0066] (1) The effect of NaCl mass fraction on the oil yield of fresh Acer palmatum leaves is as follows: Figure 1 As shown;

[0067] Depend on Figure 1It can be seen that with the increase of NaCl concentration, the yield of essential oil from fresh Acer palmatum leaves shows a trend of first increasing and then decreasing. When the NaCl concentration reaches 5%, the yield reaches its maximum of 1.15% ± 0.01%. When the NaCl concentration exceeds 5%, the yield continuously decreases and tends to reach equilibrium. The reason for the change in the yield of essential oil from fresh Acer palmatum leaves may be that the salting-out effect of NaCl increases the degree of damage to the epidermal cells of fresh Acer palmatum leaves, making it easier for the essential oil in the leaf tissue to be carried out by water vapor. It also increases the density and polarity of water, making it easier to separate from the oil, thus increasing the yield of essential oil. However, when the NaCl concentration is too high, it reduces the solubility of essential oil in water, thus reducing the extraction rate. Analysis shows that the mass fraction of NaCl in the solution affects the yield of essential oil, and the optimal NaCl mass fraction for the extraction process of essential oil from fresh Acer palmatum leaves is 5%.

[0068] (2) The effect of particle size on the oil yield of fresh Acer truncatum leaves is as follows: Figure 2 As shown:

[0069] With increasing particle size, the yield of essential oil from fresh Acer buergerianum leaves showed a trend of first increasing and then decreasing. Figure 2 It can be seen that the essential oil yield reaches its maximum of 1.09% ± 0.01% when the particle size is 0.6 mm. As the particle size exceeds 0.6 mm, the essential oil yield continuously decreases and tends towards equilibrium. Because the raw materials are pulverized, moisture can more easily penetrate into the interior, thus evaporating the essential oil. However, as the degree of raw material crushing increases and the material size becomes too small, the material expands, reducing the amount of essential oil recovered, hindering the distillation process, and leading to a decrease in oil yield. Analysis shows that particle size affects the essential oil yield, and the optimal particle size for the extraction process of fresh Acer palmatum leaf essential oil is 0.6 mm.

[0070] (3) The effect of liquid-to-solid ratio on the oil yield of fresh Acer truncatum leaves is as follows: Figure 3 As shown:

[0071] Depend on Figure 3 It can be seen that with the increase of the liquid-to-solid ratio, the oil yield of *Acer buergerianum* fresh leaves showed a trend of first slowly increasing and then gradually decreasing. The highest oil yield (1.16% ± 0.02%) was achieved at a liquid-to-solid ratio of 6 mL / g. The lower oil yield at lower liquid-to-solid ratios may be due to the raw materials not being completely submerged in the solution, resulting in a lower extraction rate. Conversely, excessively high liquid-to-solid ratios lead to excessive water, causing the essential oil to dissolve and emulsify, further reducing the oil yield. Therefore, the optimal liquid-to-solid ratio for extracting *Acer buergerianum* fresh leaves is 6 mL / g.

[0072] (4) The effect of distillation time on the yield of essential oil from fresh Acer palmatum leaves is shown in the following results. Figure 4 As shown:

[0073] Depend on Figure 4 It can be seen that the oil yield of fresh Acer palmatum leaves gradually increases with the extension of distillation time. The oil yield increases rapidly before 5 hours, and then tends to level off, with the highest oil yield reaching 0.92% ± 0.02%. Under the same power, the longer the distillation time, the more energy is consumed. Therefore, in order to find a method with high energy utilization, it is necessary to select a suitable distillation time. Thus, the results of the effect of distillation time on the oil yield show that the oil yield of fresh Acer palmatum leaves is highest after 5 hours.

[0074] Experiment 1 Result b: Analysis of response surface methodology optimization results for Maple Leaf Essential Oil:

[0075] (1) Orthogonal experiment and results on the yield of essential oil from fresh Acer palmatum leaves

[0076] Through single-factor analysis of fresh Acer buergerianum leaves, the optimal conditions and two levels near them were selected, namely, particle size (X1) 0.4 mm, 0.6 mm, 0.8 mm, distillation time (X2) 4 h, 5 h, 6 h, and liquid-to-solid ratio (X3) 5 mL / g, 6 mL / g, 7 mL / g.

[0077] Using the essential oil extraction rate (Y) of fresh Acer buergerianum leaves as the response value, and distillation time, liquid-to-solid ratio, and particle size as response factors, a three-factor, three-level experiment was designed using the Box-Benkken central composite design principle to optimize the extraction conditions of essential oil from fresh Acer buergerianum leaves. The experimental design and results are shown in Table 2. The experimental results were analyzed using Design-Expert software, and the regression equations for the essential oil extraction rate Y (%) of fresh Acer buergerianum leaves with respect to distillation time, liquid-to-solid ratio, and particle size are shown below:

[0078] Y=-2.85000+0.26625*X2+0.69250*X3+0.055125*X1+0.15000*X2X3+7.50000E-004*X2X1-2.

[0079] 50000E-003*X3X1-0.038750*X2 2 -0.061250*X3 2 -6087500E-004*X1 2

[0080] Table 2 Response Surface Experimental Design and Results

[0081]

[0082] (2) Establishment of regression model and significance analysis

[0083] The oil yield Y of fresh Acer palmatum leaves in Table 2 was analyzed using Design-Expert software through response surface methodology. The regression equations for the oil yield Y (%) of fresh Acer palmatum leaves with respect to distillation time, liquid-to-solid ratio, and particle size are shown below:

[0084] Y=-2.85000+0.26625*X2+0.69250*X3+0.055125*X1+0.15000*X2X3+7.50000E-004*X2X1-2.

[0085] 50000E-003*X3X1-0.038750*X2 2 -0.061250*X3 2 -6087500E-004*X1 2 A significance analysis was performed on the model, and the results are shown in Table 3. Table 3 shows that the analysis of variance indicates the response surface methodology for *Acer palmatum* leaf essential oil is significantly effective (P<0.01), and the lack-of-fit term is not significant (P=0.065>0.05, coefficient of determination R0.05). 2 =0.9921), indicating a good fit and small experimental error. Adjustment coefficient R 2 adj =0.9871, indicating that the model can explain 98.71% of the changes in the response values. Therefore, this model can optimize the extraction process of essential oil from fresh Acer palmatum leaves.

[0086] Table 3. Analysis of Variance for Experimental Factors

[0087]

[0088]

[0089] The F-value indicates the importance of each factor to the experimental index; a larger F-value indicates a greater impact of that factor on the yield of *Acer buergerianum* leaf essential oil. Table 3 shows that distillation time and liquid-to-solid ratio have a highly significant impact on the extraction of *Acer buergerianum* leaf essential oil, while particle size has a significant impact, with the order of influence being: X3 > X2 > X1, i.e., liquid-to-solid ratio > distillation time > particle size. Based on the experimental results, X1, X3, and X1... 2 X2 2 X3 2 The effect of X2 on the oil yield was extremely significant (P<0.001), X2X3 was highly significant (P<0.01), and the remaining items had no significant effect on the oil yield of fresh Acer palmatum leaves.

[0090] (3) Analysis of the interaction of influencing factors

[0091] The three-dimensional response surface and contour lines of the interaction of various factors on the oil yield of fresh Acer palmatum leaves were obtained using the Design-Expert software simulation. The influence of each factor on the response value and the magnitude of the interaction can be reflected by observing the response surface and contour lines. Figure 5-7 It can be seen that the contour lines are elliptical and the response surface curve is relatively steep, therefore the interaction between the three factors has a significant impact on the oil yield of fresh maple leaf essential oil.

[0092] in, Figure 5 At a liquid-to-solid ratio of 6 mL / g, the three-dimensional surface is relatively steep, indicating that the oil yield of *Acer buergerianum* leaf essential oil is affected by both the liquid-to-solid ratio and distillation time. The contour lines are nearly circular, suggesting that the interaction between the liquid-to-solid ratio and distillation time has little effect on the oil yield of *Acer buergerianum* leaf essential oil. The oil yield of *Acer buergerianum* leaf essential oil reaches its maximum when the liquid-to-solid ratio is in the range of 5–7 mL / g and the distillation time is in the range of 4–6 h. When the liquid-to-solid ratio and distillation time are increased or decreased outside the range, the oil yield of *Acer buergerianum* leaf essential oil decreases accordingly.

[0093] Figure 6 When the particle size is 0.6 mm, the three-dimensional surface is relatively steep, indicating that the oil yield of *Acer buergerianum* leaf essential oil is affected by both particle size and distillation time. The contour lines are nearly elliptical, indicating a significant interaction between particle size and distillation time. The oil yield of *Acer buergerianum* leaf essential oil reaches its maximum when the particle size is in the range of 0.4–0.8 mm and the distillation time is in the range of 4–6 h. When the particle size and distillation time are increased or decreased outside the range, the oil yield of *Acer buergerianum* leaf essential oil decreases accordingly.

[0094] Figure 7 At a liquid-to-solid ratio of 6 mL / g, the three-dimensional surface is relatively steep, indicating that the oil yield of *Acer buergerianum* leaf essential oil is affected by both the liquid-to-solid ratio and particle size. The contour lines are nearly elliptical, indicating a significant interaction between particle size and liquid-to-solid ratio. The oil yield of *Acer buergerianum* leaf essential oil reaches its maximum when the particle size is in the range of 0.4–0.8 mm and the liquid-to-solid ratio is in the range of 5–7 mL / g. When the particle size and liquid-to-solid ratio increase or decrease outside these ranges, the oil yield of *Acer buergerianum* leaf essential oil decreases accordingly.

[0095] (4) Optimization of process parameters and model validation for essential oil extraction from fresh Acer truncatum leaves

[0096] The optimal process parameters for yielding essential oil from fresh Acer palmatum leaves were determined through simulation using Design-Expert software, combined with regression model and response surface methodology. The results showed that under these conditions, the model predicted an oil yield of 1.244% for the following parameters: particle size (X1) 0.575 mm, distillation time (X2) 4.867 h, and liquid-to-solid ratio (X3) 5.584 mL / g. After three parallel experiments, the average yield of essential oil from fresh Acer palmatum leaves was found to be 1.24% ± 0.01%, which is relatively close to the model prediction. This indicates that the quadratic polynomial regression equation is reliable in predicting the oil yield of Acer palmatum leaves and has practical guiding significance. Response surface methodology showed that a higher oil yield was achieved under the following conditions: NaCl concentration of 5%, liquid-to-solid ratio of 6 mL / g, distillation time of 5 h, and particle size of 0.6 mm.

[0097] Experiment 2 Results: GC-MS Results and Analysis

[0098] like Figure 8 As shown, the chemical composition of the essential oil from fresh Acer davidii leaves obtained by steam distillation and drying was analyzed using an Agilent 88907250 gas chromatograph-mass spectrometer. With a maximum area percentage greater than 0.1%, a total of 44 compounds were identified. Using the NIST 2020 library for automatic searching, 34 chemical components were identified, accounting for 80.93% of the total peak area. The volatile oil from fresh Acer davidii leaves mainly contains terpenes and alcohols, with key components including α-Bisabolol (22.90%), (-)-α-Neoclovene (6.59%), Cedrene (5.82%), Eudesma-2,4,11-triene (4.80%), Oxacyclotetradeca-4,11-diyne (4.50%), and (Z)-sesquisabinenehydrate (4.41%). The highest content α-Bisabolol (22.90%) has anti-inflammatory and antibacterial activities, is chemically stable, and has good skin compatibility, making it suitable for use in skin care products.

[0099] The GC-MS analysis results of fresh Acer palmatum leaf essential oil are shown in Table 4:

[0100] Table 4. GC-MS Analysis Results of Fresh Maple Leaf Essential Oil

[0101]

[0102]

[0103] “RI a "Retain the exponent for the experiment; "RI b"This is the document retention index."

[0104] In summary, this application used single-factor experiments to screen NaCl concentration, distillation time, particle size, and liquid-to-solid ratio. Centering on the optimal point of the single-factor experiment, two levels were selected above and below the optimal point as the levels of the response surface. The Box-Behnken response surface design method was used to design a three-factor, three-level experiment. The oil yield of essential oil was used as the response value. The extraction conditions of fresh Acer palmatum leaf essential oil were determined by combining three-dimensional response surface plots and two-dimensional contour plots.

[0105] Example 3:

[0106] This embodiment provides the application of fresh Acer palmatum leaf essential oil, specifically using fresh Acer palmatum leaf essential oil as a raw material to prepare skin care products.

[0107] The applicant applied the *Gnaphalium affine* leaf essential oil to skincare products because, after preparing the essential oil, the applicant conducted the following tests and found that it possesses antibacterial, antioxidant, and whitening effects. The specific tests are as follows:

[0108] Experiment 3: Antibacterial test of fresh maple leaf essential oil

[0109] Staphylococcus epidermidis, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Micrococcus luteus were inoculated onto LB agar plates and cultured at 37°C for 24 h. Then, single colonies of suitable size, located away from the main bacterial community, were inoculated onto LB agar plates and cultured again to obtain second-generation bacteria. Finally, a colony with a diameter > 1 mm was selected and inoculated into LB liquid medium, cultured at 37°C and 120 rpm until the logarithmic growth phase. The OD of the bacterial suspension was measured using a microplate reader. 600 =0.7 (approximately 5 × 10) 8 (CFU / mL).

[0110] Dilute the bacterial suspension 100 times to achieve a bacterial concentration of 1×10⁻⁶. 6 -10 8 CFU / mL. Take 200 μL of the bacterial suspension to be tested and spread it evenly on a solid culture medium using a sterile triangular swab to prepare a bacterial plate. Prepare filter paper discs with a diameter of 6 mm, sterilize them in an autoclave, and then cool them to room temperature in a sterile operating room. Take small paper discs soaked in fresh Acer palmatum leaf essential oil and place them on the plate containing the bacterial suspension. Place the plate in an incubator and incubate at 37°C for 24 h. Use filter paper discs soaked in sterile water as a blank control and filter paper discs soaked in 1 mg / mL streptomycin solution as a positive control. Repeat the experiment three times. Measure the diameter of the inhibition zone using the cross-caliper method, take the average value, and calculate the inhibition rate.

[0111] Formula for calculating antibacterial rate:

[0112]

[0113] The principle of the microdilution method is to culture the antimicrobial drug and the test microorganism within a certain dilution range, typically at a suspension concentration of 1 million colony-forming units (CFU) per milliliter (mL), and then measure the growth of microorganisms at different concentrations of the antimicrobial drug. Due to the presence of microorganisms, the test system without antimicrobial activity will become turbid, while the absence of turbidity indicates that the growth of the test microorganism has been inhibited.

[0114] The minimum inhibitory concentration (MIC) of *Acer buergerianum* leaf essential oil was determined using a two-fold dilution method. *Acer buergerianum* essential oil was serially diluted halfway to obtain concentration gradients of 25%, 12.5%, 6.25%, 3.13%, 1.56%, 0.78%, 0.39%, 0.2%, and 0.1% (v / v), with 2% DMSO used as an emulsifier. 40 μL of different concentrations of *Acer buergerianum* leaf volatile oil were added to 96-well plates, followed by 150 μL of culture medium and 10 μL of bacterial suspension (1×10⁻⁶). 7 The concentrations of the final total system of fresh Acer palmatum leaf essential oil in the microplates were determined by adding CFU / mL to achieve concentrations of 10%, 5%, 2.5%, 1.25%, 0.62%, 0.31%, 0.16%, 0.08%, and 0.04%. Microplates containing only bacterial suspension and culture medium served as blank controls, while those containing only 2% DMSO and culture medium served as negative controls. After incubating the 96-well plates at 37°C for 24 hours, the concentration of volatile oil in the wells with no visible bacterial growth and a clear appearance was considered the minimum inhibitory concentration (MIC). Each experiment was performed in triplicate.

[0115] 1. Analysis of the diameter and antibacterial rate of the antibacterial zone of fresh maple leaf essential oil.

[0116] The essential oil from fresh Acer truncatum leaves showed inhibitory effects against Staphylococcus epidermidis, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Micrococcus luteus. According to Table 5, the essential oil exhibited the best antibacterial effect against Staphylococcus epidermidis, with an inhibition diameter of 12.83±0.43 mm and an inhibition rate of 53.23±1.52%. It also showed good inhibitory effects against Staphylococcus aureus, with an inhibition diameter of 12.50±0.75 mm and an inhibition rate of 52.00±2.72%. The essential oil showed moderate antibacterial effects against Micrococcus luteus and methicillin-resistant Staphylococcus aureus (MRSA), with inhibition rates of 16.78±2.68% and 23.37±3.28%, respectively. This may be due to the role of α-bisabolol, the main chemical component in the essential oil.

[0117] Table 5. Inhibitory effect (mm) of fresh maple leaf essential oil on four types of bacteria.

[0118]

[0119] Note: "-" indicates no antibacterial effect; "DEO" represents fresh maple leaf essential oil.

[0120] 2. Analysis of the results of the determination of the minimum inhibitory concentration (MIC) of fresh Acer buergerianum leaf essential oil

[0121] The results of the minimum inhibitory concentration (MIC) determination of fresh Acer buergerianum leaf essential oil are shown in Table 6. The experimental results indicate that the essential oil has a good antibacterial effect. This experiment shows that among the four bacterial species, the essential oil of fresh Acer buergerianum leaf has a better antibacterial effect against Staphylococcus epidermidis and Staphylococcus aureus, while its antibacterial effect against Micrococcus luteus and methicillin-resistant Staphylococcus aureus is moderate. The MIC value of fresh Acer buergerianum leaf essential oil against Staphylococcus epidermidis is 0.31%, against Staphylococcus aureus is 5%, against Micrococcus luteus is 10%, and against methicillin-resistant Staphylococcus aureus is greater than 10%.

[0122] Micrococcus luteus is mainly found on the skin surface of normal humans and animals. It is an opportunistic pathogen. When the skin is broken and infected with Micrococcus luteus, it may cause inflammatory reactions such as itching, redness, and rashes. Staphylococcus epidermidis is a Gram-positive coccus that thrives on the epidermis of organisms, mainly found on human skin. Staphylococcus epidermidis is prone to developing drug resistance, and infection with it often causes skin diseases such as atopic dermatitis, seborrheic dermatitis, rosacea, acne, and folliculitis. Maple leaf essential oil has a good inhibitory effect on Micrococcus luteus and Staphylococcus epidermidis, and can be used as an ingredient in skincare products.

[0123] Table 6. MIC determination results of fresh maple leaf essential oil against four bacteria.

[0124]

[0125] Experiment 4: Study on the antioxidant activity of fresh Acer palmatum leaf essential oil

[0126] 1. Ferrous ion reducing power (FRAP)

[0127] The principle of FRAP method is Fe 3+ Tripyridyl-triazine (TPTZ) has a maximum absorption peak at 593 nm. Under the action of antioxidants, it is reduced to its ferrous form, exhibiting a blue color and increasing absorbance. A higher absorbance indicates a stronger reducing ability of the antioxidant.

[0128] 2. Experimental reagents and instruments

[0129] C2H3NaO2·3H2O (crystalline sodium acetate), C2H4O2 (acetic acid), FeCl3, FeCl3·6H2O, concentrated hydrochloric acid, TPTZ

[0130] 3. Preparation of the stock solution

[0131] 0.3 mol / L acetate buffer with pH 3.6: Add 5.1 g C2H3NaO2·3H2O to 20 mL C2H4O2, then dilute with water to a final volume of 250 mL.

[0132] 20 mmol / L FeCl3 solution: 0.5606 g FeCl3·6H2O was diluted to 100 mL with distilled water.

[0133] 40 mmol / L hydrochloric acid solution: 0.333 mL of concentrated hydrochloric acid (12 mol / L) is added to water to 100 mL.

[0134] 10 mmol / L TPTZ solution: 0.0313 g of TPTZ, add 40 mmol / L hydrochloric acid solution to make up to 10 mL.

[0135] Preparation of FRAP working solution: Take 25 mL of 0.3 mol / L acetate buffer (pH 3.6), add 2.5 mL of 20 mmol / L FeCl3 solution, then add 2.5 mL of 10 mmol / L TPTZ solution. Mix well and preheat in a 37°C water bath. Note: FRAP working solution should be prepared fresh before use.

[0136] Mix 0.1 mL of the sample solution with 2.4 mL of FRAP working solution, incubate at 37 °C for 10 min, and measure the absorbance at 593 nm. Perform the experiment in triplicate. Use distilled water instead of the sample in the FRAP working solution as a blank control. Use 2,6-di-tert-butyl-p-cresol (BHT) as a positive control.

[0137] 4. Determination of total reducing power

[0138] Antioxidants (reducing agents) scavenge free radicals by donating electrons through their own reduction action; the stronger the reducing power, the stronger the antioxidant activity. In the experiment, the antioxidant in the sample reduced the ferric iron of potassium ferricyanide to ferrous iron (potassium ferrocyanide). The ferrous iron (potassium ferrocyanide) further reacted with ferric chloride to generate Prussian blue (Fe4[Fe(CN)6]3), which has the maximum absorbance at 700 nm. Therefore, the absorbance at 700 nm can indirectly reflect the reducing power of the antioxidant; the higher the absorbance, the stronger the reducing power.

[0139] The reaction mixture of 0.1 mL of the mixture (64 mg / mL DMSO) was mixed with phosphate buffer (0.2 mL, 2 mM, pH 6.6) and potassium ferric cyanide [K3Fe(CN)3] (0.2 mL, 1%, w / v). The mixture was incubated at 50 °C for 20 min. A portion (0.25 mL) of trichloroacetic acid (10%, w / v) was added to the mixture, and then centrifuged for 10 min. The supernatant (0.1 mL) was mixed with distilled water (0.5 mL) and FeCl3 (0.75 mL, 0.1% w / v), shaken vigorously, and incubated in the dark at room temperature for 30 min. The absorbance was measured at 700 nm using a spectrophotometer.

[0140] Depend on Figure 9 It was found that the antioxidant capacity of *Acer buergerianum* fresh leaf essential oil gradually increased as the concentration increased from 0.04 mg / mL to 1.28 mg / mL. At 1.28 mg / mL, *Acer buergerianum* fresh leaf essential oil exhibited approximately 50% more antioxidant capacity compared to the positive control BHT. The IC50 of the antioxidant capacity of *Acer buergerianum* fresh leaf essential oil determined by the FRAP method was calculated using GraphpadPiam 9.5 software. 50 =0.30mg / mL.

[0141] Depend on Figure 10 It was found that the total reducing power increased linearly with the increase of the concentration of *Acer buergerianum* leaf essential oil and the positive control BHT, indicating that both could inhibit free radical electron transfer and terminate free radical chain reactions. In the total reducing power determination, *Acer buergerianum* leaf essential oil, at a concentration of 6.4 mg / mL, exhibited approximately 50% more antioxidant capacity compared to the positive control BHT. The IC50 of the antioxidant capacity of *Acer buergerianum* essential oil determined by the total reducing power method was calculated using Graphpad Piam 9.5 software. 50 =2.31 mg / mL.

[0142] Experiment 5: Study on the Inhibition of Tyrosinase Activity by Fresh Maple Leaf Essential Oil

[0143] 1. Reagents and Instruments

[0144] Tyrosinase, L-DOPA, sodium dihydrogen phosphate, disodium hydrogen phosphate, positive control (kojic acid), sample, deionized water, thermostat, microplate reader, centrifuge, 96-well plate.

[0145] 2. Reagent preparation

[0146] Phosphate buffer solution (20 mmol / L, pH = 6.8): Accurately weigh 0.7957 g sodium dihydrogen phosphate and 1.7552 g disodium hydrogen phosphate, dissolve them in a small amount of deionized water, and bring the volume to 500 mL. Store at 4 °C for later use.

[0147] Preparation of 2% DMSO phosphate buffer solution: Add 2 mL of DMSO solution, then add 98 mL of pH 6.8 20 mmol / L phosphate buffer solution to dissolve it, and finally obtain 2% DMSO phosphate buffer solution.

[0148] L-DOPA solution (2 mmol / L): Accurately weigh 0.0394 g of L-DOPA powder, dissolve it in a 20 mmol / L phosphate buffer solution with pH 6.8, and bring the volume up to 100 mL to obtain a 2 mmol / L L-DOPA solution.

[0149] Test solutions: The test solutions were essential oils of Callicarpa macrocarpa and Acer palmatum, which were dissolved in 2% DMSO to prepare final system concentrations of 5.00 mg / mL, 2.50 mg / mL, 1.25 mg / mL, 0.63 mg / mL, 0.31 mg / mL, and 0.16 mg / mL.

[0150] Positive control (+CK): Accurately weigh 0.1 g of kojic acid and dissolve it in 20 mL of deionized water to obtain a 5 mg / mL test solution. Add 50 μL of PBS buffer solution containing 2% DMSO as a blank control.

[0151] Tyrosinase stock solution: 1 mg of tyrosinase is diluted to 1 mL in a volumetric flask to prepare a 1 mg / mL solution as the stock solution.

[0152] 3. Detection Method

[0153] 50 μL of sample and 50 μL of tyrosinase solution (0.06 mg / mL) were incubated at 37 °C for 10 min, and the absorbance was measured at 475 nm. Immediately afterwards, 100 μL of L-DOPA solution (0.5 mM) was added to a 96-well plate, and the plate was incubated again at 30 °C for 10 min, and the absorbance was measured at 475 nm. Kojic acid was used as a positive control.

[0154] Tyrosinase inhibition rate (%) = 1 - (A2 - A1 / B2 - B1) × 100%.

[0155] Where A1 is the absorbance value of the sample at 0 min, A2 is the absorbance value of the sample after 10 min, B1 is the absorbance value of the blank group at 0 min, and B2 is the absorbance value of the blank group after 10 min.

[0156] Depend on Figure 11The results showed that the essential oil from fresh Acer truncatum leaves had a certain inhibitory effect on mushroom tyrosinase. At a concentration of 0.05 mg / mL, the inhibitory effect of Acer truncatum leaf essential oil on mushroom tyrosinase was greater than that of the positive control kojic acid, with an inhibition rate of 52%. The inhibitory effect on mushroom tyrosinase increased with increasing concentration of Acer truncatum leaf essential oil, reaching an inhibition rate of 78% at a concentration of 12.5 mg / mL. The IC50 of the inhibitory activity of Acer truncatum leaf essential oil on mushroom tyrosinase was calculated using Graphpad Pism 9.5 software. 50 =0.48 mg / mL. Furthermore, the essential oil from fresh Acer buergerianum leaves showed good inhibitory activity against mushroom tyrosinase, and this inhibitory activity was relatively stable.

[0157] Therefore, the essential oil of fresh maple leaves has antibacterial, antioxidant and whitening effects. It is a newly discovered plant essential oil with strong comprehensive capabilities and has great potential for application in skin care products.

[0158] The above embodiments are merely examples of several implementations of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention.

Claims

1. A method for extracting essential oil from fresh leaves of Acer truncatum, characterized in that: The method comprises the following steps: (1) Crushing: Collect fresh Acer truncatum leaves free of pests and diseases, crush the Acer truncatum leaves using a crusher to obtain fresh leaf powder, and then seal and store for later use; (2) Mixing: mixing the fresh leaf powder obtained in step (1) and NaCl at a liquid-to-solid ratio of 5-7 mL / g to obtain a mixed liquid; (3) Extraction: The mixed liquid is subjected to water distillation to obtain the essential oil of fresh leaves of Acer truncatum.

2. The method according to claim 1, characterized in that The step (1) is to grind the powder to a particle size of 0.6 mm.

3. The method according to claim 1, characterized in that: The concentration of NaCl solution in step (2) is 5%.

4. The method according to claim 1, characterized in that: The liquid-to-solid ratio of fresh leaf powder to NaCl in step (2) is 6 mL / g.

5. The method according to claim 1, characterized in that The time for water distillation extraction in step (3) is 5 hours.

6. Application of the Acer truncatum fresh leaf essential oil according to any one of claims 1 to 5 in skin care products.

7. The use according to claim 6, characterized in that: The application is specifically to use the fresh leaf essential oil of Acer truncatum as a raw material for preparing skin care products.

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