Pepper stem and leaf essential oil as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to pepper stem and leaf essential oil and a preparation method and application thereof.The preparation method comprises the steps that pepper stems and leaves are taken, washed, aired, smashed and sieved, the obtained pepper stem and leaf powder is evenly mixed with distilled water, and then sodium chloride is added for soaking; carrying out ultrasonic-assisted extraction on the soaked pepper stem and leaf powder solution, then carrying out distillation extraction, and carrying out extraction separation on the obtained oil-water mixture to obtain an essential oil layer; and volatilizing the solvent in the obtained essential oil, sealing, freezing, and removing moisture in the essential oil to obtain the pepper stem and leaf essential oil. The invention further relates to application of the pepper stem and leaf essential oil as an antioxidant and an antibacterial agent. Compared with a traditional essential oil preparation method, the optimized preparation method of the pepper stem and leaf essential oil has the advantages of being short in consumed time, high in yield, stable, controllable, easy and convenient to operate and the like, and the pepper stem and leaf essential oil can also serve as a plant source antioxidant and an antibacterial agent to be applied to the fields of food, medicine and chemical engineering.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and in particular to a capsicum stem and leaf essential oil, a preparation method thereof, and its antibacterial and antioxidant applications. Background Art

[0002] The capsicum stem and leaf are the stems and leaves of Capsicum annuum L. of the genus Capsicum in the Solanaceae family. The output of capsicum stem and leaf is huge. Except for a small part used as organic fertilizer, feed and fuel, most of them are discarded in the fields. Therefore, it is urgent to explore the effective components of capsicum stem and leaf for development and utilization. The capsicum stem and leaf have a long history of medicinal use. "Compendium of Materia Medica" records that "the smell of capsicum stem and leaf is bitter, warm, and non-toxic. Taking it for a long time can make people ageless, light and energetic. It is mainly used to treat rheumatism sores, grow hair, calm the five internal organs, keep the middle energizer, not feel hungry and prolong life." Modern research shows that the extract of capsicum stem and leaf has pharmacological effects such as sedation and hypnosis, anti-aging, anti-fatigue, antioxidant, antibacterial, hypoglycemic, hypolipidemic, liver protection, anti-tumor and improving blood circulation. Its application value has been increasingly emphasized. Among them, obtaining capsicum stem and leaf essential oil from capsicum stem and leaf is expected to become an effective way for the development and utilization of capsicum stem and leaf resources.

[0003] At present, the research on capsicum essential oil at home and abroad mainly focuses on extracting essential oil from capsicum seeds and fruits, and studying chemical components, quality evaluation, production process and antioxidant activity. However, there is no report on the extraction process and biological activity of capsicum stem and leaf essential oil. At the same time, most of the existing preparation processes of plant essential oils use anhydrous sodium sulfate for water removal, so there are disadvantages such as long time consumption, relatively cumbersome operation process and large loss of essential oil. Summary of the Invention

[0004] The purpose of the present invention is to provide a capsicum stem and leaf essential oil, a preparation method thereof and an application. The preparation method is to extract capsicum stem and leaf essential oil from capsicum stem and leaf, optimize its extraction process through single factor and response surface experiments by ultrasonic-assisted steam distillation method, and optimize the water removal process of essential oil by the freezing method to obtain an optimized combination of process conditions for preparing capsicum essential oil, and study its antibacterial and antioxidant activity applications; it overcomes the disadvantages of the traditional anhydrous sodium sulfate method for essential oil water removal, such as long time consumption, relatively cumbersome operation process and large loss of essential oil; at the same time, it fills the blank of research data on the antibacterial and antioxidant activities of capsicum stem and leaf essential oil.

[0005] To solve the above technical problems, the present invention adopts the following technical solutions:

[0006] A preparation method of capsicum stem and leaf essential oil, comprising the following steps:

[0007] (1) Wash the pepper stems and leaves, dry them in the air, and crush and sieve them to obtain pepper stem and leaf powder;

[0008] (2) Mix the pepper stem and leaf powder obtained in step (1) evenly with distilled water, add sodium chloride according to the total volume of the pepper stem and leaf powder aqueous solution, and start soaking after the sodium chloride in the solution reaches a certain concentration;

[0009] (3) Perform ultrasonic-assisted extraction on the pepper stem and leaf powder solution obtained in step (2), then transfer it to an essential oil distillation device for essential oil steam distillation extraction to obtain an oil-water mixture, and then transfer the oil-water mixture to a separatory funnel, add a certain amount of n-hexane, let it stand for extraction for 19 h, and separate to obtain the essential oil layer;

[0010] (4) Evaporate the n-hexane in the essential oil obtained in step (3), then seal it and place it in a freezer, and use the freezing water removal method to remove the water in the essential oil obtained in (3), that is, obtain the pepper stem and leaf essential oil.

[0011] And calculate its yield. The specific essential oil yield calculation formula is shown in formula (1):

[0012]

[0013] Further, in step (1), the particle size of the pepper stem and leaf powder is 40 - 60 mesh.

[0014] Further, in step (2), the mass-volume ratio of the pepper stem and leaf powder to distilled water is 1:16 - 1:24 g / mL; the sodium chloride concentration is 10 - 50 g / L, and the soaking time is 2 - 5 h.

[0015] Further, in step (3), the ultrasonic-assisted extraction conditions are: power is 200 - 600 W, temperature is 30 - 70 °C, ultrasonic extraction time is 20 - 60 min; the steam distillation extraction time is 1 - 5 h.

[0016] Further, in step (3) for extraction and separation, the solvent added is n-hexane, and the volume ratio of the added amount of n-hexane to the oil-water mixture is 1:40 - 1:70.

[0017] Further, in step (4), the freezing temperature is -20 - -80 °C, and the freezing time is 0.5 - 3 h.

[0018] The present invention also relates to a pepper stem and leaf essential oil, which is prepared by the above method for preparing pepper stem and leaf essential oil.

[0019] Meanwhile, the present invention also relates to the application of the pepper stem and leaf essential oil as an antioxidant; and the application of the pepper stem and leaf essential oil as an antibacterial agent.

[0020] Preferably, the antibacterial agent has an inhibitory effect on at least one of Staphylococcus aureus (G+), Listeria monocytogenes (G+), and Escherichia coli (G-).

[0021] A kind of capsicum stem and leaf essential oil, its preparation method and application provided in the above technical solution optimize the liquid-to-material ratio of capsicum stem and leaf powder to distilled water, ultrasonic extraction time, ultrasonic extraction temperature, ultrasonic power, distillation time, etc. The preparation process of the present invention is simple, the process time is short, the yield of capsicum stem and leaf essential oil obtained is high, and it has good antibacterial and antioxidant activities, with broad market application prospects. At the same time, the present invention uses the freezing dehydration method to replace the traditional anhydrous sodium sulfate dehydration method, providing a new direction for the optimization of the essential oil preparation process. The present invention turns waste capsicum stems and leaves into treasures, reduces environmental pollution, provides methods and technical inspirations for the preparation of capsicum stem and leaf essential oil, and lays a foundation for the comprehensive utilization of capsicum stem and leaf resources and the improvement of the added value of the capsicum industry.

[0022] At the same time, capsicum stem and leaf essential oil has good antioxidant, antibacterial and bactericidal activities, which has important practical significance and application value for the research and development of capsicum stem and leaf resources and the optimization of the essential oil dehydration process. Brief Description of the Drawings

[0023] Figure 1 is the process flow chart of the present invention;

[0024] Figure 2 is the influence of ultrasonic temperature on the yield of capsicum stem and leaf essential oil;

[0025] Figure 3 is the influence of ultrasonic time on the yield of capsicum stem and leaf essential oil;

[0026] Figure 4 is the influence of ultrasonic power on the yield of capsicum stem and leaf essential oil;

[0027] Figure 5 is the influence of liquid-to-material ratio on the yield of capsicum stem and leaf essential oil;

[0028] Figure 6 is the influence of distillation time on the yield of capsicum stem and leaf essential oil;

[0029] Figure 7 is the influence of sodium chloride concentration on the yield of capsicum stem and leaf essential oil;

[0030] Figure 8 is the influence of the interaction between ultrasonic power and ultrasonic time on the yield of capsicum stem and leaf essential oil;

[0031] Figure 9It is the influence of the interaction between ultrasonic power and liquid-to-material ratio on the essential oil yield of pepper stems and leaves;

[0032] Figure 10 It is the influence of the interaction between ultrasonic time and liquid-to-material ratio on the essential oil yield of pepper stems and leaves;

[0033] Figure 11 It is the result graph of the DPPH free radical scavenging by pepper stems and leaves essential oil;

[0034] Figure 12 It is the result graph of the ABTS free radical scavenging by pepper stems and leaves essential oil. Specific implementation manners

[0035] In order to make the purpose and advantages of the present invention clearer, the present invention will be specifically described below in conjunction with embodiments. It should be understood that the following text is only used to describe one or several specific implementation manners of the present invention, and does not strictly limit the scope of protection specifically claimed by the present invention.

[0036] As Figure 1 shown, a preparation method of pepper stems and leaves essential oil includes the following steps:

[0037] Step 1 Raw material collection and treatment: Take the pepper stems and leaves collected in November, wash them with water, dry the pepper stems and leaves at room temperature, crush and sieve them with a pulverizer, and store the obtained pepper stems and leaves powder sealed in a -20°C refrigerator for later use;

[0038] Step 2 Preparation of pepper stems and leaves powder aqueous solution: Mix the pepper stems and leaves powder obtained in Step 1 evenly with distilled water, add sodium chloride according to the total volume of the pepper stems and leaves powder aqueous solution to make the sodium chloride concentration in the solution 20 g / L, and soak for 3 h;

[0039] Step 3 Single-factor experiment: Perform single-factor experiments on the pepper stems and leaves powder solution obtained in Step 2 according to five factors: ultrasonic power of 200 - 600 W, temperature of 30 - 70°C, ultrasonic extraction time of 20 - 60 min, liquid-to-material ratio of 16:1 - 24:1, and distillation time of 1 - 5 h to obtain an oil-water mixture. Then transfer the oil-water mixture to a separatory funnel, add n-hexane at a ratio of 1:60, let it stand for extraction for 19 h, separate to obtain the essential oil layer and evaporate the n-hexane. Then collect the essential oil layer containing a small amount of water into a reagent bottle, seal it and put it into a -20°C refrigerator. After freezing, take it out, collect the upper layer of essential oil into a clean sample bottle, weigh it, and calculate the essential oil yield of each experimental level according to formula (1) to provide a basis for the selection of the experimental levels of each factor in the later response surface experiment.

[0040] Step 4 Optimization of the preparation process of capsicum stem and leaf essential oil by response surface experiment: On the basis of single-factor experiments, the ultrasonic temperature was selected as 60 °C. According to the central composite design principle of Box-Behnken, with ultrasonic power, ultrasonic extraction time, and liquid-to-solid ratio as the independent variables (represented by X1, X2, and X3 respectively), and the yield of capsicum stem and leaf essential oil as the response value, a response surface analysis experiment with 17 experimental points of three factors and three levels was designed. The experimental points were divided into 14 factorial points and 3 zero points. After ultrasonic extraction was completed according to each level condition, constant-temperature distillation was carried out at 120 °C for 3 h in a distillation device to obtain an oil-water mixture. The same optimized extraction process conditions of capsicum stem and leaf essential oil were operated 21 times to prepare 21 portions of oil-water mixture.

[0041] Step 5 Separation and purification of capsicum stem and leaf essential oil: Transfer the oil-water mixture obtained in Step 4 to a separating funnel, add n-hexane in a ratio of 1:60, let it stand for extraction for 19 h, and separate to obtain 18 portions of essential oil containing a small amount of water. Then, 3 portions of the essential oil containing a small amount of water were dried by the freezing method. Another 3 portions of the essential oil containing a small amount of water were dried by the anhydrous sodium sulfate method. Then, calculate the essential oil yield of each experimental level according to Equation (1) above to obtain the optimal preparation process conditions of capsicum essential oil.

[0042] The specific operation process is as follows:

[0043] 1. Optimization of the extraction process of capsicum stem and leaf essential oil

[0044] On the basis of single-factor experiments (the results can be seen Figures 2 to 7 ), three factors of liquid-to-solid ratio, ultrasonic extraction time, and ultrasonic power were selected for response surface optimization experiments.

[0045] (1) Response surface experiment design table:

[0046] Table 1 Response surface analysis experiment design table of three factors and three levels

[0047]

[0048] The experiments were carried out in a random order. The yield of capsicum stem and leaf essential oil obtained from the experiments was analyzed using the Design-Expert 12 program, and the response surface analysis diagram, regression fitting equation, and variance analysis table were obtained. The response surface experiment design and results are shown in Table 2.

[0049] (2) Response surface central composite design (CCD) design and experimental results table:

[0050] Table 2 Response surface CCD design scheme and experimental results of capsicum stem and leaf essential oil

[0051]

[0052] (3) Establishment and variance analysis of the model

[0053] According to the Box-Behnken central composite design principle, the Design-Expert 12 software was used to perform multiple regression fitting on the experimental data in Table 2, and a quadratic multiple regression equation for the relationship between ultrasonic power, ultrasonic extraction time, solid-liquid ratio, and the yield of essential oil from pepper stems and leaves was obtained:

[0054] Yield of essential oil from pepper stems and leaves (%) = -1.38334 + 0.000939X1 + 0.005114X2 + 0.134104X3 + 1.72500E -06 X1X2 - 7.75000E -06 X1X3 + 0.000029X2X3 - 1.12900E -06 X1 2 -0.000111X2² - 0.003885X3 2 .

[0055] In the formula, the absolute value of the coefficients of X1, X2, and X3 directly reflects the influence degree of each factor on the yield of essential oil from pepper stems and leaves, and the positive or negative of the coefficient reflects the increase or decrease of the yield of essential oil from pepper stems and leaves. The experimental results in Table 2 were statistically analyzed, and the analysis results are shown in Table 3.

[0056] Table 3 Analysis of variance table of the response surface regression model

[0057]

[0058] Note: "**" indicates P < 0.01, with extremely significant difference; "*" indicates P < 0.05, with significant difference; "--" indicates P > 0.05, with no significant difference.

[0059] As can be seen from Table 3, among the first-order terms of the model, the solid-liquid ratio (X3) has the most significant influence on the essential oil from pepper stems and leaves; among the second-order terms, ultrasonic power (X1), ultrasonic extraction time (X2), and solid-liquid ratio (X3) all have extremely significant effects on the essential oil from pepper stems and leaves (p < 0.01). From the F value, the order of the influence of each factor on the yield of essential oil from pepper stems and leaves is: solid-liquid ratio (X3) > ultrasonic extraction time > ultrasonic power (X1). The Prob > F value of the overall model is less than 0.01, indicating that the regression model reaches an extremely significant level; the correlation coefficient R of the regression model 2 = 0.9245, and the lack-of-fit term is not significant (the Prob > F value is 0.1379, greater than 0.05), indicating that the fit between the model and the data is very good. Therefore, this regression equation can be used to analyze the experimental results instead of the actual experimental points.

[0060] At the same time, the response surface contour plots of the interaction of the three groups of factors obtained from the central composite design experiment were made, seeFigures 8 to 10 。

[0061] (4) Prediction and Verification of the Optimal Extraction Conditions for Capsicum Stem and Leaf Essential Oil

[0062] The response surface graph is a contour map on a two-dimensional plane formed by factors X1, X2, and X3 corresponding to the response value of the yield of capsicum stem and leaf essential oil in a three-dimensional space ( Figures 8 to 10 ), which can intuitively reflect the interaction between factors and the influence on the response value. It can be seen from the graph that the fitted surface has a maximum value. By taking the partial derivative of the fitted equation, the maximum value of the model can be obtained, which is the optimal experimental scheme.

[0063] Within the experimental range, through the analysis and prediction of the regression equation by Design-Expert 12 software, the optimal extraction process conditions for capsicum stem and leaf essential oil are obtained as follows: ultrasonic power 499.32 W, ultrasonic extraction time 29.19 min, liquid-to-solid ratio 17.87. Under these optimized conditions, three groups of parallel experiments are conducted. The average yield of capsicum stem and leaf essential oil obtained is 0.1827%, which is only 2.3% different from the theoretical predicted value of 0.187%. This shows that there is a good degree of fit between the predicted value of the regression equation and the experimental value. Therefore, the optimized process parameters obtained based on the response surface method are accurate and reliable and have practical value.

[0064] According to the operation procedures in steps 4 and 5, 21 portions of essential oil containing a small amount of water are extracted using the optimal extraction process conditions for capsicum stem and leaf essential oil, preparing for the subsequent separation and preparation of essential oil.

[0065] 2. Separation and Preparation of Capsicum Stem and Leaf Essential Oil

[0066] 2.1 Water Removal and Preparation of Essential Oil by Freezing Method: Transfer 18 portions of essential oil containing a small amount of water into reagent bottles respectively, seal them, and place them in a -20°C refrigerator. Take them out after testing different freezing times (0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h), collect the upper-layer essential oil into clean sample bottles and weigh it. Calculate the yield of capsicum stem and leaf essential oil according to formula (1). The experimental results are shown in Table 4.

[0067] Table 4 Influence of Different Freezing Times on the Yield of Capsicum Stem and Leaf Essential Oil

[0068]

[0069] Note: When the freezing time in Table 4 is 0.5 h, the water in the essential oil is not completely frozen, and it is impossible to obtain the water-removed essential oil. Therefore, the essential oil yield under the condition of freezing for 0.5 h is not obtained.

[0070] 2.2 Essential oil dehydration and preparation with anhydrous sodium sulfate: Another 3 portions of essential oil containing a small amount of water were dried using the traditional anhydrous sodium sulfate method. That is, anhydrous sodium sulfate accounting for 3% of the weight of the essential oil was added to the essential oil while stirring. After stirring evenly, the mixture was allowed to stand for 24 h, centrifuged, and the upper-layer essential oil was collected into a clean sample bottle and weighed. Then, the yield of the essential oil was calculated according to the above formula (1), as shown in Table 5.

[0071] Table 5 Yield of capsicum stem and leaf essential oil obtained by dehydration with anhydrous sodium sulfate

[0072]

[0073] From the above results, it can be seen that under the optimized extraction process conditions, the yield of capsicum stem and leaf essential oil obtained by combining the optimized freezing dehydration process (-20 °C, freezing time 1 h) was 0.1887‰; while under the same optimized extraction process conditions, by combining the commonly used anhydrous sodium sulfate dehydration method, the yield of capsicum stem and leaf essential oil was 0.162‰. Under the optimized extraction process conditions, the yield of capsicum essential oil by the freezing dehydration process was 16.5% higher than that by the traditional anhydrous sodium sulfate dehydration process.

[0074] 2.3 Chemical composition identification of capsicum stem and leaf essential oil obtained by freezing dehydration method: The components of capsicum stem and leaf essential oil were identified by gas chromatography - mass spectrometry (GC-MS). The component determination was carried out on an HP-5MS (30 m × 0.25 mm id, film thickness 0.25 μm, Agilent) fused silica capillary column. The experimental conditions were as follows: inlet temperature 250 °C, flow rate 1.2 mL / min, sample injection volume 1 μL, splitless. The initial column temperature was 40 °C, held for 2 min, then increased to 110 °C at a rate of 5 °C / min and immediately increased to 250 °C at a rate of 10 °C / min, and held for 10 min. Ionization was carried out in the EI mode, electron energy 70 eV, ion source temperature 230 °C, interface temperature 250 °C, mass scanning range 45 - 550 amu, and scanning voltage 1420 V. Finally, the chemical composition analysis results of the capsicum stem and leaf essential oil prepared by the freezing dehydration method were obtained, as shown in Table 6 specifically.

[0075] Table 6 Chemical composition analysis results of capsicum stem and leaf essential oil

[0076]

[0077]

[0078]

[0079]

[0080]

[0081] Example

[0082] According to the optimized method for preparing capsicum stem and leaf essential oil, it includes the following steps:

[0083] Step 1: Wash 10 kg of capsicum stem and leaf collected in November with water, dry the surface water at room temperature, then dry the capsicum stem and leaf at room temperature, crush it with a pulverizer, sieve it through a 50-mesh sieve, and store the obtained capsicum stem and leaf powder sealed in a -20°C freezer for later use.

[0084] Step 2: Add distilled water to the capsicum stem and leaf powder (with a total weight of 2.97 kg) at a ratio of 17.87:1 (mL:g), add sodium chloride to make its concentration 20 g / L, and then soak for 3 h. Then, perform ultrasonic extraction for 29.2 min at an ultrasonic power of 499 W and an extraction temperature of 60°C. Distill the obtained oil-water mixture at 120°C for 3 h, then transfer the oil-water mixture to a separating funnel, add n-hexane at a ratio of 1:60, let it stand for extraction for 19 h, and separate to obtain the essential oil layer.

[0085] Step 3: Collect the essential oil layer containing a small amount of water into a reagent bottle, seal it and put it into a -20°C refrigerator, take it out after freezing for 1 h, collect the upper-layer essential oil into a clean sample bottle and weigh it, obtain 594.5 mg of capsicum stem and leaf essential oil, and the yield of the obtained capsicum stem and leaf essential oil is 0.185‰.

[0086] Test Example

[0087] 1. Antioxidant effect of capsicum stem and leaf essential oil

[0088] (1) Determination of DPPH scavenging rate

[0089] First, weigh 5 mg of DPPH and add it to a brown volumetric flask, then add 167 mL of methanol to obtain the DPPH reagent with the required concentration. Use a pipette to suck 3.9 mL of the prepared DPPH reagent and put it into a clean brown volumetric flask, and accurately and quickly add 0.1 mL of the capsicum stem and leaf essential oil sample solution dissolved in dimethyl sulfoxide (DMSO) with different concentration gradients, where the capsicum stem and leaf essential oil is from the example. After quickly mixing evenly, react in the dark at about 25°C for 30 min, and then measure the OD value with a UV spectrophotometer at a wavelength of 750 nm to obtain the A sample value. Take another 3.9 mL of DPPH reagent, add 0.1 mL of solvent, shake it evenly quickly in the same way, react at room temperature (about 25°C) and in the dark for 30 min, and then measure the absorbance value at a wavelength of 415 nm to obtain the A blank value.

[0090]

[0091] (2) Determination of ABTS radical scavenging rate

[0092] First, prepare 50 mL each of 7 mmol / L ABTS solution and 2.45 mmol / L potassium persulfate solution, mix them in a volume ratio of 2:1, add an appropriate amount of distilled water, and let it stand in the dark for 12 h to form a stock solution. Before use, dilute it with absolute ethanol. At 30 °C and a wavelength of 734 nm, the absorbance value is about 0.07 to form the ABTS working solution. Prepare samples with different concentrations using dimethyl sulfoxide as the solvent, and prepare VC with different concentrations as the positive control using distilled water as the solvent. In an environment of 30 °C, take 3.8 mL of the ABTS working solution, add 0.2 mL of the sample, react in a water bath for 6 min, and measure OD734 and record it as A1; measure with the solvent instead of the sample and record it as A0. The calculation formula is as follows:

[0093]

[0094] (3) Experimental results

[0095] The antioxidant ability of the essential oil was demonstrated by measuring its ability to scavenge free radicals. Through the analysis of the experimental data measured by Origin 2018 software, the DPPH and ABTS free radical scavenging activities of the essential oil from pepper stems and leaves are as Figure 10 、 Figure 11 shown.

[0096] Here, the concentration of the essential oil corresponding to a scavenging rate of 50% is denoted as IC 50 , and the smaller the value of IC 50 , the stronger its antioxidant ability. The IC 50 values of the two antioxidant methods were obtained through analysis by Origin 2018 software as shown in Table 7. Compared with the existing essential oils, they are all stronger than the antioxidant activities of the perilla essential oil reported by Zeng Yanru et al. (the DPPH and ABTS scavenging rates of perilla essential oil are 7.00 and 2.66 mg·mL -1 ) and the antioxidant activity of the Artemisia annua essential oil reported by Chen Wendan et al. (the IC 50 value of Artemisia annua essential oil for scavenging DPPH is 6.76 mg·mL -1 ). Therefore, the essential oil from pepper stems and leaves has strong antioxidant activity. Combining the component analysis results in Table 6 and the existing literature reports on the antioxidant activity determination of the corresponding components, it is preliminarily inferred that the active substances playing the antioxidant role are mainly phenolic acids and alcohols, and the representative compounds are 10(E),12(Z)-conjugated linoleic acid, 9,12,15-octadecatrienoic acid, (Z,Z,Z), 7,10,13-hexadecatrienoic acid, (Z,Z,Z), linalool, methyl 9,12,15-octadecatrienoate, (Z,Z,Z), and phytol, etc. The results of the essential oil from pepper stems and leaves scavenging DPPH free radicals and ABTS free radicals are as Figure 11 、 Figure 12 and Table 7 shown.

[0097] Table 7 The IC 50 value

[0098]

[0099] Note: *This result is referenced from the article reported by Zeng Yanru et al. in Fujian Agricultural Science and Technology; **This result is referenced from the article reported by Chen Wendan et al. in Science and Technology of Food Industry.

[0100] 2. Antibacterial effect of essential oil from pepper stems and leaves

[0101] 2.1 Specific test method

[0102] (1) Test strains:

[0103] Staphylococcus aureus (ATCC), Escherichia coli (ATCC), Listeria monocytogenes (ATCC).

[0104] (2) Preparation of culture medium: Weigh 8.0 g of LB medium and add distilled water to prepare 800 mL of liquid medium. Prepare 4 conical flasks, each filled with 50 mL of LB liquid medium, and seal them. Weigh 12 g of agar powder again and dissolve it in the remaining 600 mL of LB liquid medium. Heat it to completely melt the agar, and then dispense it into 2 conical flasks of 500 mL each and seal them.

[0105] (3) Sterilization: Sterilize 4 bottles of LB liquid medium, 2 bottles of LB solid medium, 24 culture dishes (6 in each pack, 4 packs in total), 2 bottles of sterile water, 4 empty conical flasks, 1 box of pipette tips and filter paper by autoclaving.

[0106] (4) Preparation of bacterial suspension: One day before the inhibition zone experiment, use an inoculation loop to inoculate the four bacteria into the previously sterilized LB liquid medium in a ultra-clean workbench (do a good job in sterilization to avoid cross-contamination between different strains). Incubate at 37 °C and 200 rpm for 16 h. Before the inhibition zone experiment, take out a small amount of bacterial liquid into a sterilized empty conical flask and dilute it with sterile water. Use sterile water as a blank control to make the absorbance value of the diluted bacterial liquid at a wavelength of 600 nm in a spectrophotometer about 0.1, that is, obtain the bacterial suspension required for the experiment.

[0107] (5) Antibacterial circle experiment: Start the laminar flow hood, wipe the workbench surface with cotton soaked in 70% alcohol solution, then put the required items into the laminar flow hood, turn on the ultraviolet lamp for sterilization for 20 - 30 min. After sterilization, pour the culture medium into plates. After the culture medium has completely solidified, add 200 μL of the bacterial suspension into the plate and spread it with a spreader. Use forceps to pick up single-layer filter paper discs and stick them onto the culture medium. Inject enrofloxacin solution (positive control), n-hexane (negative control), and the sample at the corresponding positions respectively, and mark the basic information such as time, bacterial strain, and sample with a marker pen. Finally, incubate it upside down in an incubator at 37 °C for about 16 h, observe the results, and measure the diameter of the antibacterial circle with a ruler. The larger the diameter of the antibacterial circle, the stronger the antibacterial activity of the essential oil.

[0108] (6) For the experimental principles and methods of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), determine the following experimental plan: Accurately weigh 12.5 g of LB medium powder, dissolve it in 500 mL of distilled water, and stir with a glass rod to accelerate dissolution. Prepare 4 conical flasks, each filled with 50 mL of LB liquid medium, and the rest are filled in 500 mL conical flasks, wrap and seal them. Then, together with 200 μL pipette tips, sterilize them by high-pressure steam at 121 °C for 15 min. The method for preparing the bacterial suspension is basically the same as that in 2.2.3. This time, use the culture medium for dilution to make the absorbance value OD600 of the bacterial suspension reach about 0.1. At the same time, use n-hexane as the solvent and negative control, and dilute the sample by the gradient dilution method. The concentration gradients are (mg / ml): 20, 18, 16, 14, 12, 10, 8, 6, 4, 2, 0, with enrofloxacin as the positive control. Take a 96-well plate, mark the basic information on the lid, add 100 μL of samples with different concentrations to it respectively (3 parallel tests for each concentration), then add the same volume of bacterial suspension to each well, shake the 96-well plate and place it in an incubator at 37 °C. Observe the results after 16 h. The lowest concentration in the clear holes is the MIC. Spread the culture medium in the wells with a concentration greater than the MIC on a plate, and the lowest sample concentration without colony growth after incubation is the MBC.

[0109] 2.2 Test results

[0110] 2.2.1 The results of antibacterial circle determination are shown in Table 8.

[0111] Table 8 Results of antibacterial circle diameter (mm)

[0112]

[0113] Note: The sample concentration is 5 mg / mL for all; the antibacterial circle diameter includes the filter paper disc diameter of 6.0 mm.

[0114] 2.2.2 The results of MIC and MBC determination are shown in Table 9.

[0115] Table 9 MIC and MBC of essential oil from pepper stems and leaves against tested bacteria

[0116]

[0117] Note: *This result is referenced from the article reported by Feng Yue et al. in the "Journal of Sichuan University of Science & Engineering (Natural Science Edition)", and the result is converted according to the conventional density of essential oil, 1mg / μL; **This result is referenced from the article reported by Zhang Linhui et al. in "China Condiment"; -- indicates that the antibacterial effect of this kind of bacteria is not reported in this literature.

[0118] As can be seen from Table 8 and Table 9, the essential oil from pepper stems and leaves obtained in the examples has strong antibacterial activities against Staphylococcus aureus, Escherichia coli and Listeria monocytogenes, among which the antibacterial activity against Escherichia coli is the strongest.

[0119] The MIC and MBC values of the essential oil from pepper stems and leaves against the three tested bacteria are the same. Combining with the antibacterial circle test results of the essential oil from pepper stems and leaves in Table 8, it can be inferred that the antibacterial effects of the essential oil from pepper stems and leaves against Escherichia coli, Listeria monocytogenes and Staphylococcus aureus are basically the same. The results show that the essential oil has good antibacterial effects (superior to the antibacterial effects of the essential oil from Cinnamomum longepaniculatum reported by Feng Yue et al. and the essential oils from Cinnamomum subavenium, Cunninghamia lanceolata, Cupressus funebris, Curcuma longa, Rosmarinus officinalis and Illicium verum reported by Zhang Linhui et al.). Combining with the component analysis results in Table 6 and the existing literature reports on the antibacterial activities of the corresponding components, it is preliminarily inferred that the main types of compounds exerting antibacterial effects are alcohols, acids and phenolic compounds, and the representative compounds are hexadecanoic acid, linalool, palmitic acid, 4-bromo-2-chlorophenol, tetradecanoic acid, pentadecanoic acid, etc.

[0120] Through the above examples, it can be found that the optimized process conditions for the preparation of the essential oil from pepper stems and leaves of the present invention have the characteristics of high yield, reliability and simplicity in operation. At the same time, it also has good antioxidant, antibacterial and bactericidal activities, which has important practical significance and application value for the research and development of pepper stem and leaf resources and the optimization of the essential oil dehydration process.

[0121] In summary, the method of the present invention has the advantages of short time consumption, high yield, stability and controllability, and simplicity in operation compared with the traditional essential oil preparation methods. Experimental verification shows that the essential oil from pepper stems and leaves has good antioxidant and antibacterial activities. Through GC-MS determination and combined with the existing literature reports, the main antibacterial and antioxidant active components of the essential oil from pepper stems and leaves are preliminarily inferred. The essential oil from pepper stems and leaves of the present invention can be used as a plant-derived antioxidant and antibacterial agent in the fields of food, medicine and chemical industry.

[0122] The embodiments of the present invention have been described in detail in combination with the above embodiments. However, the present invention is not limited to the above embodiments. For those of ordinary skill in the art, after learning the content recorded in the present invention, without departing from the principle of the present invention, several equivalent transformations and substitutions can still be made, and these equivalent transformations and substitutions should also be regarded as belonging to the protection scope of the present invention.

Claims

1. A preparation method of capsicum stem and leaf essential oil, characterized in that, It includes the following steps: (1) Wash the pepper stems and leaves, dry them in the air, and crush and sieve them to obtain pepper stem and leaf powder; (2) Mix the pepper stem and leaf powder obtained in step (1) evenly with distilled water, and then add sodium chloride for soaking; (3) Perform ultrasonic-assisted extraction on the pepper stem and leaf powder solution after soaking in step (2), and then perform essential oil steam distillation extraction to obtain an oil-water mixture; then perform extraction and separation on the oil-water mixture to obtain an essential oil layer; (4) Evaporate the solvent in the obtained essential oil, and then seal it and freeze it to remove the water in the essential oil, thus obtaining pepper stem and leaf essential oil.

2. The preparation method of capsicum stem and leaf essential oil according to claim 1, characterized in that: In step (1), the particle size of the pepper stem and leaf powder is 40-60 mesh.

3. The preparation method of capsicum stem and leaf essential oil according to claim 1, wherein: In step (2), the mass-volume ratio of the pepper stem and leaf powder to distilled water is 1:16-1:24 g / mL; the concentration of sodium chloride is 10-50 g / L, and the soaking time is 2-5 h.

4. The preparation method of capsicum stem and leaf essential oil according to claim 1, characterized in that: In step (3), the conditions for ultrasonic-assisted extraction are: power 200-600 W, temperature 30-70 °C, ultrasonic extraction time 20-60 min; the time for steam distillation extraction is 1-5 h.

5. The preparation method of capsicum stem and leaf essential oil according to claim 1, characterized in that: In the extraction and separation in step (3), the solvent added is n-hexane, and the volume ratio of the added amount of n-hexane to the oil-water mixture is 1:40-1:

70.

6. The preparation method of capsicum stem and leaf essential oil according to claim 1, wherein: In step (4), the freezing temperature is -20--80 °C, and the freezing time is 0.5-3 h.

7. A pepper stem and leaf essential oil prepared by the method for preparing pepper stem and leaf essential oil according to any one of claims 1-6.

8. Use of the pepper stem and leaf essential oil according to claim 7 as an antioxidant.

9. Use of the pepper stem and leaf essential oil according to claim 7 as an antibacterial agent.

10. The application according to claim 9, wherein: The antibacterial agent has an inhibitory effect on at least one of Staphylococcus aureus, Listeria, and Escherichia coli.

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