Mulberry leaf volatile oil and preparation method and application thereof

Abstract

The application discloses mulberry leaf volatile oil and a preparation method and application thereof, and relates to the technical field of plant extraction. The preparation method of the mulberry leaf volatile oil comprises the following steps: (1) crushing mulberry leaves to obtain mulberry leaf powder; (2) mixing the mulberry leaf powder with deionized water, and then using a microwave-assisted hydro-distillation (MAHD) method to obtain a distillate; and (3) using an organic solvent to extract the distillate to obtain the mulberry leaf volatile oil. The mulberry leaf volatile oil is extracted from mulberry leaves by using the MAHD method, the extraction process is optimized through single-factor experiments and a response surface design, and the optimal extraction process is obtained. It is verified that the mulberry leaf volatile oil prepared by the application has antibacterial activity on bacillus subtilis, salmonella paratyphi B, pseudomonas aeruginosa, escherichia coli and staphylococcus aureus, and has a strong scavenging capacity on ABTS and DPPH free radicals.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant extraction, in particular to a mulberry leaf volatile oil and a preparation method and application thereof. BACKGROUND

[0002] Mulberry leaf is the leaf of Morus alba L. The leaf blade is wide ovate with serrate margin, and the leaf veins are densely covered with white soft hair. It is widely distributed in the middle and lower reaches of the Yangtze River, and is rich in resources. It is mainly used in the fields of animal husbandry and medicine. Mulberry leaf has the effects of dispersing wind-heat, clearing lung and moistening dryness, and clearing liver and brightening eyesight. It can be used for treating wind-heat cold, lung-heat dry cough, dizziness and headache, and red eyes and blurred vision. In addition, mulberry leaf is also used as a kind of vegetable in folk, and is also the first batch of Chinese medicine materials with the same origin of food and medicine. At present, mulberry leaf is applied in animal production to improve animal production performance and improve the quality of livestock and poultry products. Since mulberry leaf contains rich flavonoids, animals can be fed with mulberry leaf to improve the digestion and utilization of fiber, thereby increasing the milk yield of ruminants. Mulberry leaf is a traditional Chinese medicine material with good biological activity, and its use in medicine was first recorded in Han Dynasty in Shennong's Herbal Classic. There are many researches on the research and application of mulberry leaf, but there are few researches on mulberry leaf volatile oil (MLEO). Therefore, there is great development potential for mulberry leaf volatile oil. SUMMARY

[0003] The present application aims to provide a mulberry leaf volatile oil and a preparation method and application thereof to solve the problems in the prior art. The mulberry leaf volatile oil is extracted by a microwave-assisted steam distillation method, and has antibacterial and antioxidant activities.

[0004] To achieve the above object, the present application provides the following solutions.

[0005] The present application provides a preparation method of a mulberry leaf volatile oil, which comprises the following steps:

[0006] (1) grinding mulberry leaves to obtain mulberry leaf powder;

[0007] (2) mixing the mulberry leaf powder with deionized water, and then obtaining a distillate by a microwave-assisted steam distillation method;

[0008] (3) extracting the distillate by an organic solvent to obtain the mulberry leaf volatile oil.

[0009] Further, in step (2), the mass-volume ratio of the mulberry leaf powder to the deionized water is 1g:8mL.

[0010] Further, in step (2), the microwave extraction time of the microwave-assisted steam distillation method is 55min.

[0011] Further, in step (2), the power of the microwave is 500 W.

[0012] Further, in step (3), the organic solvent is dichloromethane.

[0013] The application further provides a mulberry leaf volatile oil prepared according to the preparation method.

[0014] The application further provides application of the mulberry leaf volatile oil in preparation of an antibacterial product.

[0015] Further, the strains inhibited by the antibacterial product are Staphylococcus aureus, Bacillus subtilis, Salmonella paratyphi B, Escherichia coli and / or Pseudomonas aeruginosa.

[0016] The application further provides application of the mulberry leaf volatile oil in preparation of an antioxidant product.

[0017] Further, the antioxidant is ABTS and / or DPPH free radical scavenging.

[0018] The application discloses the following technical effects:

[0019] The application extracts the volatile oil of mulberry leaves by a microwave-assisted hydro-distillation (MAHD) method, takes the extraction rate of the volatile oil as an index, optimizes the extraction process through single-factor experiments and a response surface design, and the optimal process combination is: extraction time (55 min), power (500 W) and liquid-solid ratio (8 mL / g). Under the process condition, the extraction rate of the volatile oil of mulberry leaves is 0.079±0.009%, which is close to the predicted value 0.079% (obtained according to the response surface optimization experiment by using DE8 software). The chemical composition of the volatile oil of mulberry leaves is identified by a gas chromatography-mass spectrometry (GC-MS), and 74 kinds of compounds are identified, the main components are 3-methyl-1-butanol (3.26 wt%), furanmethanol (3.15 wt%), 2-furanmethanol (7.08 wt%), 3-decyl-5-ketone (3.2 wt%), α,5,6,7,7-tetrahydro-α,4,4,7-trimethyl-2(4H)-benzoxazolone (3.23 wt%), L-(+)-ascorbic acid-2,6-hexacosanoate (8.45 wt%), plant alcohol (5.89 wt%) and (E,E,E)-7,10,13-hexadecatriene (4.87 wt%). The MLEO is subjected to an in-vitro antibacterial experiment by using a filter paper diffusion method, and the antibacterial activity of the MLEO on Bacillus subtilis, Salmonella paratyphi B, Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus is investigated. The experimental results show that the MLEO has antibacterial effects on the five kinds of bacteria and has broad-spectrum antibacterial activity. From the antibacterial effect of the MLEO, the antibacterial capacity from large to small is Staphylococcus aureus > Bacillus subtilis > Salmonella paratyphi B > Escherichia coli > Pseudomonas aeruginosa. The in-vitro antioxidant activity of the MLEO is detected, and the results show that the MLEO has a strong scavenging capacity on ABTS and DPPH free radicals, and the scavenging activity on DPPH free radicals is the strongest. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0021] Figure 1 The device diagram for extracting MLEO by using a microwave-assisted hydro-distillation method;

[0022] Figure 2 The influence of different single factors on the extraction rate of MLEO; wherein, A: extraction time; B: microwave power; C: liquid-solid ratio;

[0023] Figure 3Three-dimensional graphs of the effects of the interactions between extraction time and microwave power (A), liquid-to-material ratio and microwave power (C), and extraction time and liquid-to-material ratio (E) on the extraction yield of MLEO; contour plots of the effects of the interactions between extraction time and microwave power (B), liquid-to-material ratio and microwave power (D), and extraction time and liquid-to-material ratio (F) on the extraction yield of MLEO;

[0024] Figure 4 The antibacterial activity of five kinds of bacteria, A is Bacillus subtilis, a is its negative control; B is Salmonella paratyphi B, b is its negative control; C is Pseudomonas aeruginosa, c is its negative control; D is Escherichia coli, d is its negative control; E is Staphylococcus aureus, e is its negative control; unit: mm;

[0025] Figure 5 The antibacterial activity of 400 μL / mL MLEO on Bacillus subtilis (A), Salmonella paratyphi B (B), Pseudomonas aeruginosa (C), Escherichia coli (D), and Staphylococcus aureus (E); unit: mm;

[0026] Figure 6 The antibacterial activity of 500 μL / mL MLEO on Bacillus subtilis (A), Salmonella paratyphi B (B), Pseudomonas aeruginosa (C), Escherichia coli (D), and Staphylococcus aureus (E); unit: mm;

[0027] Figure 7 The antibacterial activity of 600 μL / mL MLEO on Bacillus subtilis (A), Salmonella paratyphi B (B), Pseudomonas aeruginosa (C), Escherichia coli (D), and Staphylococcus aureus (E); unit: mm;

[0028] Figure 8 The antibacterial activity of 700 μL / mL MLEO on Bacillus subtilis (A), Salmonella paratyphi B (B), Pseudomonas aeruginosa (C), Escherichia coli (D), and Staphylococcus aureus (E); unit: mm;

[0029] Figure 9 The antibacterial activity of 800 μL / mL MLEO on Bacillus subtilis (A), Salmonella paratyphi B (B), Pseudomonas aeruginosa (C), Escherichia coli (D), and Staphylococcus aureus (E); unit: mm;

[0030] Figure 10 The scavenging activity of MLEO on ABTS free radicals;

[0031] Figure 11 The scavenging effect of MLEO on DPPH free radicals. DETAILED DESCRIPTION

[0032] The following detailed description of various example embodiments of the application is not to be considered limiting of the scope or spirit of the application, but rather as a description of certain aspects, features and embodiments of the application.

[0033] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Additionally, for the purposes of the present application, the term "about" means plus or minus 10% of the specified value. Furthermore, the use of the term "or" in the detailed description or in the claims is used to mean "and / or" unless explicitly indicated to refer to an alternative exclusive conjunction. In addition, the use of the term "example" is intended to represent one example, but not an exhaustive list of possibilities. As it is used herein for purposes of the present disclosure and the appended claims, the conjunction "and" is not intended to be limiting of a particular recited feature, method or process, but rather intended to mean "and / or" unless explicitly indicated to the contrary.

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All patents, patent applications, publications, and descriptions mentioned herein are incorporated by reference to the extent allowed by law for the disclosure and description thereof. In the case of conflict between the specifications of any incorporated reference and that of the present specification, the present specification shall control.

[0035] Various modifications and changes can be made to the specific embodiments of the application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples presented herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application.

[0036] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having" or variants thereof are open-ended, and specifically do not exclude additional, unrecited elements or method steps.

[0037] Example 1

[0038] I. Experimental Materials

[0039] Fresh mulberry leaves were collected from Dongba Town, Yun'an County, Yunfu City. The main components of the leaves are flavonoids, polyphenols, alkaloids and pigments, and the content of volatile oil is low.

[0040] II. Methods

[0041] 2.1 Extraction of mulberry leaf volatile oil

[0042] The MLEO was extracted by microwave-assisted hydro-distillation (MAHD) method using a combination of microwave synthesis extractor and traditional distillation apparatus. First, the fresh mulberry leaves were dried, then according to the People's Republic of China Pharmacopoeia 2005 edition, the national standard R40 / 3 series was selected, the mulberry leaves were crushed by a plant crusher, then sieved by an 80 mesh pharmaceutical sieve to make mulberry powder, which was sealed and stored in a cool place. 200 g of mulberry powder was mixed with 1600 mL of deionized water and heated by microwave. The obtained mulberry volatile oil was collected in a brown bottle and extracted with an equal amount of dichloromethane three times. The dichloromethane was evaporated by a rotary evaporator. The mulberry volatile oil was collected and the volume was detected, and stored at 4°C for standby.

[0043] The MLEO device extracted by the MAHD method is as shown in Figure 1 and the MLEO extraction rate calculation method is as follows:

[0044]

[0045] N (m / v) is the MLEO extraction rate, V MLEO is the volume of MLEO, V ML is the volume of mulberry powder.

[0046] 2.2 Optimization of mulberry volatile oil extraction process

[0047] 2.2.1 Single factor experiment

[0048] 2.2.1.1 Extraction time

[0049] 500 mL of mulberry powder was placed in a round-bottom flask, and the extraction time was set to 45, 55, 65, 75 and 85 min, respectively, and other extraction parameters: liquid-solid ratio was 8 mL / g, microwave power was 500 W, the effect of different extraction times on MLEO extraction rate was determined.

[0050] 2.2.1.2 Microwave power

[0051] 500 mL of mulberry powder was placed in a round-bottom flask, and the microwave power was set to 400, 450, 500, 550, 600 and 650 W, respectively, and other extraction parameters: liquid-solid ratio was 8 mL / g, extraction time was 55 min, the effect of different microwave power on MLEO extraction rate was determined.

[0052] 2.2.1.3 Liquid-solid ratio

[0053] 500 mL of mulberry powder was placed in a round-bottom flask, and the liquid-solid ratio was set to 6, 7, 8, 9, 10 and 11 mL / g, respectively, and other extraction parameters: extraction time was 55 min, microwave power was 500 W, the effect of different liquid-solid ratio on MLEO extraction rate was determined.

[0054] 2.2.2 Response surface design

[0055] Based on the single factor experiment, the response surface design was carried out by using Design-Expert 8.0.6 software. According to the Box-Behnken central composite design principle, the MLEO extraction rate was selected as the response value, and the liquid-solid ratio, extraction time and microwave power were selected for three-factor and three-level response surface design, as shown in Table 1. The experimental results were fitted to a second-order polynomial model using the equation as follows:

[0056]

[0057] where Y is the predicted MLEO extraction rate, β0is the constant coefficient, β i , β ii and β ij are the first-order coefficients, second-order coefficients and interaction effect coefficients of X i , respectively; and ε is the experimental error.

[0058] Table 1 Response surface factor and level design

[0059]

[0060] 2.3 GC-MS analysis of MLEO

[0061] The chemical components in MLEO were identified and analyzed by GC-MS. The chromatographic conditions were as follows: a flexible quartz capillary chromatographic column DB-5MS (0.25 nm x 30 m, 0.25 μm), programmed temperature: initial temperature of 60℃, holding for 1 min, programmed temperature to 220℃ at 2.5℃ / min, injection port temperature of 240℃, holding for 15 min, interface temperature of 250℃, pre-column pressure of 52.7 kPa. Carrier gas: high-purity helium (purity of 99.99%), injection amount of 0.1 μL, split ratio of 20:1, carrier gas flow rate of 1 ml / min. Mass spectrometry conditions: ion source EI, ion source temperature of 200℃. Quadrupole rod temperature of 150℃. Electron energy of 70 eV, scan range of 35-800 amu.

[0062] III. Results

[0063] 3.1 Results of single factor experiment

[0064] The effects of three parameters (extraction time, microwave power, liquid-solid ratio) on the MLEO extraction rate are shown in Figure 2 , which shows that the MLEO extraction rate is closely related to these three factors.

[0065] 3.1.1 Effect of extraction time on MLEO extraction rate

[0066] The results of the effect of extraction time on the MLEO extraction rate are shown inFigure 2 As shown in Figure A, with the microwave power set to 500W and the liquid-to-solid ratio set to 8mL / g, the effect of extraction time between 45min and 85min on the MLEO extraction rate was investigated. When the extraction time was between 45min and 65min, the MLEO extraction rate continuously increased; however, when the extraction time exceeded 65min, the MLEO extraction rate continuously decreased. This indicates that excessively long extraction times lead to the loss of volatile substances in the product; therefore, 55min is the optimal extraction time for MLEO.

[0067] 3.1.2 Effect of microwave power on MLEO extraction rate

[0068] The effect of microwave power on MLEO extraction rate is as follows: Figure 2 As shown in Figure B, the effect of microwave power between 400W and 650W on the MLEO extraction rate was studied when the extraction time was set to 55 min and the liquid-to-solid ratio was set to 8 mL / g. When the microwave power increased from 400W to 550W, the MLEO extraction rate gradually increased, reaching its maximum at 550W. However, when the microwave power increased from 550W to 650W, the MLEO extraction rate decreased. Excessively high extraction power can reduce the MLEO extraction rate, possibly due to the loss of volatile compounds and degradation of essential oils caused by excessive microwave power. Therefore, the microwave power needs to be controlled within an appropriate range. Thus, 500W is the optimal microwave power.

[0069] 3.1.3 Effect of liquid-to-solid ratio on MLEO extraction rate

[0070] The effect of liquid-to-solid ratio on MLEO extraction rate is as follows: Figure 2 As shown in Figure C, the extraction time was set to 55 min, the microwave power to 500 W, and the liquid-to-solid ratios were set to 6, 7, 8, 9, 10, and 11 mL / g to study the effect of different liquid-to-solid ratios on the MLEO extraction rate. As the liquid-to-solid ratio increased from 6 mL / g to 11 mL / g, the MLEO extraction rate continuously increased. Therefore, 8 mL / g is the optimal liquid-to-solid ratio.

[0071] 3.2 Optimization of MLEO Extraction Process Using Response Surface Methodology

[0072] To obtain the optimal MLEO extraction process, a three-factor, three-level Box-Behnken design was conducted based on single-factor experiments to optimize the interaction and effect of three extraction parameters: extraction time, microwave power, and liquid-to-solid ratio. The design and results are shown in Table 2. Using the MLEO extraction rate as the response value, a multiple regression was performed on the data in Table 2 using Design-Expert 8.0.6 software to obtain the quadratic polynomial regression equation for the response value Y:

[0073] Y(%) = 0.079 + 3.750 x 10 -4 X1+ 1.875 x 10 -3 X2- 2.750 x 10 -3 X3+ 1.750 x 10 -3 X1X2+ 1.500 x 10 -3 X1X3- 1.000 x 10 -3 X2X3- 0.017X1 2 - 0.020X2 2 - 9.175 x 10 -3 X3 2

[0074] where X1, X2 and X3 represent the coded factors for extraction time, microwave power and liquid-to-material ratio, respectively. The regression equation was validated by analysis of variance (ANOVA) and the results are shown in Table 3. Table 3 shows a low P-value of 0.0005 (significant) and a high F-value of 18.13, which indicates that the resulting quadratic polynomial equation is ideal and the model is highly significant, indicating that the experimental method is reliable. 2 (0.9589) indicates that the predicted values agree well with the experimental values, and the adjusted coefficient of determination (R 2 adj) is 0.9060, which is relatively close to 1, indicating that the quadratic polynomial equation fits the experimental values very well. In addition, R 2 and R 2 adj demonstrate that the extraction yield of MLEO is highly correlated with the three variables of extraction time, microwave power and liquid-to-material ratio. The lack of fit F-value is 0.64, indicating that the lack of fit is not significant relative to the pure error, indicating that the regression model agrees well with the measured values. The Adeq Precision is 11.005, which is significantly higher than 4, indicating that the regression model has sufficient signal-to-noise ratio. These experimental results demonstrate that the experimental design of the regression model is effective and feasible. Finally, according to Table 3, it can be seen that the P-values of the quadratic coefficients X1 2 , X2 2 and X3 2 are significant, but the other values are not significant. Therefore, the factors that affect the extraction yield of MLEO in descending order are: liquid-to-material ratio (X3) > microwave power (X2) > extraction time (X1).

[0075] Table 2 Box-Behnken design experimental scheme and results

[0076]

[0077] Table 3 Simplified analysis of variance of the regression model

[0078]

[0079]

[0080] The impact of the interaction between the two variables on MLEO extraction was verified by plotting three-dimensional (3D) response curves and contour plots. Figure 3 As shown, A, C, and E are response curves, and B, D, and F are contour lines. According to... Figure 3 It can be seen that by increasing the extraction time or microwave power, the extraction efficiency of MLEO initially increases gradually, and then gradually decreases. Figure 3 (A and B); By increasing microwave power and liquid-to-solid ratio, the extraction rate of MLEO initially increased gradually, then gradually decreased. Figure 3 (C and D); By increasing the extraction time and liquid-to-solid ratio, the extraction rate of MLEO also gradually increased first, and then gradually decreased. Figure 3 (E and F in the middle). Furthermore, the shape of the contour plot demonstrates the significance of the interaction between the two extracted parameters. Elliptical or saddle-shaped contour plots indicate significance, while circular contour plots indicate insignificance. Figure 3 China B and Figure 3 The results showed that the interaction between extraction time, microwave power, extraction time and liquid-to-solid ratio was significant (P < 0.05).

[0081] Based on the above experimental results, the optimal extraction conditions for MLEO were: extraction time of 55.07 min, microwave power of 502.52 W, and liquid-to-solid ratio of 7.85 mL / g. Finally, the optimal extraction process for MLEO was adjusted according to actual conditions: extraction time of 55 min, microwave power of 500 W, and liquid-to-solid ratio of 8 mL / g. Under these conditions, the extraction rate of MLEO was 0.079 ± 0.009%, close to the predicted value of 0.079%, and the difference from the predicted value was not significant. These experimental results demonstrate that RSM is a highly effective mathematical statistical tool that can assess the interactions between multiple parameters in a response variable and provide a high-precision regression equation.

[0082] 3.3 GC-MS Analysis of MLEO

[0083] The MLEO obtained from the optimal extraction conditions (extraction time of 55 min, microwave power of 500 W, and liquid-to-material ratio of 8 mL / g) in 3.2 was analyzed and identified in GC-MS by matching the retention index, retention time, and mass spectrum. Finally, 74 chemical components were identified, and the relative content of each chemical component in MLEO was calculated by area normalization. The results are shown in Table 4, and the main components include alcohols, ketones, hydrocarbons, heterocyclic compounds, phenols, aldehydes, and esters. According to the data, the main components in MLEO are 3-methyl-1-butanol (3.26 wt%), ethyl lactate (2.35 wt%), furfuryl alcohol (3.15 wt%), 2-furfuryl alcohol (7.08 wt%), 2,5-dimethylpyrazine (2.84 wt%), benzyl alcohol (2.64 wt%), 2,5-dimethyl-4-hydroxy-3-(2-hydrogen)-furanone (2.26 wt%), 1-(1-hydrogen-pyrrole-2-yl) ethanone (2.30 wt%), phenyl ethanol (2.69 wt%), 6,10-dimethyl-5,9-undecadiene-2-ketone (2.9 wt%), 4-(2,6,6-trimethyl-1-cyclohexene-1-yl)-3-buten-2-ketone (2.13 wt%), 3-decyl-5-ketone (3.2 wt%), α,5,6,7,7-tetrahydro-α,4,4,7-trimethyl-2(4H)-benzoxazolone (3.23 wt%), L-(+)-ascorbic acid-2,6-hexacosanoate (8.45 wt%), phytol (5.89 wt%), (E, E, E)-7,10,13-hexadecatriene (4.87 wt%), and the total content of these compounds accounts for 59.24 wt% of the total MLEO. The amount of each chemical component and the percentage of the relative content in MLEO are shown in Table 5.

[0084] Alcohols, ketones, and heterocyclic compounds are the main components in MLEO, with 11, 16, and 19 compounds detected, accounting for 68.94 wt% of the total content of MLEO. Phytol has a certain inhibitory effect on Plasmodium berghei and can reduce some pathological changes caused by Plasmodium berghei. Although the polyphenol content in mulberry leaves is relatively small, it is one of the important components of the antioxidant activity of mulberry leaves.

[0085] Table 4 Chemical components of MLEO extracted by microwave-assisted steam distillation

[0086]

[0087]

[0088]

[0089]

[0090] Table 5: Types and percentage of contents of compounds in MLEO

[0091]

[0092] In summary, the following conclusions were drawn from the present example:

[0093] (1) MADH method was used to extract MLEO, and the extraction time (min), microwave power (W) and liquid-to-solid ratio (mL / g) were selected as the extraction parameters. Single-factor experiment and response surface three-factor three-level Box-Behnken design were used to optimize the extraction process of MLEO by MAHD method. The final results showed that the optimal extraction process of MLEO by response surface design optimization was: extraction time of 55 min, microwave power of 500 W, and liquid-to-solid ratio of 8 mL / g. Under the optimal extraction process conditions, the extraction rate of MLEO was 0.079 ± 0.009%, which was close to the predicted value of 0.079% and had no significant difference.

[0094] (2) The chemical components of MLEO extracted by MADH method were identified by GC-MS, and 74 chemical components were identified, mainly including alcohols, ketones, heterocyclic compounds, and esters, etc. The contents of hydrocarbons, phenols, and aldehydes were relatively small. 3-methyl-1-butanol (3.26%), furfuryl alcohol (3.15%), 2-furfuryl alcohol (7.08%), 3-decyl-5-ketone (3.2%), α, 5, 6, 7, 7-tetrahydro-α, 4, 4, 7-trimethyl-2(4H)-benzoxazolone (3.23%), L-(+)-ascorbic acid-2, 6-hexacosanoate (8.45%), plant alcohol (5.89%), and (E, E, E)-7, 10, 13-hexadecatriene (4.87%) were the main chemical components of MLEO.

[0095] Example 2: Antibacterial activity

[0096] I. Experimental materials

[0097] The experimental strains are shown in Table 6:

[0098] Table 6: Experimental strains

[0099]

[0100] II. Methods

[0101] 2.1 Preparation of culture medium and activation of bacterial strains

[0102] Recovery, activation and preservation of each strain requires the use of nutrient agar medium and nutrient broth medium, while antibacterial experimental research requires the use of hydrolyzed casein peptone agar medium and hydrolyzed casein peptone broth medium. The medium dry powder is heated and dissolved in a conical flask, then sterilized at 121°C for 15 min, and then used. When the solid medium is cooled to about 50°C, pour the culture dish under sterile operating environment, about 20 mL per culture dish. The medium used in the experiment needs to be prepared and used immediately.

[0103] Under sterile conditions, the five strains (Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Salmonella paratyphi B, Pseudomonas aeruginosa) stored at low temperature were naturally thawed, then the freeze-dried powder was dissolved in physiological saline, and the five strains were inoculated into the nutrient broth solid medium using the three-line method, and finally placed in a 37°C biochemical incubator for 24 hours. After 24 hours, the medium was taken out, the strain grew well, and then placed in a 4°C environment for preservation.

[0104] Under sterile conditions, use a loop to pick a single colony in nutrient broth medium, then place it in an air-powered shaker, set the temperature to 37°C, and the speed to 120 r / min, incubate for 8 to 10 hours, then place it in a 4°C environment for preservation.

[0105] During the experiment, under sterile conditions, the bacterial solution was diluted to 0.5 McFarland turbidity, equivalent to 1 x 10 8 CFU / mL, then diluted to 10 5 CFU / mL with physiological saline and used.

[0106] 2.2 Test of antibacterial activity of MLEO

[0107] The antibacterial activity of MLEO was tested, which was extracted by the optimal extraction process conditions (extraction time 55 min, microwave power 500 W, liquid-solid ratio 8 mL / g) of 3.2 in Example 1. In a sterile environment, 0.02% dimethyl sulfoxide was used as a diluent to dilute MLEO to 400, 500, 600, 700 and 800 μL / mL concentration solutions, respectively, and then 6 mm diameter round filter paper was immersed in five concentrations of MLEO for 2 hours, then the excess liquid was absorbed with filter paper and used. Add 100 μL of 10 5The bacterial solution was diluted to 1 x 105CFU / mL, then evenly spread using a sterile cotton swab, the filter paper was gently pasted on the flat plate, and levofloxacin was used as a positive control and sterile water as a negative control. The plates were incubated in a biochemical incubator at 37°C for 24 hours. After 24 hours, the diameters of the different inhibition zones were recorded. The standard for antibacterial activity was: high activity (inhibition zone > 20 mm), medium activity (inhibition zone 15-19 mm), weak to medium activity (9-15 mm), and weak activity (inhibition zone < 9 mm).

[0108] III. Results

[0109] The antibacterial bioactivity of MLEO against five common typical pathogenic bacteria, Bacillus subtilis, Salmonella paratyphi B, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus, was tested by the filter paper diffusion method, and the results are shown in Tables 6, Figure 4 and 7. Sterile water was the negative control group, and the inhibition zone was 0; levofloxacin was the positive control group, and the inhibition zone of the five bacteria was greater than 20 mm, indicating that the antibacterial activity of levofloxacin against the five bacteria was high, among which the antibacterial activity against Bacillus subtilis was the strongest, and the antibacterial activity against Staphylococcus aureus was the weakest. The antibacterial activity of MLEO at five concentrations of 400, 500, 600, 700, and 800 μL / mL against the five bacteria is shown in Table 8. The results show that as the concentration of MLEO increases, the inhibition of the five bacteria gradually increases.

[0110] When the concentration of MLEO was 400 μL / mL, the antibacterial activity against Bacillus subtilis and Staphylococcus aureus was weak to medium; the antibacterial activity against Salmonella paratyphi B was weak; and the antibacterial activity against Pseudomonas aeruginosa and Escherichia coli was weak, as shown in Table 6. Figure 5

[0111] When the concentration of MLEO was 500, 600, and 700 μL / mL, respectively, the antibacterial activity against Bacillus subtilis, Salmonella paratyphi B, and Staphylococcus aureus was weak to medium; the antibacterial activity against Pseudomonas aeruginosa and Escherichia coli was weak, as shown in Tables 7, Figure 6 , 7 and 8.

[0112] When the concentration of MLEO was 800 μL / mL, the antibacterial activity against Staphylococcus aureus was medium; the antibacterial activity against Bacillus subtilis and Salmonella paratyphi B was weak to medium; and the antibacterial activity against Pseudomonas aeruginosa and Escherichia coli was weak, as shown in Table 8. Figure 9

[0113] ​​In summary, MLEO has antibacterial activity on Bacillus subtilis, Salmonella paratyphi B, Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus, but it has certain selectivity in terms of antibacterial effect, and the antibacterial ability from large to small is Staphylococcus aureus > Bacillus subtilis > Salmonella paratyphi B > Escherichia coli > Pseudomonas aeruginosa.

[0114] Table 7 antibacterial activity test of five kinds of bacteria

[0115]

[0116]

[0117] Table 8 antibacterial effect of MLEO of five concentrations on five kinds of bacteria

[0118]

[0119] Example 3 in vitro antioxidant activity of MLEO

[0120] I. Method

[0121] 1.1 Determination of in vitro antioxidant activity of MLEO

[0122] The MLEO was extracted by the optimal extraction process conditions of 3.2 in Example 1 (extraction time was 55 min, microwave power was 500 W, and liquid-to-material ratio was 8 mL / g).

[0123] 1.1.1 ABTS free radical scavenging experiment

[0124] At room temperature, equal volumes of 7 mmol / L ABTS and 2.45 mmol / L potassium persulfate solution were mixed in the dark, and then allowed to stand at 4°C for 12 to 16 hours. The ABTS solution was diluted with methanol and prepared into ABTS working solution with an absorbance of 0.70±0.02 at 734 nm. Then MLEO was diluted with methanol to five concentrations of 100, 200, 300, 400 and 500 μL / mL, and 50 μL of MLEO sample solution and 3 mL of ABTS working solution were mixed in each of the five concentrations of solution. After 10 minutes of light protection, the absorbance was measured at 734 nm. Ascorbic acid was used as a positive control, and ascorbic acid was also diluted to five concentrations of 100, 200, 300, 400 and 500 μL / mL, and then ABTS free radical scavenging experiments were performed. The calculation formula of MLEO for ABTS free radical scavenging activity is:

[0125]

[0126] A0 is the control absorbance, A is the sample absorbance.

[0127] 1.1.2 DPPH radical scavenging experiment

[0128] The MLEO was diluted with methanol to five concentrations of 100, 200, 300, 400 and 500 μL / mL, and 3 mL of DPPH solution was mixed with MLEO sample solution at five concentrations, respectively, and the concentration was 20 mL / L. Incubate in the dark for 30 minutes, detect the absorbance at 517 nm. DPPH needs to be dissolved with methanol before use, and it is prepared and used immediately. Ascorbic acid is used as a positive control, and the calculation formula of MLEO's DPPH radical scavenging activity is:

[0129]

[0130] A0 is the control absorbance, A is the sample absorbance. i A is the sample absorbance, A j is the sample absorbance without DPPH.

[0131] II. Results

[0132] ABTS and DPPH radicals have strong oxidizing properties and can accept hydrogen radicals or electrons. ABTS and DPPH radical scavenging experiments are one of the effective means to detect the antioxidant capacity of compounds and natural products. The ABTS radical scavenging activity of MLEO is shown in Table 9 and Table 10, and the DPPH radical scavenging activity of MLEO is shown in Table 10. Figure 10 Figure 11 The results show that with the increase of the concentration of MLEO, its ABTS and DPPH radical scavenging ability gradually increases, and the DPPH radical scavenging ability is the strongest. According to the experimental results, it can be seen that MLEO has strong antioxidant capacity, and has potential application value as an antioxidant in alleviating oxidative stress and delaying aging, etc.

[0133] Table 9 ABTS radical scavenging effect of MLEO and ascorbic acid

[0134]

[0135] Table 10 DPPH radical scavenging effect of MLEO and ascorbic acid

[0136]

[0137] The above-described embodiments are only to describe the preferred modes of the present application, and not to limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.​

Claims

1. Use of a volatile oil of mulberry leaf in the preparation of an antioxidant product, characterized in that, The antioxidant is to scavenge ABTS and / or DPPH free radicals; The preparation method of the mulberry leaf volatile oil comprises the following steps: (1) crushing mulberry leaves to obtain mulberry leaf powder; (2) mixing the mulberry leaf powder with deionized water, and then using a microwave-assisted steam distillation method to obtain a distillate; (3) extracting the distillate with an organic solvent to obtain the mulberry leaf volatile oil; In step (2), the mass-to-volume ratio of the mulberry leaf powder to the deionized water is 1 g:8 mL; In step (2), the microwave extraction time of the microwave-assisted steam distillation method is 55 min; In step (2), the power of the microwave is 500 W; In step (3), the organic solvent is dichloromethane.

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