Efficient mining method for antithrombotic active components in orange peel essential oil

Abstract

The invention discloses an efficient mining method for antithrombotic active components in orange peel essential oil. The invention discloses the efficient mining method for the antithrombotic active components in the orange peel essential oil. The method specifically comprises the following steps: step 1, taking peels of citrus plants as experimental materials, and effectively extracting oxygen-containing compounds with remarkable antithrombotic activity, such as esters, acids, aldehydes and phenols, contained in the peels through a portable simultaneous distillation and extraction technology; 2, carrying out accurate qualitative and quantitative analysis on the active components by adopting a gas chromatography-mass spectrometry (GC-MS) technology; and 3, exploring an active mechanism in the citrus peel essential oil by virtue of a computer simulation experiment. The method has the characteristics of efficient preparation, comprehensive detection and rapid and clear component activity, and can provide guidance for deep development and application of the citrus peel essential oil.

Description

Technical Field

[0001] The present invention focuses on the field of extracting anti - thrombotic active components from citrus peel essential oil, and specifically introduces an efficient method for exploring anti - thrombotic active components in orange peel essential oil. This method combines gas chromatography - mass spectrometry (GC - MS) for accurate qualitative and quantitative analysis of active ingredients, and deeply explores the active mechanism of citrus peel essential oil through computer simulation experiments. Background Art

[0002] Currently, the processing of citrus products mainly focuses on the fruits, resulting in a large amount of by - products such as peels being discarded, which not only wastes resources but also causes environmental pollution problems.

[0003] Plant essential oil, a volatile oily liquid with a strong aroma, widely exists in parts such as the roots, leaves, seeds and flowers of plants. Citrus peel essential oil is a complex mixture extracted from citrus peels, composed of various secondary metabolites, and its biological activity is directly affected by chemical components. Different extraction methods and conditions will change the chemical composition of the essential oil, and thus affect its biological activity. Therefore, it is crucial to select an appropriate extraction method. Currently, the cold pressing method is a commonly used extraction method, but the content of terpenes and alcohols in the obtained essential oil is relatively high, while the content of esters, acids, aldehydes and phenols is relatively low; in contrast, the hot processing method can extract citrus peel essential oil rich in active components such as esters, acids and aldehydes. With the increasing popularity of plant essential oils, their efficient extraction technology has become a research hotspot. A portable simultaneous distillation extraction device for plant essential oils that combines steam distillation and solvent extraction method is not only easy to operate, but also can achieve high - efficiency extraction with a small amount of raw materials, meeting the dual requirements of equipment simplicity and high extraction rate.

[0004] Plant essential oils have beneficial effects on the cardiovascular system such as anti - thrombotic, anti - platelet, protecting vascular endothelium, antioxidant, improving vascular elasticity and lowering blood pressure. They play a significant role in improving cardiovascular function and reducing risk factors for cardiovascular diseases. Phenolic substances in essential oils have strong antioxidant effects, can scavenge oxygen free radicals in the body and protect vascular endothelium. In addition, some volatile ketones, alcohols, aldehydes and phenolic substances also have the effect of protecting the cardiovascular system. Thrombosis is an important cause of cardiovascular diseases, and anti - thrombosis treatment has become an important strategy for treating cardiovascular diseases. Research shows that essential oils of Rutaceae plants have certain anti - thrombotic functions.

[0005] The concept of molecular docking can be traced back to Fischer's "receptor theory" 100 years ago. This theory holds that there is a recognition relationship between small molecules and macromolecular proteins in the body, similar to that of a "key and lock". Subsequently, in 1958, Koshland proposed the concept of induced fit in molecular recognition, that is, the structure of the active center of the receptor molecule does not exactly match that of the substrate, but is flexible and plastic. According to the characteristics of the ligand-receptor being docked, molecular docking methods can be divided into rigid docking, semi-flexible docking, and flexible docking. This technology can predict the binding mode and affinity between proteins and ligands through calculations using software such as AutoDock and Discovery Studio. This technology not only has the advantages of a short cycle and strong operability. Currently, it has become an important means for screening the active components of plant essential oils.

[0006] Currently, the research on the preparation of orange peel essential oil mainly focuses on cold pressing extraction, while the research on using thermal processing technology to extract orange peel essential oil rich in antithrombotic active components such as esters, acids, aldehydes, and phenols is relatively scarce, and there is a lack of means for quickly and efficiently mining the antithrombotic active components in orange peel essential oil. In view of this, developing a method that can efficiently prepare orange peel essential oil, comprehensively detect its components, and conveniently mine its active components has important theoretical guiding significance for promoting the comprehensive utilization and in-depth development of orange peel essential oil. This study proposed a convenient method for preparing orange peel essential oil by simultaneous distillation extraction, combined with gas chromatography-mass spectrometry (GC-MS) for component analysis, and used molecular docking technology to deeply mine the antithrombotic active components of orange peel essential oil. This method not only has the advantages of efficient preparation, comprehensive detection, and convenient exploration, but also is simple to operate and has a low cost, making it more suitable for practical application and promotion. Summary of the Invention

[0007] The purpose of the present invention is to develop an efficient and convenient method for mining antithrombotic active components from citrus peel essential oil. This method not only has high preparation efficiency, comprehensive substance detection ability, and convenient activity exploration characteristics, but also has the advantages of simple operation and low cost, aiming to solve the preparation problems of functional products of citrus peel essential oil and provide useful guidance for the development and utilization of citrus peel essential oil.

[0008] The technical solution of the present invention is as follows: This method uses the citrus peel of citrus plants as the starting material and adopts a convenient simultaneous distillation extraction method to extract the essential oil from the citrus peel. Subsequently, gas chromatography-mass spectrometry (GC-MS) is used to accurately determine the extracted essential oil. Finally, molecular docking technology is used to further mine the antithrombotic active components of citrus peel essential oil.

[0009] In the present invention, preferably, a portable simultaneous distillation extraction device, which is composed of a round-bottom flask, an extractor, and a condenser tube, combines the steam distillation method and the solvent extraction method. The upper layer is an organic solvent layer, and the lower layer is an aqueous layer.

[0010] In the present invention, preferably, detection is carried out using an HP-5 capillary column (30 m × 0.25 mm × 0.25 µm); the carrier gas is high-purity helium, with a flow rate of 0.8 mL / min, a split ratio of 50:1, and an injection port temperature of 280 °C; the mass spectrometry interface temperature is 250 °C, the ionization method is an electron impact source (EI), the ion energy is 70 eV, and the ion source temperature is 250 °C.

[0011] In the present invention, preferably, the temperature programming of GC-MS is as follows: the initial temperature is 80 °C, held for 2 min; heated to 200 °C at a rate of 4 °C / min and held for 5 min; finally heated to 280 °C at a rate of 20 °C / min and held for 2 min.

[0012] In the present invention, preferably, the computer-aided molecular docking technology is adopted to select the proteins that control blood coagulation and inhibit thrombosis, and dock them with the oxygen-containing active components detected quantitatively above to explore the anti-thrombotic activity of citrus peel essential oil.

[0013] In the present invention, preferably, the extraction and preparation steps of the sample are as follows: (1) After crushing the peel sample with a pulverizer, weigh an appropriate amount of peel powder and put it into a round-bottom flask, add distilled water, and let it stand at room temperature.

[0014] (2) Set up a portable simultaneous distillation extraction device, add distilled water to the lower layer and an organic solvent to the upper layer. After heating to boiling, continue distillation.

[0015] (3) Separate the organic solvent layer, and filter the sample with anhydrous sodium sulfate to dehydrate it.

[0016] (4) Use a rotary evaporator to remove the organic solvent to obtain the peel essential oil.

[0017] (5) Use the obtained peel essential oil for GC-MS detection and analysis.

[0018] (6) Screen key anti-thrombotic proteins as macromolecular receptors.

[0019] (7) Conduct a molecular docking experiment between the active components of citrus peel essential oil and the macromolecular receptor.

[0020] In step (1), the ratio of the peel powder to the distilled water is 1:20 - 1:30. The mass unit of the peel powder is grams (g), the volume unit of the distilled water is milliliters (mL), and the standing time at room temperature is 10 - 12 hours (h).

[0021] In step (2), the organic solvent is n - hexane, and the duration of continuous distillation is 5 - 7 hours.

[0022] In step (3), the ratio of the organic solvent layer to anhydrous sodium sulfate is 20:1 - 10:1, where the volume unit of the organic solvent layer is milliliters and the mass unit of anhydrous sodium sulfate is grams.

[0023] In step (4), the rotation speed of the rotary evaporator increases from slow to fast, with a rotation speed range of 20 - 80 revolutions per minute (rpm), and the water bath temperature is 30 - 40 °C.

[0024] In step (5), external standard method is used for quantitative analysis in GC - MS.

[0025] In step (6), the screened thrombomodulin (P07204), tissue factor (P13726), and antithrombin - III (P01008) are active proteins that inhibit thrombus formation.

[0026] In step (7), the molecular docking experiment mainly uses Discovery Studio software. With the main active components of essential oil as the docking ligands and the key anti - thrombus protein molecules as the docking receptors, semi - flexible docking is carried out to obtain binding energy data.

[0027] The beneficial effects of the present invention are as follows: First, high - efficiency preparation. The present invention adopts a highly efficient and portable simultaneous distillation and extraction device, which realizes an innovative combination of steam distillation method and solvent extraction method, and significantly improves the extraction efficiency of essential oil.

[0028] Second, convenient screening. The present invention uses computer - simulated molecular docking technology to deeply explore the anti - cancer active components in orange peel essential oil in a more convenient and scientific way.

[0029] Third, low cost. The portable simultaneous distillation and extraction device in the present invention only requires a small amount of raw materials to complete the extraction process of essential oil. On the premise of ensuring high sensitivity and accurate quantification, compared with liquid chromatography - mass spectrometry technology, the cost of detection using GC - MS is significantly reduced. Description of the Drawings

[0030] Figure 1 It is a flow chart for the preparation of citrus peel essential oil and exploration of active components in the present invention Figure 2 It is the total ion current chromatogram of kumquat peel essential oil detected by GC - MS Figure 3 It is the total ion current chromatogram of ponkan peel essential oil detected by GC - MS Figure 4 It is the 3D structure diagram of thrombomodulin (P07204) and its binding site Figure 5 For the 3D structure diagram of tissue factor and its binding sites Figure 6 For the 3D structure diagram of antithrombin-III and its binding sites Figure 7 For the 2D structure diagram of the interaction between palmitic acid and thrombomodulin Figure 8 For the 2D structure diagram of the interaction between palmitic acid and tissue factor Figure 9 For the 2D structure diagram of the interaction between palmitic acid and antithrombin-III Among them, Figures 4 to 6 For the 3D structure diagram of the key antithrombotic protein and the schematic diagram of its binding sites, Figures 7 to 9 For the 2D structure diagram of the interaction between palmitic acid and the key antithrombotic protein Specific implementation manners

[0031] The following further describes the preparation method of citrus peel essential oil rich in antithrombotic active components of the present invention in conjunction with the accompanying drawings and specific implementation manners, but the protection scope of the present invention is not limited to the above content. Examples

[0032] The preparation method of kumquat peel essential oil described in this example specifically includes the following steps: Crush the peel with a pulverizer, weigh 20 g of the powder with a spatula, and place it in a 1000 ml round-bottom flask.

[0033] Add 500 mL of distilled water to the round-bottom flask and soak for 10 h.

[0034] Set up a portable simultaneous distillation and extraction device, add 10 ml of n-hexane to the organic solvent layer, and heat for reflux for 6 h.

[0035] Separate the n-hexane layer, and take 2 g of anhydrous sodium sulfate to filter and dehydrate the sample.

[0036] Use a rotary evaporator to remove n-hexane at 40 °C, and the rotation speed is increased from 20 rpm to 80 rpm slowly.

[0037] The essential oil yield is 2.32 ± 0.16%. Examples

[0038] The preparation method of ponkan peel essential oil described in this example specifically includes the following steps: Crush the peel with a pulverizer, weigh 20 g of the powder with a spatula, and place it in a 1000 ml round-bottom flask.

[0039] Add 600 mL of distilled water to the round-bottom flask and soak for 12 h.

[0040] Set up a portable simultaneous distillation extraction device, add 10 ml of n-hexane to the organic solvent layer, and heat under reflux for 7 h.

[0041] Separate the n-hexane layer, and take 1 g of anhydrous sodium sulfate to filter the sample for dehydration.

[0042] Use a rotary evaporator to remove n-hexane at 35 °C, with the rotation speed increasing gradually from 20 rpm to 80 rpm.

[0043] The essential oil yield is 2.30 ± 0.26%. Example

[0044] The preparation method of ponkan citrus peel essential oil described in this example specifically includes the following steps: Crush the fruit peel with a pulverizer, weigh 20 g of the powder with a spatula, and place it in a 1000 ml round-bottom flask.

[0045] Add 400 mL of distilled water to the round-bottom flask and soak for 11 h.

[0046] Set up a portable simultaneous distillation extraction device, add 10 ml of n-hexane to the organic solvent layer, and heat under reflux for 5 h.

[0047] Separate the n-hexane layer, and take 1.5 g of anhydrous sodium sulfate to filter the sample for dehydration.

[0048] Use a rotary evaporator to remove n-hexane at 30 °C, with the rotation speed increasing gradually from 20 rpm to 80 rpm.

[0049] The essential oil yield is 2.26 ± 0.21%. Example

[0050] The detection method of the active components in kumquat peel essential oil described in this example specifically includes the following steps: GC-MS detection conditions Use a Shimadzu GCMS-2010Ultra gas chromatograph-mass spectrometer, with an HP-5 type capillary column (30 m × 0.25 mm × 0.25 µm). The initial temperature is 80 °C and is held for 2 min; it is heated to 200 °C at a rate of 4 °C / min and held for 5 min; finally, it is heated to 280 °C at a rate of 20 °C / min and held for 2 min. The carrier gas is high-purity helium with a flow rate of 1 mL / min. Split injection is used with a split ratio of 50:1. The inlet temperature is 280 °C. Set the electron energy of the electron impact (EI) ion source to 70 eV; the electron multiplier voltage is 1.00 kV, and the mass scanning range is 30 - 550 m / z. The ion source temperature is 250 °C, and the interface temperature is 250 °C.

[0051] The results of the total ion current chromatogram of kumquat peel essential oil detected by GC-MS are shown in Figure 2, the components obtained by GC-MS extraction were subjected to structural identification, and further screened, retrieved and analyzed in the information database, and the content was calculated by external standard. The oxygen-containing active components and contents of kumquat peel essential oil are shown in Table 1.

[0052] Table 1 Oxygen-containing active components and contents of ponkan peel essential oil Number Active ingredient CAS number Component content (mg / kg) 1 Neryl acetate 141-12-8 3.51 2 Lavandulyl acetate 25905-14-0 3.01 3 Diisobutyl phthalate 84-69-5 3.40 4 Vinyl 10 - undecenoate 5299-57-0 27.10 5 Methyl linoleate 112-63-0 6.38 6 Octanal 124-13-0 1.79 7 Neral 106-26-3 1.72 8 Citral 5392-40-5 2.30 9 Perillaldehyde 2111-75-3 5.52 10 2 - Pentyl - 2 - aldehyde 3021-89-4 20.41 11 β - Isophorone 471-01-2 1.86 12 (-)-Carvone 6485-40-1 4.45 13 Methyl benzylideneacetone 80-57-9 3.78 14 Nootkatone 4674-50-4 20.69 15 Sinensetin 2306-27-6 11.01 16 Linoleic acid 60-33-3 5.87 17 p - Vinylguaiacol 7786-61-0 25.31 Example

[0053] The detection method for active components in the ponkan peel essential oil described in this example specifically includes the following steps: GC-MS detection conditions A Shimadzu GCMS-2010Ultra gas chromatograph-mass spectrometer was used. The chromatographic column was an HP-5 type capillary column (30 m × 0.25 mm × 0.25 µm). The initial temperature was 80 °C and it was held for 2 min; it was heated to 200 °C at a rate of 4 °C / min and held for 5 min; finally, it was heated to 280 °C at a rate of 20 °C / min and held for 2 min. The carrier gas was high-purity helium with a flow rate of 1 mL / min. Split injection was used with a split ratio of 50:1. The inlet temperature was 280 °C. The electron energy of the electron impact (EI) ion source was set to 70 eV; the electron multiplier voltage was 1.00 kV, and the mass scanning range was 30 - 550 m / z. The ion source temperature was 250 °C and the interface temperature was 250 °C.

[0054] The results of the total ion current chromatogram of ponkan peel essential oil detected by GC-MS are shown in Figure 3 , the components obtained by GC-MS extraction were subjected to structural identification, and further screened, retrieved and analyzed in the information database, and the content was calculated by external standard. The oxygen-containing active components and contents of ponkan peel essential oil are shown in Table 2.

[0055] Number Active ingredient CAS number Component content (mg / kg) 1 Neryl acetate 141-12-8 1.64 2 Lavandulyl acetate 25905-14-0 2.06 3 Methyl linolenate 301-00-8 3.01 4 Ethyl palmitate 628-97-7 11.33 5 Ethyl linolenate 1191-41-9 43.87 6 Ethyl oleate 111-62-6 2.64 7 p - Mentha - 1 - en - 9 - aldehyde 29548-14-9 1.38 8 2 - Pentyl - 2 - aldehyde 3021-89-4 2.76 9 Nootkatone 4674-50-4 9.80 10 Palmitic acid 57-10-3 34.48 11 Linoleic acid 60-33-3 17.27 12 Linolenic acid 463-40-1 31.11 Example

[0056] The exploration of anti-thrombotic active components in the kumquat peel essential oil described in this example specifically includes the following steps: Treatment of small molecule ligands Open http: / / www.chemspider.com / Default.aspx. Enter the name or CAS number of the active components of kumquat peel essential oil, search and download the 3D structures of the oxygen-containing active components in kumquat peel essential oil, and use the Open Babel GUI software to perform small molecule ligand format transcoding, and output it in mol2 format for subsequent docking experiments with key anti-thrombotic proteins.

[0057] Use the Discovery Studio 2019 Client software to preprocess the small molecule ligands to minimize their energy and save them in dsv format.

[0058] Screening and processing of key antithrombotic protein molecules Open https: / / www.genecards.org / , enter "Antithrombotic" for keyword search to obtain the UniProt ID of the key antithrombotic protein molecule. Enter the UniProt ID in https: / / www.uniprot.org / , select the target protein molecule, and save its 3D structure in ent format. Information on the key antithrombotic protein molecules is shown in Table 3.

[0059] Serial number Protein name Protein name UniPort ID 1 Thrombomodulin Thrombomodulin P07204 2 Tissue factor Tissue factor P13726 3 Antithrombin - III Antithrombin - III P01008 Import the receptor protein into the Discovery Studio 2019 Clie program, remove the excess small molecule ligands and water molecules in the protein, add hydrogen atoms, and perform "Clean Protein" so that it can dock with the small molecule ligand in the receptor active site region according to the principles of spatial structure complementarity and energy minimization. The 3D structure of the key antithrombotic active protein and its binding site are shown in Figure 7 .

[0060] Molecular docking experiment In this study, the Discovery Studio 2019 Clien software was used to perform semi-flexible docking with the key antithrombotic protein molecule as the receptor for this docking and the main component molecule of kumquat peel essential oil as the ligand for this docking. The process of molecular docking is a process of inputting energy when atoms bind, so the binding energy is negative, and the larger the absolute value, the stronger the binding. The results of the docking binding energy between the active components of kumquat peel essential oil and the key antithrombotic protein are shown in Table 4.

[0061] Table 4 Results of the docking binding energy between the active components of kumquat peel essential oil and the key antithrombotic protein Number Active ingredient CAS number -Binding energy P07204 -Binding energy P13726 -Binding energy P01008 1 Neryl acetate 141-12-8 -19.39 -14.76 -11.88 2 Lavandulyl acetate 25905-14-0 -10.89 -6.40 -6.17 3 Methyl linolenate 301-00-8 -8.24 -5.22 4.06 4 Ethyl palmitate 628-97-7 37.58 39.73 46.32 5 Ethyl linolenate 1191-41-9 -6.38 -0.53 7.30 6 Ethyl oleate 111-62-6 23.95 26.36 31.94 7 p - Mentha - 1 - en - 9 - aldehyde 29548-14-9 -5.58 -3.84 -1.85 8 2 - Pentyl - 2 - aldehyde 3021-89-4 11.15 15.49 18.38 9 Nootkatone 4674-50-4 -22.95 -15.49 -32.34 10 Palmitic acid 57-10-3 39.04 43.68 55.89 11 Linoleic acid 60-33-3 9.25 14.09 26.64 12 Linolenic acid 463-40-1 -7.21 -1.85 5.93 The components with a negative binding energy and an absolute value greater than 20 are the main contributing components to antithrombotic activity. From the results, it can be observed that ethyl palmitate, palmitic acid, and ethyl oleate in kumquat peel essential oil contribute significantly to antithrombotic activity. Especially palmitic acid, which has a high absolute value of the binding energy with 3 key antithrombotic proteins and has strong antithrombotic activity.

[0062] There is a literature study on the in vitro antithrombotic activity experiment of palmitic acid. The in vitro coagulation activity of blackberry seeds was studied using activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), and fibrinogen (FIB). The results are shown in Table 5.

[0063] Table 5 In vitro antithrombotic activity test of palmitic acid Component APTT(s) PT(s) TT(s) FIB(g / L) Palmitic acid 20.41±0.42 9.82±0.26 32.64±0.15 1.29±0.01 The in vitro experimental results show that palmitic acid has a strong coagulation function and is an important active component in antithrombotic activity. Further study the interaction mechanism between palmitic acid and key antithrombotic proteins. The interaction between ligand and receptor is a process of molecular recognition, mainly including electrostatic interaction, hydrogen bond interaction, hydrophobic interaction, van der Waals interaction, etc. The 2D structure diagram of the interaction between palmitic acid and key antithrombotic proteins is shown in Figure 5 。

[0064] Obviously, the above embodiments are only examples for clear illustration and not limitations on the implementation manners. Any person skilled in the art can make some changes or modifications to equivalent embodiments with equivalent changes within the scope of the technical solution of the present invention without departing from the technical solution of the present invention. However, any simple modification, equivalent change, and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. The present invention provides an efficient method for mining antithrombotic active components in orange peel essential oil. The method uses the peel of citrus plants as raw materials and extracts oxygen-containing compounds such as esters, acids, aldehydes and phenols through a convenient synchronous distillation extraction technology. These compounds have significant antithrombotic activity. Subsequently, these active ingredients are qualitatively and quantitatively analyzed using gas chromatography-mass spectrometry (GC-MS), and the antithrombotic mechanism of citrus peel essential oil is deeply studied through computer simulation experiments.

2. As described in claim 1, the method adopts a convenient simultaneous distillation extraction technology, and the device consists of a round-bottom flask, an extractor and a condenser. The device combines steam distillation and solvent extraction to form a structure of an upper organic solvent layer and a lower water layer.

3. As described in claim 1, the method uses an HP-5 capillary column (30 m×0.25 mm×0.25 µm) for analysis; the carrier gas is high-purity helium, the flow rate is set to 0.8 mL / min, the split ratio is 50:1, and the injection port temperature is 280°C; the mass spectrometer interface temperature is 250°C, the ionization mode is an electron impact source (EI), the ion energy is 70eV, and the ion source temperature is 250°C. The initial temperature is set to 80°C and maintained for 2 minutes; then the temperature is increased to 200°C at a rate of 4°C / min and maintained for 5 minutes; finally, the temperature is increased to 280°C at a rate of 20°C / min and maintained for 2 minutes.

4. As described in claim 1, the method uses molecular docking technology to select active proteins that control blood coagulation and inhibit thrombosis, and docks the oxygen-containing active components obtained by quantitative analysis with anti-thrombotic proteins to explore the anti-thrombotic active components of citrus peel essential oil.

5. As described in claim 1, the steps of extracting, detecting and mining the antithrombotic active components of the sample in the method are as follows: (1) After the peel sample is crushed with a grinder, an appropriate amount of peel powder is weighed and placed in a round-bottom flask, distilled water is added, and the mixture is placed at room temperature. (2) A convenient synchronous distillation extraction device is built, distilled water is added to the lower layer, and an organic solvent is added to the upper layer. After heating to boiling, the distillation is continued. (3) The organic solvent layer is separated, and the sample is filtered using anhydrous sodium sulfate to dehydrate. (4) The organic solvent is removed using a rotary evaporator to obtain the peel essential oil. (5) The obtained peel essential oil is used for GC-MS detection and analysis. (6) Key antithrombotic proteins are screened as macromolecular receptors. (7) Molecular docking experiments are conducted between the active components of citrus peel essential oil and macromolecular receptors.

6. As described in claim 5, in the steps of extracting, detecting and mining the antithrombotic active components of the sample in the method, in step (1), the ratio of peel powder to distilled water is 1:20-1:30, the mass unit of peel powder is gram (g), the volume unit of distilled water is milliliter (mL), and the time of standing at room temperature is 10-12 hours (h). In step (2), the organic solvent is n-hexane, and the distillation time is 5-7 hours. In step (3), the ratio of organic solvent layer to anhydrous sodium sulfate is 20:1-10:1, the volume unit of organic solvent layer is milliliter, and the mass unit of anhydrous sodium sulfate is gram. In step (4), the speed of the rotary evaporator is from slow to fast, the speed range is 20-80 revolutions per minute (rpm), and the water bath temperature is 30-40°C. In step (5), GC-MS uses the external standard method for quantitative analysis, and the specific experimental conditions are as described in claim 3. In step (6), three active proteins that control coagulation and inhibit thrombosis are screened: thrombomodulin (P07204), tissue factor (P13726), and antithrombin-III (P01008). In step (7), the molecular docking experiment mainly uses Discovery Studio software, with the main active components of the essential oil as docking ligands and the antithrombotic key protein molecules as docking receptors, and semi-flexible docking is performed to obtain the binding energy data between the ligand and the receptor, and further analyze the antithrombotic activity of orange peel essential oil.

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