Prunus humilis leaf essential oil as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of plant leaf essential oil extraction, and particularly relates to cerasus humilis leaf essential oil and a preparation method and application thereof. The preparation method of the cerasus humilis leaf essential oil comprises the following steps: mixing cerasus humilis leaves with water, and carrying out first distillation to obtain cerasus humilis leaf hydrolat; mixing the cerasus humilis leaf hydrolat with cerasus humilis leaves, and performing secondary distillation to obtain the cerasus humilis leaf essential oil. Through the preparation method of the cerasus humilis leaf essential oil provided by the invention, the cerasus humilis leaf essential oil containing various volatile components such as benzaldehyde, methylcyclopentane, n-heptane and the like can be prepared. According to the record of the embodiment, the obtained cerasus humilis leaf essential oil can have a remarkable antibacterial effect on escherichia coli, staphylococcus aureus and bacillus subtilis and has scavenging activity on free radicals such as DPPH and ABTS, and the highest antioxidant capacity can reach 3.86 mmol / L FeSO4 through iron ion reduction antioxidant capacity (FRAP) detection.

Description

Technical Field

[0001] The invention belongs to the technical field of plant leaf essential oil extraction, and particularly relates to a Prunus cerasifera leaf essential oil and a preparation method and application thereof. Background Art

[0002] Essential oil is a general term for volatile aromatic substances extracted from aromatic plants or aromatic animals. It is usually produced by seeds, flowers, leaves, buds, stems, fruits, roots, wood or bark of plants and stored in oil ducts, resin ducts, glands or trichomes of plants. Essential oils have strong antioxidant properties and aromatic smells and are widely used in medicine, food flavors and cosmetics. In addition, essential oils have strong antibacterial activity and are considered to be a natural alternative to antibiotic resistance.

[0003] Cerasus humilis belongs to the genus Prunus in the Rosaceae family and is a short woody shrub. Cerasus humilis is an important traditional medicinal plant with high medicinal value. Its kernel is called "Yu Li Ren" in traditional Chinese medicine. It contains high levels of polyunsaturated fatty acids and can be used to treat symptoms such as large intestine qi stagnation, dryness and astringency, urination difficulties, abdominal edema, limb edema, and athlete's foot. However, as an emerging plant resource, Cerasus humilis has little research on its leaves, with insufficient depth and breadth, few results, and insufficient comprehensive development. In particular, Cerasus humilis leaf essential oil has not yet been developed and marketed. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a preparation method and application of Prunus cerasifera leaf essential oil. The method provided by the present invention can not only prepare Prunus cerasifera leaf essential oil containing various volatile components such as benzaldehyde, methylcyclopentane, n-heptane, etc., which makes up for the vacancy of Prunus cerasifera leaf essential oil products on the market, but also the prepared Prunus cerasifera leaf essential oil has significant antibacterial effect and strong antioxidant ability.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing Prunus cerasifera leaf essential oil, comprising the following steps:

[0007] The leaves of Prunus cerasifera are mixed with water and subjected to a first distillation to obtain a hydrosol of the leaves of Prunus cerasifera; the hydrosol of the leaves of Prunus cerasifera is mixed with leaves of Prunus cerasifera and subjected to a second distillation to obtain the essential oil of the leaves of Prunus cerasifera.

[0008] Preferably, during the first distillation, the mass volume ratio of the Prunus armeniaca leaves to water is 1:5 to 2:5 g / mL; during the second distillation, the mass volume ratio of the Prunus armeniaca leaves to the hydrosol is 1:3 to 1:5 g / mL.

[0009] Preferably, the temperature of the first distillation is 90°C to 100°C, and the time is 60 min; the temperature of the second distillation is 100°C to 110°C, and the time is 120 min.

[0010] The present invention provides Prunus humilis Bunge leaf essential oil prepared by the above preparation method. By mass percentage, the Prunus humilis Bunge leaf essential oil includes 58.67% to 61.18% benzaldehyde, 12.48% to 13.99% methylcyclopentane, 8.74% to 9.37% n-heptane, 5.54% to 5.78% 2-methylhexane, 4.50% to 4.34% 3-methylhexane, and the balance of other substances.

[0011] The present invention also provides the application of the Prunus humilis Bunge leaf essential oil described in the above technical solution in the preparation of antibacterial products.

[0012] Preferably, the bacteria include one or more of Escherichia coli, Staphylococcus aureus, and Bacillus subtilis.

[0013] Preferably, the product includes a repellent and / or a bactericide.

[0014] The present invention also provides the application of the Prunus humilis Bunge leaf essential oil described in the above technical solution in the preparation of antioxidant products.

[0015] Preferably, the antioxidant includes scavenging of free radicals and / or reduction of iron ions; the free radicals include DPPH and / or ABTS.

[0016] Preferably, the product includes an antioxidant.

[0017] Beneficial effects:

[0018] The present invention provides a preparation method of Prunus humilis Bunge leaves, comprising the following steps: mixing Prunus humilis Bunge leaves with water for the first distillation to obtain Prunus humilis Bunge leaf hydrosol; mixing the Prunus humilis Bunge leaf hydrosol with Prunus humilis Bunge leaves for the second distillation to obtain the Prunus humilis Bunge leaf essential oil. The method provided by the present invention has a simple process and is easy to operate, and can prepare Prunus humilis Bunge leaf essential oil containing various volatile components such as benzaldehyde, methylcyclopentane, and n-heptane. According to the records of the examples, the Prunus humilis Bunge leaf essential oil obtained by the preparation method provided by the present invention includes 20 compounds, among which benzaldehyde has the largest relative abundance, accounting for 60.32%, and it is one of the most important molecules in the spice industry.

[0019] In addition, the eucalyptus leaf essential oil prepared by the present invention contains multiple volatile components such as benzaldehyde, methylcyclopentane, and n-heptane, and can have significant antibacterial effects on Escherichia coli, Staphylococcus aureus, and Bacillus subtilis. Moreover, the eucalyptus leaf essential oil prepared by the present invention has strong antioxidant capacity. According to the examples, the eucalyptus leaf essential oil obtained by the present invention has scavenging activity of free radicals such as DPPH and ABTS, and the highest antioxidant capacity can reach 3.86mmol / LFeSO4 by using iron ion reduction antioxidant capacity (FRAP) detection.

[0020] The preparation method of the Prunus cerasifera leaf provided by the present invention can successfully prepare the Prunus cerasifera leaf essential oil, and the obtained Prunus cerasifera leaf essential oil can be used to prepare antibacterial and antioxidant products, which makes up for the vacancy of the Prunus cerasifera leaf essential oil products on the market and provides a theoretical basis for the development and research of the Prunus cerasifera leaf. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0022] Figure 1 Different chromatograms formed for Example 2;

[0023] Figure 2 This is a graph showing the evaluation results of the antibacterial activity of the Prunus cerasifera leaf essential oil prepared in Example 1 against Escherichia coli, Bacillus subtilis and Staphylococcus aureus in Example 2;

[0024] Figure 3 The figure is a result of evaluating the antibacterial activity of ampicillin against Escherichia coli, Bacillus subtilis and Staphylococcus aureus in Example 3;

[0025] Figure 4 The inhibition zone sizes (mm) of the Prunus oleifera leaf essential oil prepared in Example 1 at different concentrations (50, 100, 200, 500 μL / mL) against Escherichia coli, Bacillus subtilis and Staphylococcus aureus;

[0026] Figure 5 The inhibition zone size (mm) of ampicillin against Escherichia coli, Bacillus subtilis and Staphylococcus aureus in Example 3. DETAILED DESCRIPTION

[0027] The invention provides a method for preparing Prunus cerasifera leaf essential oil, comprising the following steps: mixing Prunus cerasifera leaf with water and performing a first distillation to obtain Prunus cerasifera leaf hydrosol; and mixing the Prunus cerasifera leaf hydrosol with Prunus cerasifera leaf and performing a second distillation to obtain the Prunus cerasifera leaf essential oil.

[0028] In the present invention, the Prunus cerasifera leaves are preferably crushed to obtain Prunus cerasifera leaf fragments. The present invention has no special limitation on the crushing method, and the conventional crushing method in the art can be used.

[0029] After obtaining the Euphorbia pulex leaf fragments, the present invention mixes the Euphorbia pulex leaf fragments with water for the first distillation to obtain Euphorbia pulex leaf hydrosol. The mass volume ratio of the Euphorbia pulex leaf and water in the present invention is preferably 1:5 to 2:5 g / mL, more preferably 2:5 g / mL. The first distillation of the present invention preferably adopts a steam distillation method, and specifically preferably places the Euphorbia pulex leaf fragments in a distillation device containing water for heating and distillation, collects the condensed distillate, and obtains the Euphorbia pulex leaf hydrosol. The steam distillation method used in the present invention has relatively mature technologies in terms of process, equipment, operation, etc., with low cost and high output, and can effectively extract the essential oil components in the Euphorbia pulex leaves, and the equipment and operation are relatively simple. The temperature of the first distillation of the present invention is preferably 90°C to 110°C, more preferably 100°C, and the distillation time is preferably 60min. The present invention does not specifically limit the device for the first distillation, and a conventional distillation device in the art can be used.

[0030] After obtaining the Prunus armeniaca leaf hydrosol, the present invention mixes the Prunus armeniaca leaf hydrosol with the Prunus armeniaca leaf for a second distillation to obtain the Prunus armeniaca leaf essential oil. The mass volume ratio of the Prunus armeniaca leaf and the Prunus armeniaca leaf hydrosol of the present invention is preferably 1:3 to 1:5 g / mL, more preferably 1:4 g / mL. The second distillation of the present invention preferably adopts a steam distillation method, specifically placing the Prunus armeniaca leaf in a distillation device containing the Prunus armeniaca leaf hydrosol and heating and distilling, collecting the condensed distillate, and obtaining the Prunus armeniaca leaf essential oil. During the second distillation of the present invention, no additional water is required. The temperature of the second distillation of the present invention is preferably 100°C to 110°C, and more preferably 100°C; the distillation time is preferably 120min. The present invention does not specifically limit the device for the second distillation, and a conventional distillation device in the art can be used. The advantage of the second distillation of the present invention is that it is easy to extract and can reduce the extraction time.

[0031] The preparation method of the Prunus cerasifera leaf provided by the invention has simple process and convenient operation, and can prepare the Prunus cerasifera leaf essential oil containing various volatile components such as benzaldehyde, methylcyclopentane, n-heptane, etc.; it makes up for the vacancy of the Prunus cerasifera leaf essential oil products on the market, and provides a theoretical basis for the development and research of the Prunus cerasifera leaf.

[0032] The present invention provides the essential oil of Prunus humilis Bunge leaves prepared by the above preparation method. By mass percentage, it includes 58.67% - 61.18% benzaldehyde, 12.48% - 13.99% methylcyclopentane, 8.74% - 9.37% n-heptane, 5.54% - 5.78% 2-methylhexane, 4.50% - 4.34% 3-methylhexane and the balance of other substances. Preferably, it is 60.32% benzaldehyde, 13.14% methylcyclopentane, 8.99% n-heptane, 5.64% 2-methylhexane, 4.39% 3-methylhexane and the balance of other substances. The essential oil of Prunus humilis Bunge leaves described in the present invention contains various volatile components such as benzaldehyde, methylcyclopentane, and n-heptane, has a significant antibacterial effect on Escherichia coli, Staphylococcus aureus, and Bacillus subtilis, and has strong antioxidant ability, such as strong scavenging activity against free radicals such as DPPH and ABTS.

[0033] Based on the above advantages, the present invention also provides the application of the essential oil of Prunus humilis Bunge leaves described in the above technical solution in the preparation of antibacterial products. The bacteria described in the present invention preferably include one or more of Escherichia coli, Staphylococcus aureus, and Bacillus subtilis, more preferably Escherichia coli and / or Bacillus subtilis, and even more preferably Bacillus subtilis. The products described in the present invention preferably include insect repellents and / or fungicides, more preferably fungicides.

[0034] The present invention also provides the application of the essential oil of Prunus humilis Bunge leaves described in the above technical solution in the preparation of antioxidant products. The antioxidant described in the present invention preferably includes the scavenging of free radicals and / or the reduction of iron ions, more preferably the scavenging of free radicals. The free radicals described in the present invention preferably include DPPH and / or ABTS, more preferably DPPH. The products described in the present invention preferably include antioxidants.

[0035] The essential oil of Prunus humilis Bunge leaves prepared in the present invention is used in antibacterial products, and has a significant antibacterial effect on bacteria such as Escherichia coli and Bacillus subtilis; while used in antioxidant products, it has strong scavenging activity against free radicals such as DPPH and ABTS, and the highest antioxidant ability detected by the ferric ion reducing antioxidant power (FRAP) can reach 3.86 mmol / L FeSO4.

[0036] In order to further illustrate the present invention, the essential oil of Prunus humilis Bunge leaves and its preparation method provided by the present invention will be described in detail below with reference to the drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

[0037] Example 1

[0038] A preparation method of the essential oil of Prunus humilis Bunge leaves is as follows:

[0039] Material selection: From August to October 2022, the leaves of 'Nongda No. 4' Prunus humilis were collected from the Prunus humilis germplasm resource nursery as experimental materials.

[0040] Specific operation: Break the Prunus humilis leaves, take 1200 g of leaves and place them in a distillation device containing 3000 mL of water for heating. Distill at 100 °C for 60 min, collect the condensed distillate, and obtain the pure dew of Prunus humilis leaves. Then take 90 g of Prunus humilis leaves and place them in a distillation device containing 360 mL of the pure dew of Prunus humilis leaves for heating. Distill at 100 °C for 120 min, collect the condensed distillate, and obtain the essential oil of Prunus humilis leaves.

[0041] Example 2

[0042] Gas chromatography - mass spectrometry (GC - MS) analysis

[0043] Before GC - MS analysis, take 50 μL of the essential oil of Prunus humilis leaves prepared in Example 1 and dissolve it in 3 mL of n - hexane. Pipette 0.5 mL of the essential oil solution into a 2 - mL vial.

[0044] Gas chromatography: The temperature - rising program is as follows: Hold at 45 °C for 2 min, then rise to 240 °C at a rate of 5 °C / min and hold for 8 min. The inlet temperature is 240 °C, and the transfer line temperature is 240 °C. Use He as the carrier gas, without splitting, and the flow rate is 1.5 mL / min.

[0045] Mass spectrometry: The ion source is an EI source, and the ion source temperature is 200 °C. The scanning range is 40 - 500 amu, and the solvent delay is 1.5 min. Each volatile substance is separated by gas chromatography to form different chromatographic peaks, as Figure 1 shown.

[0046] Using the NIST mass spectrometry database, export data according to the template. Based on the principle that the positive and negative matching values (SI&RSI) of each compound are greater than 800 (the maximum value is 1000) or the possibility is greater than 20%, the data is sorted out, and the non - meeting ones are excluded. The specific results are shown in Table 1.

[0047] Table 1 Chemical components of the essential oil of Prunus humilis leaves in Example 1

[0048]

[0049]

[0050]

[0051] As can be seen from Table 1, in this embodiment, the relative abundances of the components in the essential oil sample were compared by relative peak area percentage (RA%). In addition, Table 1 also provides information on the identified components in the essential oil, their corresponding retention times, molecular formulas, and CAS numbers. The results in Table 1 show that a total of 20 compounds were successfully identified in the Cerasus humilis leaf essential oil prepared by the present invention. By comparing and analyzing different components of the essential oil sample through RA%, it is shown that the relative abundance of benzaldehyde is relatively large, accounting for 60.32%. It is one of the most important molecules in the perfume industry, and its natural smell can be used as a flavor enhancer. At the same time, it has antifungal and antioxidant activities and has been found to be the main volatile component of Photinia serrulata, the leaf essential oil of Prunus persica (L.) Batsch, cherries, and other natural fruit flavors. In addition, methylcyclopentane (13.14%), heptane (8.99%), 2-methylhexane (5.64%), and 3-methylhexane (4.39%) in the essential oil sample are four alkanes with relatively high RA%.

[0052] Example 3

[0053] Antibacterial experiment of the Cerasus humilis leaf essential oil prepared in Example 1

[0054] Selection of bacterial strains

[0055] Gram-negative bacteria were selected: Escherichia coli (CICC 10899); Gram-positive bacteria were selected: Staphylococcus aureus (CCTCC AB 91093), Bacillus subtilis (CCTCC AB 90008).

[0056] Evaluation of the antibacterial activity of the Cerasus humilis leaf essential oil by the agar disc diffusion method

[0057] The Escherichia coli, Staphylococcus aureus, and Bacillus subtilis strains cryopreserved in a -80 °C low-temperature storage box were taken out and restored to room temperature. Under sterile conditions, an appropriate amount of the bacterial strains was taken with an inoculation loop and inoculated onto an LB solid medium (preparation method for 1 L: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 15 g of agar, 1000 mL of distilled water) by the streak plate method. Escherichia coli, Staphylococcus aureus, and Bacillus subtilis were all cultured in an inverted position in a 37 °C incubator for 12 h.

[0058] Transfer the well - activated plate to pick a single colony into the corresponding LB liquid medium (preparation method for 1 L: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 1000 mL of distilled water) under a sterile environment. The single colonies of Escherichia coli, Staphylococcus aureus, and Bacillus subtilis are cultured in a constant - temperature shaker at 37 °C for 12 h at a rotation speed of 200 rpm / min. When the cultured bacterial suspension is under the OD600 condition, the concentration of the bacterial suspension is 1x10 7 CFU / mL, and it is reserved for use.

[0059] Take 200 μL of the bacterial suspension (1×10 7 CFU / mL) and spread it on the culture dish with the pre - prepared solid LB medium. The Cerasus humilis leaf essential oil prepared in Example 1 is dissolved in acetone at four concentration gradients of 500 μL / mL, 200 μL / mL, 100 μL / mL, and 50 μL / mL respectively. Place a circular filter paper with a diameter of 8 mm (containing 10 μL of essential oil) on the surface of the culture dish. Place ampicillin with concentrations of 500 μL / mL, 200 μL / mL, 100 μL / mL, and 50 μL / mL on the surface of the culture dish as the positive control group. Use the pure acetone solution without essential oil as the negative control. After incubating at 37 °C for 24 hours, use a vernier caliper to measure the diameter of the inhibition zone. The results are shown in Figure 2 and Figure 3 .

[0060] As can be seen from Figure 2 and Figure 3 , the antibacterial effects of Cerasus humilis leaf essential oil and ampicillin are found in Escherichia coli, Bacillus subtilis, and Staphylococcus aureus, and the concentration is positively correlated with the antibacterial activity. In addition, compared with Bacillus subtilis and Staphylococcus aureus, Cerasus humilis leaf essential oil has better antibacterial activity against Escherichia coli, and the inhibition zone of pure Cerasus humilis essential oil is larger. However, the positive control (ampicillin) has better antibacterial activity against Staphylococcus aureus than the essential oil.

[0061] To further study the antibacterial effect of Cerasus humilis leaf essential oil, the antibacterial evaluation of ampicillin and the Cerasus humilis leaf essential oil prepared in Example 1 was carried out by the diameter of the inhibition zone. The results are shown in Table 2 and Figure 4 , 5 . Group the inhibition effects according to the size of their inhibition zones, including strong (20 mm), medium (12 - 20 mm), and non - inhibition (<12 mm). In Table 2, 90 represents the diameter size of the culture dish.

[0062] Table 2 Inhibition zones (mm) of Cerasus humilis leaf essential oil on bacterial growth

[0063]

[0064] Note: Values in the table with letters superscripted in the upper right corner represent significant differences.

[0065] Figure 4 , 5 The results in Table 2 and others showed that when using the essential oil concentration of pure essential oil, the three bacteria were extremely sensitive to the essential oil of Cerasus humilis leaves. In addition, when the essential oil concentration was 500 μL / mL and 200 μL / mL, the essential oil of Cerasus humilis leaves had a moderate inhibitory effect on the three bacteria; when the essential oil concentration was 100 μL / mL, it showed a moderate inhibitory effect on Bacillus subtilis, while no inhibitory effect was observed on the other two bacteria; when the essential oil concentration was diluted to 50 μL / mL, it had no inhibitory effect on the three bacteria. Based on the inhibition zones at the same concentration, the antibacterial activity of the essential oil of Cerasus humilis leaves against Escherichia coli and Bacillus subtilis was stronger than that of ampicillin. However, the inhibitory effect of different concentrations of essential oil on Staphylococcus aureus was not significant, while different concentrations of ampicillin had a strong antibacterial effect on Staphylococcus aureus. It can be speculated that the essential oil of Cerasus humilis leaves can resist bacteria that cannot be well inhibited by ampicillin, but the inhibition of Staphylococcus aureus is not as strong as that of ampicillin. Many studies have shown that the antibacterial activity of essential oils is closely related to their chemical compositions. This study showed that the essential oil of Cerasus humilis leaves was composed of compounds such as benzaldehyde, methylcyclopentane, heptane, 2-methylhexane, and 3-methylhexane, which may be the main reasons for the antibacterial activity of this essential oil. Plant essential oils contain various antibacterial components, which may lead to a decrease in the sensitivity of microorganisms to drug resistance.

[0066] Determination of MIC and MBC of the essential oil of Cerasus humilis leaves by broth dilution method

[0067] Add 100 μL of broth medium prepared in a 96-well plate, and then add 100 μL of the essential oil acetone solution with a concentration of 500 μL / mL to the first well in each row. Then, pipette 100 μL of the essential oil acetone solution from the first well into the second well, and pipette 100 μL of the solution in the second well into the third well, and so on for serial dilution. After the second-to-last well is diluted, discard 100 μL of the liquid, and do not add essential oil to the last well as a blank control. Add the adjusted solution (20 μL of bacterial suspension at 1×10 7 CFU / mL) to each well. After covering and incubating at 37 °C for 18 - 20 hours, the MIC value was determined as the lowest concentration of the essential oil that completely inhibited the growth of the microorganism. Take 5 μL of the culture solution from each well without turbidity change and culture it on the broth solid medium on a coated plate for 24 hours. The minimum sample concentration without any colony growth on the plate was defined as the MBC of the sample against the bacteria. Each experiment was repeated three times, with acetone at a concentration of 500 μL / mL as the negative control and ampicillin at a concentration of 500 μL / mL as the positive control.

[0068] Based on the above broth dilution method, the values of MIC and MBC were determined through the analysis of the above three consecutive experiments. The observed MIC and MBC values vary with the types of all bacterial strains. The results of the changes in Prunus humilis Bunge leaf essential oil are shown in Table 3.

[0069] Table 3 Minimum Inhibitory Concentration (MIC) (μL / mL) and Minimum Bactericidal Concentration (MBC) (μL / mL) of Prunus humilis Bunge leaf essential oil and ampicillin against bacterial growth

[0070]

[0071] Among them, ">" indicates that the highest concentration of this experiment has been exceeded.

[0072] As can be seen from Table 3, the Prunus humilis Bunge leaf essential oil prepared in Example 1 showed strong antibacterial activity against Escherichia coli, Bacillus subtilis, and Staphylococcus aureus. The MIC values were 15.63 μL / mL, 62.50 μL / mL, 31.25 μL / mL, and 62.50 μL / mL respectively. However, the positive control ampicillin showed strong antibacterial activity against Staphylococcus aureus, with an MIC of 15.63 μL / mL and an MBC of 250.00 μL / mL; it showed weak antibacterial activity against Escherichia coli and Bacillus subtilis, with MIC values of 125.00 μL / mL, 31.25 μL / mL respectively, and MBC > 500.00 μL / mL. These results are consistent with those obtained by the above agar disc diffusion method.

[0073] Example 4

[0074] Antioxidant experiment of Prunus humilis Bunge leaf essential oil prepared in Example 1

[0075] DPPH free radical scavenging activity:

[0076] The DPPH free radical is a stable organic free radical. The scavenging ability of essential oil samples against DPPH free radicals can effectively evaluate the intensity of their antioxidant ability. The specific measurement method was slightly modified according to the literature (Mollaei, S., Sedighi, F., Habibi, B., Hazrati, S., Asgharian, P., 2019. Extraction of essential oils of Ferulago angulata with microwave-assisted hydrodistillation. Ind. Crop. Prod. 137, 43-51.). Essential oils (anhydrous ethanol as the solvent) and DPPH solution (0.2 mM) with concentrations of 50, 100, 200, 500 μL / mL were taken in a volume ratio of 1:1 and mixed to form a reaction mixture. Then the reaction mixture was left to stand in the dark for 30 minutes. A mixture of essential oil and anhydrous ethanol with concentrations of 50, 100, 200, 500 μL / mL in a 1:1 ratio was used as a positive control, and a mixture of DPPH solution and anhydrous ethanol solution in a 1:1 ratio was used as a blank control. The absorbance of the reaction mixture was measured at 517 nm. Each treatment was repeated 3 times. The inhibition percentage of DPPH free radicals was calculated as inhibition (%) = [1 - (A1 - A2) / A0] * 100%, where A1 represents the absorbance of the reaction mixture, A2 represents the absorbance of the positive control, and A0 represents the absorbance of the blank control. The test results are shown in Table 4.

[0077] Table 4 Scavenging activity of essential oil extracts from Cerasus humilis leaves against DPPH free radicals

[0078]

[0079]

[0080] As can be seen from Table 4, the Cerasus humilis essential oil prepared in Example 1 has a strong ability to scavenge DPPH free radicals, with a scavenging rate of over 90%. When the essential oil concentration is 200 μL / mL, the highest scavenging rate is 99.19%. Compared with other highly antioxidant plants (Atractylodes macrocephala; Eucalyptus globulus), the Cerasus humilis leaf essential oil provided by the present invention shows a very high ability to scavenge DPPH free radicals. For example, in the literature

Feng, H., Wei, W., Meichan, W., Fu, X., 2020. Antioxidant and antibacterial activities of essential oil from Atractylodes lancea rhizomes. Industrial Crops & Products (C)

[0081] ABTS free radical scavenging activity:

[0082] The ABTS stock solution was prepared by mixing potassium persulfate (2.6 mM) with ABTS solution (7.4 mM) in the dark for 16 h. The ABTS solution was diluted with ethanol to obtain an accurate absorbance at 734 nm. Different concentrations of essential oils (pure essential oil, 500 μL / mL, 200 μL / mL, 100 μL / mL, and 50 μL / mL) and ABTS solution (0.1 mM) were taken at a volume ratio of 1:10 and mixed as the reaction mixture. Then the reaction mixture was allowed to stand in the dark for 30 min. Essential oils at concentrations of 500 μL / mL, 200 μL / mL, 100 μL / mL, and 50 μL / mL were mixed with absolute ethanol at a ratio of 1:10 as the positive control, and ABTS solution and absolute ethanol were mixed at a ratio of 1:10 as the blank control. The absorbance of the reaction mixture was measured at 734 nm. Each treatment was repeated 3 times. The inhibition percentage of ABTS radicals was calculated as inhibition (%) = [1 - (A1 - A2) / A0] * 100%, where A1 represents the absorbance of the test sample, A2 represents the absorbance of the positive control, and A0 represents the absorbance of the blank control. The test results are shown in Table 5.

[0083] Table 5 Scavenging activity of Cerasus humilis leaf essential oil extract on ABTS radicals

[0084]

[0085]

[0086] As can be seen from Table 5, the Cerasus humilis essential oil prepared in Example 1 has a strong ability to scavenge ABTS radicals, and the scavenging rate is above 95%. When the essential oil concentration is 100 μL / mL, the highest scavenging rate is 98.91%.

[0087] Ferric reducing antioxidant power (FRAP):

[0088] As a rapid, simple, reproducible, and inexpensive detection method, FRAP mainly reduces Fe 3+ to Fe 2+To achieve the characterization of antioxidant capacity. Although simple, this method is generally regarded as a measure of total antioxidant capacity. In the dark, 100 μL of essential oil samples with different concentrations (pure essential oil, 500 μL / mL, 200 μL / mL, 100 μL / mL, and 50 μL / mL) were reacted in a 37 °C water bath for 20 minutes. Then, 1.8 mL of FRAP solution and 3.1 mL of distilled water were added. The FRAP solution consisted of 25 mL of acetate buffer (300 mmol / L, pH 3.6), 2.5 mL of TPTZ solution (10 mmol / L, with a solvent of 40 mmol / L HCl solution), and 2.5 mL of ferric chloride solution (20 mmol / L). After the reaction was completed, the ultraviolet absorbance of the essential oil samples and the positive control at 593 nm was measured. Each treatment had 3 replicates. 0.2, 0.4, 0.8, and 1.6 mmol / L of FeSO4 were added respectively, along with 0.1 mL, 1.8 mL of FRAP solution and 3.1 mL of distilled water. After a 30-minute water bath at 37 °C, the absorbance was measured at 593 nm, and a standard curve (curve equation: y = 0.3589x + 0.0066) was plotted as shown in Table 6. The concentration of FeSO4 was calculated based on the standard curve and the absorbance value. The total antioxidant capacity of the sample was expressed as the FRAP value, where 1 FRAP = 1 mmol / L FeSO4, that is, the antioxidant capacity of the sample was equivalent to the concentration of FeSO4. The results are shown in Table 7.

[0089] Table 6 Absorbance and FeSO4 Concentration

[0090] <![CDATA[FeSO4 concentration]]> Absorbance 0 0 0.2 0.07 0.4 0.1585 0.8 0.3089 1.6 0.5722

[0091] Table 7 Ferric Ion Reducing Antioxidant Power of Cerasus humilis Leaf Essential Oil Extract

[0092]

[0093]

[0094] As can be seen from Table 7, the ferric ion reducing antioxidant power of the Cerasus humilis essential oil prepared in Example 1 was greater than 3.5 mmol / L FeSO4. When the essential oil concentration was 100 μL / mL, the highest antioxidant capacity was 3.86 mmol / L FeSO4.

[0095] From the above examples, it can be seen that the Cerasus humilis leaf essential oil provided by the present invention not only contains various beneficial volatile components such as benzaldehyde, methylcyclopentane, and n-heptane, has a significant antibacterial effect on Escherichia coli, Staphylococcus aureus, and Bacillus subtilis, but also has strong antioxidant capacity, mainly manifested as the scavenging activity of free radicals such as DPPH and ABTS. The highest antioxidant capacity detected by the ferric ion reducing antioxidant power (FRAP) can reach 3.86 mmol / L FeSO4.

[0096] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A preparation method of Cerasus humilis leaf essential oil, characterized in that, It includes the following steps: Mix the Cerasus humilis leaves with water for the first distillation to obtain the pure dew of Cerasus humilis leaves; Mix the pure dew of Cerasus humilis leaves with the Cerasus humilis leaves for the second distillation to obtain the essential oil of Cerasus humilis leaves.

2. The preparation method according to claim 1, wherein During the first distillation, the mass-volume ratio of Cerasus humilis leaves to water is 1:5 - 2:5 g / mL; during the second distillation, the mass-volume ratio of Cerasus humilis leaves to the pure dew is 1:3 - 1:5 g / mL.

3. The preparation method according to claim 1 or 2, characterized in that, The temperature of the first distillation is 90°C - 100°C, and the time is 60 min; the temperature of the second distillation is 100°C - 110°C, and the time is 120 min.

4. The essential oil of Cerasus humilis leaves prepared by the preparation method according to any one of claims 1 to 3, characterized in that, By mass percentage, the essential oil of Cerasus humilis leaves includes 58.67% - 61.18% benzaldehyde, 12.48% - 13.99% methylcyclopentane, 8.74% - 9.37% n-heptane, 5.54% - 5.78% 2-methylhexane, 4.50% - 4.34% 3-methylhexane and the balance of other substances.

5. Application of the essential oil of Cerasus humilis leaves prepared by the preparation method according to any one of claims 1 - 3 in the preparation of antibacterial products.

6. The application according to claim 5, wherein, The bacteria include one or more of Escherichia coli, Staphylococcus aureus and Bacillus subtilis.

7. The application according to claim 5 or 6, characterized in that, The product includes a repellent and / or a bactericide.

8. Application of the essential oil of Cerasus humilis leaves prepared by the preparation method according to any one of claims 1 - 3 in the preparation of antioxidant products.

9. The application according to claim 8, wherein The antioxidant includes the scavenging of free radicals and / or the reduction of iron ions; the free radicals include DPPH and / or ABTS.

10. The application according to claim 8 or 9, characterized in that, The product includes an antioxidant.

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