Application of platycladus orientalis essential oil in field of anti-oxidation and immunity-enhancing medicines / foods

Abstract

The invention discloses an application of platycladus orientalis essential oil in the field of anti-oxidation and immunity-enhancing medicines / foods, and belongs to the technical field of food health care, the main component of the adopted platycladus orientalis essential oil is cedrol, the relative content is 33.7%, and other components comprise esters, cuparene, thujopsene and the like. The administration dosage is determined through an acute toxicity test, and the platycladus orientalis essential oil is determined to have the functions of resisting oxidation and enhancing immunity by measuring the content of SOD, GSH, protein carbonyl and lipid oxidation product MDA in animal tissues and through an immune experiment.

Description

Technical Field

[0001] The invention belongs to the technical field of food health care, and particularly relates to application of Platycladus orientalis essential oil in the field of anti-oxidation and immunity-enhancing medicines / foods. Background Art

[0002] Under normal circumstances, free radicals in the human body are in a dynamic balance between generation and removal. However, as the body ages, the rate of free radical removal may slow down, disrupting the dynamic balance and causing pathological changes in the body. Therefore, supplementing with an appropriate amount of antioxidants is beneficial to maintaining the body's health. Subhealthy conditions are mainly manifested as fatigue, lack of concentration, and reduced immunity. Improving immunity can be used as a way to prevent subhealth. Improving immunity can enhance the body's immune defense function and maintain the body's immune homeostasis. Timely supplementation of immunity-enhancing substances can help maintain the body's health.

[0003] Therefore, it is very necessary to provide a food that can improve immunity. Summary of the invention

[0004] The invention aims to provide application of Platycladus orientalis essential oil in anti-oxidation and immunity enhancement.

[0005] To achieve the above object, the scheme of the present invention is as follows:

[0006] The invention provides application of Platycladus orientalis essential oil in the field of anti-oxidation and immunity-enhancing medicines / foods.

[0007] Furthermore, the arborvitae essential oil includes the following components in relative content: 33.677% of cedarwood alcohol, esters, thujacene and thujacene.

[0008] Further, the arborvitae essential oil specifically includes the following components in relative content: 33.677% of cedar alcohol, 11.503% of 2.10.10-Trimethyllric yclo|7.1.1.0[2,7]|undec-6-en-8-one, 14.125% of 1-(1,3-benzodioxol-5-ylmethyl)-2-oxocyclopentanecarboxylic acid ethyl ester, 9.365% of thujapentene, 6.982% of thujapentene, 4.096% of 2,6'-dimethyl-5-isopropyl-2,6'-[7]oxabicyclo[4.1.0]heptane]-5'-one, 2.347% of β-Vatirenene, 3.432% of α-cedarene, 3.401% of Aromadendrene oxide-[1], 2.103% Aristolene epoxide, 1.235% β-cedrene.

[0009] Furthermore, the drug delivery preparation includes the following components by volume fraction: 1% Tween 80, 0.25% to 1% Thuja orientalis essential oil and 98% to 98.75% saline. Among them, Tween 80 is polysorbate 80. The proportion of Thuja orientalis essential oil in the drug delivery preparation is adjusted to 0.25%, 0.5% and 1% (vt%) according to low, medium and high doses. Specifically, the amount of Thuja orientalis essential oil is 0.25%, 0.5% or 1% (vt%); correspondingly, the amount of saline is 98.75%, 98.5% or 98% (vt%).

[0010] Furthermore, the drug preparation is first subjected to ultrasonic treatment before use to ensure that the drug preparation is fully mixed.

[0011] Furthermore, the preparation method of the Thuja orientalis essential oil includes: crushing fresh Thuja orientalis wood into 1-2 mm fragments to increase the surface area and improve the extraction efficiency; then drying the Thuja orientalis wood to remove excess moisture to prevent moisture from affecting the extraction efficiency during the extraction process; further crushing the Thuja orientalis wood to a particle size of 0.1-0.5 mm to obtain pretreated Thuja orientalis wood, and storing it in a cool place for standby use; setting the extraction conditions to a pressure of 20 MPa and a temperature of 50°C, and extracting the pretreated Thuja orientalis wood for 65 minutes using a supercritical CO2 extraction method to obtain the Thuja orientalis essential oil.

[0012] Furthermore, the Platycladus orientalis essential oil is used to reduce protein carbonyl levels, increase antioxidant enzyme activity and improve glutathione content.

[0013] Furthermore, the Platycladus orientalis essential oil is used to promote delayed-type allergic reaction and enhance the activity of natural killer cells.

[0014] Compared with the prior art, this application has the following beneficial effects:

[0015] The present invention provides an application of oriental arborvitae essential oil in the field of anti-oxidation and immunity-enhancing drugs / foods. The main component of the oriental arborvitae essential oil is cedarwood alcohol, with a relative content of 33.7%, and other components include esters, thujacene, thujacene and the like. The present invention determines the dosage of the oriental arborvitae essential oil through an acute toxicity test, and determines that the oriental arborvitae essential oil has the function of anti-oxidation and immunity-enhancing functions through the determination of the content of SOD, GSH, protein carbonyl and lipid oxidation product MDA in animal tissues and immune experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the survival of mice within 7 days in the acute toxicity test;

[0017] Figure 2 This is the change in mouse body weight within 7 days in the acute toxicity test;

[0018] Figure 3 The weight changes of mice during 30 days of oral administration of essential oils;

[0019] Figure 4 The results of the determination of the antioxidant index malondialdehyde (MDA) content in different treatment groups after intragastric administration;

[0020] Figure 5 The results of the determination of protein carbonyl content, an antioxidant index in different treatment groups after intragastric administration;

[0021] Figure 6 The results of the determination of superoxide dismutase (SOD) enzyme activity, an antioxidant index, in different treatment groups after intragastric administration;

[0022] Figure 7 The results of the determination of glutathione (r-glutamylcysteingl+glycine, GSH) content in different treatment groups after intragastric administration;

[0023] Figure 8 Body weight changes of mice during oral administration of Platycladus orientalis essential oil in the immune experiment. DETAILED DESCRIPTION

[0024] Platycladus orientalis, a plant of the genus Platycladus in the Cupressaceae family, is widely distributed in southern Inner Mongolia, Liaoning, Yunnan, Hubei and other regions in my country. Platycladus orientalis, also known as yellow withered, fragrant cedar, flat shoot, flat inspection, fragrant tree, etc. In addition to being used directly as a wood raw material, Platycladus orientalis still has great application value in other aspects. Platycladus orientalis extracts have pharmacological activities such as antibacterial, antioxidant and anti-tumor activities.

[0025] Essential oils are concentrated liquids extracted from different parts of plants, containing the essence of plants. Currently, many kinds of essential oils are widely used as cosmetics or health products. Lavender essential oil can promote skin cell regeneration and repair dry and damaged skin. Tea tree essential oil has natural antibacterial and anti-inflammatory effects. Platycladus orientalis essential oil has anti-insect, antibacterial, antioxidant and anti-cancer effects, and has broad application prospects in pesticides, preservatives and health products.

[0026] The inventors found that the main components of Platycladus orientalis essential oil include natural ingredients such as cedarwood alcohol, thujacene, thujacene, and cedrene, which have a woody aroma and insecticidal and antibacterial effects. Platycladus orientalis essential oil is extracted from Platycladus orientalis plants and may contain natural antioxidants. In order to expand its role as a health food, its application in anti-oxidation and immunity enhancement was explored.

[0027] The present application is further described in detail below in conjunction with the examples. It is particularly noted that if no specific conditions are specified in the following examples, the experiments are carried out according to conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, the raw materials used in the following examples can be obtained from common commercial sources.

[0028] Example 1

[0029] Preparation process of arborvitae essential oil:

[0030] Supercritical CO2 extraction was used to extract the essential oil of Platycladus orientalis. Fresh Platycladus orientalis wood was crushed into 1-2 mm fragments to increase the surface area and improve the extraction efficiency. The Platycladus orientalis wood was dried to remove excess moisture to prevent moisture from affecting the extraction efficiency during the extraction process; the wood was further crushed to a particle size of 0.1-0.5 mm and stored in a cool place. According to the results of the literature review, the extraction conditions were set to a pressure of 20 MPa and a temperature of 50 ° C for 65 minutes. The extracted essential oil was used for subsequent experiments.

[0031] Example 2

[0032] Analysis of components of Platycladus orientalis essential oil:

[0033] GC-MS detection method: The essential oils were prepared into solutions with a mass concentration of 0.04 g / mL using n-hexane (chromatographic grade), and the essential oils of Platycladus orientalis were analyzed by gas chromatography-mass spectrometry using GC-MS (Agilent, USA).

[0034] The above analysis and detection methods were used to analyze the components of Platycladus orientalis essential oil, and its volatile components are shown in Table 1.

[0035] Table 1 Volatile components in Platycladus orientalis essential oil

[0036]

[0037] As can be seen from Table 1, the highest relative content is cedarwood alcohol at 33.677%, followed by ester components and terpenes such as thujase. These components often have a relatively fragrant plant aroma and woody aroma, without irritating odor, and smell more comfortable.

[0038] It should be noted that the relative content is the peak area of ​​a compound divided by the sum of all integrated peak areas, which is its relative content in percentage.

[0039] Example 3

[0040] The dosage needs to be determined before conducting in vivo experiments, and the median lethal dose is determined through acute toxicity tests to screen the appropriate dosage.

[0041] LD50 50Value determination: 70 healthy adult Balb / c male mice (6-8 weeks old), weighing 17-22g, were randomly divided into seven groups, 10 mice in each group. Fasting but not water for 12h, one-time oral administration of Platycladus orientalis essential oil 10mL / kg. According to the preliminary experimental data, six dose groups (0.116g / kg, 0.231g / kg, 0.462g / kg, 0.924g / kg, 1.848g / kg, 3.696g / kg) and a solvent control group were set up, and the survival rate of mice within 7d was continuously observed and recorded. Since the essential oil is oily and relatively viscous, it was emulsified with Tween 80 and prepared into an emulsion for subsequent oral administration. The specific emulsion prescription is shown in Table 2.

[0042] Table 2 Preparation prescription

[0043]

[0044] As can be seen from Table 2, the dosages of DMSO and Tween 80 selected in the preparation prescriptions were within a reasonable range, and a solvent control group was set up to determine the toxic effects of the excipients on mice; the ratio between the doses of Platycladus orientalis essential oil was maintained at 0.5:1; the preparation prescription groups 1-6 corresponded to 0.116 g / kg, 0.231 g / kg, 0.462 g / kg, 0.924 g / kg, 1.848 g / kg, and 3.696 g / kg dosage groups, respectively.

[0045] The survival of mice was observed within 7 days and LD was calculated 50 The survival of mice and the changes in body weight within 7 days are shown in Figure 1 , Figure 2 Chinese data. Figure 1 The data showed that mice survived after being gavaged with 0.924g / kg or less of Platycladus orientalis essential oil, only half of the mice survived after being given 1.848g / kg, and all mice died on the second day after being given the highest dose, indicating that this dose was highly toxic. The weight of mice did not change significantly within 7 days. The LD50 of Platycladus orientalis essential oil was obtained by modified Koch method. 50 Calculate the LD based on the dosage and animal survival. 50 The value is 0.864g / kg, and the 95% confidence interval is 0.636-1.091g / kg. The median lethal dose LD of Platycladus orientalis essential oil 50 The subsequent dosing doses were set at 1 / 30 (28.8 mg / kg), 1 / 60 (14.4 mg / kg), and 1 / 120 (7.2 mg / kg) LD 50 The high, medium and low doses were administered orally for 30 days.

[0046] Example 4

[0047] Antioxidant function of arborvitae essential oil:

[0048] The mice were modeled with ethanol oxidative damage. Excessive intake of ethanol will activate oxygen molecules to produce free radicals, which will lead to cellular peroxidation and depletion of reduced glutathione in the body. This experiment observed the degree of peroxidation and reduced glutathione concentration in the model control group and the drug-treated group to determine the antioxidant capacity of the samples.

[0049] Balb / c healthy adult male mice (6-8 weeks old) were selected and the experiment was started after one week of adaptation to the experimental environment. The mice were randomly divided into five groups, 10 in each group, namely, blank no-drug group, model group, low-dose group (7.2 mg / kg), medium-dose group (14.4 mg / kg) and high-dose group (28.8 mg / kg). The drug was administered by gavage continuously for 30 days. After the last administration, the model group and the three dose groups fasted for 16 hours, and then gavaged with 50% ethanol 12 mL / kg once. The samples were taken after 6 hours. The blank group did not need to fast to take the samples. The liver was taken to determine the content of lipid oxidation product malondialdehyde (MDA), protein carbonyl content, reduced glutathione (GSH) content, and antioxidant enzyme (superoxide dismutase SOD) activity.

[0050] Balb / c mice were gavaged continuously for 30 days, and the mice were weighed every five days during the administration period. Figure 3 The results showed that there was no decreasing trend in body weight, indicating that administration of Platycladus orientalis essential oil did not affect the body weight of mice.

[0051] All experimental mice were killed by cervical dislocation, and their livers were obtained for tissue homogenization.

[0052] MDA is one of the end products of cell membrane lipid peroxidation. The degree of lipid peroxidation can be indirectly reflected by measuring its content. MDA and TBA (thiobarbituric acid) molecules are co-heated under acidic conditions to form a pink complex, and the absorbance is measured at 532nm. The determination of MDA content in liver tissue is carried out according to the specified operation of the MDA kit. Figure 4 The results showed that compared with the blank group, the MDA content in the model group increased, indicating that acute alcohol intoxication induced oxidative damage in mice. The MDA results of the mouse tissues in the drug-treated group showed that the administration of essential oils could not reduce the generation of lipid oxidation products in the tissues.

[0053] Protein carbonyl content can directly reflect the degree of protein damage. Protein carbonyl formation is an early sign of many amino acids in the process of protein oxidation modification, and the content increases with age. The carbonyl group of oxidized protein increases. The carbonyl group is re-dissolved by reacting the carbonyl group with 2,4-dinitrophenylhydrazine to produce a precipitate, and then its absorbance at 370nm is measured to determine the protein carbonyl content. Figure 5The results showed that compared with the blank group, the protein carbonyl content in the model group increased, indicating that acute alcohol intoxication induced oxidative damage in mice. High-dose essential oil administration significantly reduced the protein carbonyl content to the level of normal mice.

[0054] The content of SOD in certain organs of animals and human red blood cells has obvious changes with age, and the enzyme activity is inversely proportional to the increase of biological age. The ability to eliminate free radicals is proportional to the enzyme activity. The final product of O2·ˉ oxidation of hydroxylamine is nitrite, which appears purple-red under the action of p-aminobenzenesulfonic acid and naphthylamine, and has a maximum absorption peak at a wavelength of 530nm. It can be measured by spectrophotometry. When SOD eliminates O2·ˉ, the nitrite formed decreases. Figure 6 The results showed that compared with the blank group, the SOD enzyme activity in the model group was significantly reduced, indicating that acute alcohol intoxication induced oxidative damage in mice, indicating that the model was successful. The administration of essential oils could increase the SOD enzyme activity, indicating that essential oils have the effect of increasing the activity of antioxidant enzymes.

[0055] Glutathione is a low molecular weight scavenger that can remove O2ˉ, H2O2, and LOOH. It is the main non-protein sulfhydryl compound in tissues. It can stabilize sulfhydryl-containing enzymes and prevent hemoglobin and other auxiliary factors from being damaged by oxidation. The lack or depletion of GSH will cause many chemicals or environmental factors to produce poisoning effects. The amount of GSH is an important factor in measuring the body's antioxidant capacity. GSH and 5,5'-dithio-p-nitroformic acid (DTNB) react under the catalysis of GSH-Px to generate yellow 5-thio-2-nitroformate anion, which has a maximum absorption peak at a wavelength of 420nm. By measuring the concentration of this ion, the content of GSH can be calculated. Figure 7 The results showed that the GSH content in the model group decreased compared with the blank group, indicating that acute alcohol intoxication induced oxidative damage in mice. GSH content was significantly increased by administration of low-dose essential oils. See Table 3.

[0056] Table 3 Results

[0057]

[0058] As can be seen from Table 3, according to the results of the antioxidant part in the "Health Food Function Test and Evaluation Method (2023 Edition)", three of the four indicators of lipid oxidation products, protein oxidation products, antioxidant enzymes, and antioxidant substances are positive, and it can be determined that the test sample is positive for the antioxidant animal test results. Combined with the above experimental results, it can be seen that Platycladus orientalis essential oil can play an antioxidant role in the body by reducing protein carbonyl levels, increasing antioxidant enzyme activity, and increasing GSH content.

[0059] Example 5

[0060] Immune-boosting properties of arborvitae essential oil:

[0061] Reagent preparation:

[0062] Sheep red blood cells (SRBC): Take defibrinated sheep blood and store it at 4°C. Shake it well every day. Prepare it to the corresponding concentration with physiological saline or SA buffer.

[0063] Complete culture medium: Use sterile RMPI1640 culture medium, add 10% FBS, penicillin (100U / mL), and streptomycin (100μg / L) before use, and adjust the pH to 7.0-7.2.

[0064] ConA solution: Prepare a 0.1 mg / mL solution with double distilled water, filter and sterilize, and store at -20°C until use.

[0065] MTT solution: Dissolve MTT in physiological saline to prepare 5 mg / mL MTT solution, prepare it before use.

[0066] Acidic isopropanol: Add 4 mL of 1M HCl to 96 mL of isopropanol. Prepare immediately before use.

[0067] DNFB solution: weigh 50 mg of DNFB and dissolve it in 5 mL of acetone sesame oil solution (v / v=1:1), prepare it before use.

[0068] Ink for injection: Dilute the India ink stock solution 4 times with normal saline before use. Prepare it before use.

[0069] Na2CO3 solution: Take 0.1g Na2CO3 and add distilled water to 100mL.

[0070] Preparation of chicken erythrocyte suspension: Take defibrinated chicken blood, wash it with physiological saline for 2-3 times, centrifuge it at 2000r / min for 10min, discard the supernatant, and prepare a 20% (v / v) chicken erythrocyte suspension with physiological saline.

[0071] Preparation of LDH matrix solution: 50 mM lithium lactate, 0.66 mM iodononitroblue tetrazolium chloride (INT), 0.28 mM phenazine dimethyl sulfate (PMS), 1.3 mM oxidized coenzyme I (NAD), and the above reagents were dissolved in 0.2 M Tris-HCl buffer (pH 8.2).

[0072] Determination of organ / body weight ratio: The mice were weighed and then killed, and the thymus and spleen were taken out and weighed to calculate the organ / body weight ratio. Figure 8As shown, the weight of mice in each dosage group increased, and there was no significant difference compared with the control group (P>0.05), indicating that Platycladus orientalis essential oil had no significant effect on the weight gain of mice; the organ coefficients of the immune organs of mice 30 days after oral administration of different doses of Platycladus orientalis essential oil to mice are shown in Table 4.

[0073] Table 4 Effects of Platycladus orientalis essential oil on organ coefficients of mice ( n=10)

[0074]

[0075] As can be seen from Table 4, there was no significant difference in the thymus coefficient and spleen coefficient of mice in each dosage group compared with the control group (P>0.05), indicating that Platycladus orientalis essential oil had no significant effect on the quality of immune organs of mice.

[0076] 1. Cellular immune function assay:

[0077] (1) ConA-induced mouse spleen lymphocyte transformation experiment (MTT method)

[0078] After continuous gavage for 30 days, the mice were killed by dislocating the cervical vertebrae, and the spleen of the mice was aseptically removed and placed in a six-well plate containing an appropriate amount of sterile Hank's solution. The spleen was gently ground with a 5mL syringe pusher to make a single cell suspension. The filtrate was collected after filtering through a 200-mesh sieve, and then washed twice with Hank's solution, centrifuged at 1000r / min for 10min each time. The cells were resuspended in RMPI1640 complete medium, and the number of live cells was counted by trypan blue staining, and the cell density was adjusted to 3 million / mL. The cell suspension was then added to two wells of a 24-well plate, 1mL per well, 75μL ConA solution was added to one well, and the other well was used as a control, and incubated in a 5% CO2, 37°C incubator for 72h. 4 hours before the end of the culture, 700 μL of supernatant was discarded from each well, 700 μL of RMPI1640 basal medium (without fetal bovine serum) was added, and 50 μL / well of MTT (5 mg / mL) was added to each well. After continuing to culture for 4 hours, 1 mL of acidic isopropanol was added to each well, and the purple crystals at the bottom were completely dissolved by blowing and mixing. Then it was divided into 96-well plates, and multiple parallel groups were set in each well. The optical density (OD) was measured at 570 nm with the help of an enzyme reader. The proliferation ability of lymphocytes was expressed by subtracting the optical density of the well without ConA from the optical density of the well with ConA. The results are shown in Table 5.

[0079] Table 5 Effect of Platycladus orientalis essential oil on ConA-induced transformation ability of mouse spleen lymphocytes ( n=10)

[0080]

[0081] As can be seen from Table 5, there was no significant difference in the proliferation ability of mouse lymphocytes in each dose group of Platycladus orientalis essential oil compared with the control group (P>0.05), indicating that Platycladus orientalis essential oil had no significant effect on the transformation ability of mouse spleen lymphocytes induced by ConA.

[0082] (2) Dinitrofluorobenzene-induced delayed-type hypersensitivity (DTH) reaction in mice (ear swelling method)

[0083] After continuous gavage for 30 days, the abdominal skin of the mice was depilated, with an area of ​​about 3cm×3cm, and 50μL of DNFB solution was evenly applied to the depilated area of ​​the abdomen for sensitization. After 5 days of sensitization, 10μL of DNFB solution was evenly applied to the right ear (both sides) of the mice for attack. 24 hours after the attack, the mice were killed by cervical dislocation, the left and right ear shells were cut off, and the ear pieces with a diameter of 8mm were taken with a punch and weighed. The difference in the weight of the left and right ears was used to represent the degree of DTH. The results are shown in Table 6.

[0084] Table 6 Effect of Platycladus orientalis essential oil on delayed-type hypersensitivity (DTH) in mice ( n=10)

[0085]

[0086] Note: Compared with the control group, *P<0.05, **P<0.01

[0087] As can be seen from Table 6, the difference in the weight of the left and right ear pieces of mice in the low and medium dose groups of Platycladus orientalis essential oil was significantly higher than that in the control group (*P<0.05, **P<0.01), indicating that Platycladus orientalis essential oil has the effect of promoting delayed-type hypersensitivity in mice.

[0088] 2. Humoral immune function test:

[0089] Determination of serum hemolysin (hemagglutination method)

[0090] Take defibrinated sheep blood, wash it three times with saline, and centrifuge it at 2000r / min for 10min each time. Prepare a 2% (v / v) cell suspension of packed SRBC with saline, and inject 0.2mL into the mouse intraperitoneal cavity for immunization. 5 days after immunization, remove the eyeball and collect blood in a centrifuge tube. After standing at room temperature for 1h, centrifuge at 4℃, 2000r / min, 10min to collect serum. Dilute the serum in multiples with saline, and place the serum of different dilutions in a micro-hemagglutination test plate, 100μL per well, and then add 100μL of 0.5% (v / v) SRBC suspension, mix well, put into a moist flat plate, cover, and incubate in a 37℃ incubator for 3h, observe the degree of hemagglutination, and calculate the anti-volume number.

[0091] Anti-volume number = (S1+2S2+3S3...nS n )

[0092] Wherein 1, 2, 3...n is the index of multiple dilution, and S is the level of agglutination.

[0093] The results are shown in Table 7.

[0094] Table 7 Effect of Platycladus orientalis essential oil on serum hemolysin in mice ( n=10)

[0095]

[0096] As can be seen from Table 7, there was no significant difference in the anti-volume of mice in each dose group of Platycladus orientalis essential oil compared with the control group (P>0.05), indicating that Platycladus orientalis essential oil had no significant effect on mouse serum hemolysin.

[0097] 3. Function of monocytes and macrophages

[0098] (1) Carbon clearance experiment in mice

[0099] After 30 days of oral gavage, mice were injected with four times diluted Indian ink (10 mL / kg) with physiological saline through the tail vein. 2 and 10 minutes after the injection of the ink, blood was collected from the inner canthus of the mouse eye and immediately added to 2 mL of 0.1% Na2CO3 solution. The Na2CO3 solution was used as a blank control, and the optical density (OD) was measured at a wavelength of 600 nm using an enzyme marker. The results are shown in Table 8.

[0100] Table 8 Effect of Platycladus orientalis essential oil on carbon clearance of mouse monocytes and macrophages ( n=10)

[0101]

[0102] As can be seen from Table 8, there was no significant difference in the phagocytic index of mice in each dose group of Platycladus orientalis essential oil compared with the control group (P>0.05), indicating that Platycladus orientalis essential oil had no significant effect on the carbon clearance function of mouse monocyte-macrophages.

[0103] (2) Experiment on phagocytosis of chicken red blood cells by mouse peritoneal macrophages (ex vivo method)

[0104] 30 days after oral gavage, mice were intraperitoneally injected with 1 mL of 20% chicken red blood cell suspension. 1.5 hours later, the mice were killed by cervical dislocation, fixed in a supine position on a mouse board, the abdominal skin was cut open, 2 mL of normal saline was injected through the peritoneal cavity, the abdomen was gently rubbed, 1 mL of peritoneal washing fluid was taken out, and dropped on two slides, respectively, and placed in an enamel box with wet gauze, and incubated in a 37°C incubator for 30 minutes. The slides were rinsed with normal saline and dried, fixed with a 1:1 acetone-methanol solution, stained with 4% Giemsa-phosphate buffer for 3 minutes, then rinsed with distilled water and dried, macrophages were counted under an oil immersion lens, and the phagocytic percentage and phagocytic index were calculated as follows.

[0105]

[0106] The results are shown in Table 9.

[0107] Table 9 Effect of Platycladus orientalis essential oil on the ability of mouse peritoneal macrophages to phagocytose chicken red blood cells ( n=10)

[0108]

[0109] As can be seen from Table 9, there were no significant differences in the phagocytic percentage and phagocytic index of mice in each dose group of Platycladus orientalis essential oil compared with the control group (P>0.05), indicating that Platycladus orientalis essential oil had no significant effect on the ability of mouse peritoneal macrophages to phagocytose chicken red blood cells.

[0110] 4. Natural killer cell (NK cell) activity

[0111] 30 days after oral administration, mice were killed by cervical dislocation, and their spleens were removed aseptically and placed in a six-well plate containing an appropriate amount of sterile Hank's solution. The spleen was gently ground with a 5mL syringe pusher to make a single cell suspension. Filtered through a 200-mesh sieve, washed twice with Hank's solution, and centrifuged for 10 minutes each time (1000r / min). The supernatant was discarded, the cell slurry was flicked, 0.5mL of sterile water was added for 20s, 0.5mL of 2x Hank's solution and 8mL of Hank's solution were added after lysing the red blood cells, and the supernatant was discarded, and the cell density was adjusted to 2×10 with 1mL RPMI1640 complete medium, diluted with 1% glacial acetic acid, and counted. The viable cells were counted with trypan blue staining, and finally the cell density was adjusted to 2×10 with RMPI1640 complete medium. 7 24 h before the experiment, mouse lymphoma (YAC-1) cells were passaged, washed three times with Hank's solution, and the cell density was adjusted to 4×10 cells / mL with RMPI1640 complete medium. 5 / mL, which is the target cell. Take 100μL of target cells and effector cells (50:1 effector-target ratio) respectively, add them to the U-shaped 96-well plate as the reaction well; add 100μL of target cells and culture medium to the target cell natural release well; add 100μL of target cells and 1% NP40 to the target cell maximum release well. Set up multiple groups of parallel wells above, culture in a 37℃, 5% CO2 incubator for 4h, then centrifuge the 96-well plate at 1500r / min for 5min, aspirate 100μL of supernatant from each well and place it in a flat-bottomed 96-well plate, add 100μL of LDH matrix solution to each well, react at room temperature for 5min, add 30μL of 1M HCl to each well, and measure its optical density (OD) at 490nm with the help of an enzyme reader.

[0112] The NK cell activity was calculated according to the following formula.

[0113]

[0114] The results are shown in Table 10.

[0115] Table 10 Effect of Platycladus orientalis essential oil on NK cell activity in mice ( n=10)

[0116]

[0117] Note: Compared with the control group, *P<0.05

[0118] As can be seen from Table 10, the NK cell activity of mice in the low- and high-dose groups of Platycladus orientalis essential oil was significantly higher than that in the control group (*P<0.05), indicating that Platycladus orientalis essential oil has the effect of improving the NK cell activity of mice.

[0119] According to the "Health Food Function Test and Evaluation Method (2023 Edition)", the test method that helps to enhance immunity, if the results are positive in any two of the four aspects of cellular immune function, humoral immune function, monocyte-macrophage function, and NK cell activity, it can be determined that the test sample has the effect of helping to enhance immunity. Among them, the results of the two dose groups of the delayed-type hypersensitivity experiment in the cellular immune function determination project were positive, which can be determined that the cellular immune function results are positive; the results of the two dose groups of the NK cell activity determination experiment were positive, which can be determined that the NK cell activity results are positive. Therefore, it can be determined that Platycladus orientalis essential oil has the function of enhancing immunity. Combined with the above experimental results, it can be seen that Platycladus orientalis essential oil can promote delayed-type hypersensitivity in mice and increase NK cell activity, indicating that Platycladus orientalis essential oil has the function of enhancing immunity.

[0120] In conclusion, by measuring the content of SOD, GSH, protein carbonyl and lipid oxidation product malondialdehyde (MDA) in mice and comparing it with the control group data, Platycladus orientalis essential oil has antioxidant function, can reduce protein carbonyl level, increase antioxidant enzyme activity and increase GSH content; in addition, the immune function of Platycladus orientalis essential oil was measured in terms of cellular immune function, humoral immune function, monocyte-macrophage function and NK cell activity. Platycladus orientalis essential oil has a certain effect of enhancing immunity, can promote delayed-type hypersensitivity in mice and increase NK cell activity. It also shows that Platycladus orientalis essential oil has important application prospects in health food.

[0121] The above is only an exemplary embodiment of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present application should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. Application of Platycladus orientalis essential oil in the field of antioxidant and immunity-enhancing drugs / foods.

2. The use according to claim 1, characterized in that: The arborvitae essential oil comprises the following components in relative content: 33.677% of cedarwood alcohol, esters, thujacene and thujacene.

3. The use according to claim 1, characterized in that: The arborvitae essential oil specifically includes the following components in relative content: 33.677% of cedarwood alcohol, 11.503% of 2.10.10-Trimethyllric yclo| 7.1.1.0[2,7]|undec-6-en-8-one, 14.125% 1-(1,3-benzodioxol-5-ylmethyl)-2-oxocyclopentanecarboxylic acid ethyl ester, 9.365% thujacene, 6.982% thujacene, 4.096% 2,6'-dimethyl-5-isopropyl-2,6'-[7]oxabicyclo[4.1.0]heptane]-5'-one, 2.347% β-Vatirenene, 3.432% α-Cedrene, 3.401% Aromadendrene oxide-[1], 2.103% Aristolene epoxide, 1.235% β-Cedrene.

4. The use according to claim 1, characterized in that: The drug administration preparation comprises the following components by volume fraction: 1% Tween 80, 0.25% to 1% oriental arborvitae essential oil and 98% to 98.75% physiological saline.

5. The use according to claim 4, characterized in that: Before use, the drug delivery preparation is first subjected to ultrasonic treatment to ensure that the drug delivery preparation is fully mixed.

6. The use according to claim 1, characterized in that: The preparation method of the arborvitae essential oil comprises: The fresh arborvitae wood is crushed into 1-2 mm pieces, and then the arborvitae wood is dried, and then the arborvitae wood is further crushed to a particle size of 0.1-0.5 mm to obtain pre-treated arborvitae wood, which is stored in a cool place for later use; The extraction conditions were set at a pressure of 20 MPa and a temperature of 50° C., and a supercritical CO 2 extraction method was used to extract the pretreated Platycladus orientalis wood for 65 minutes to obtain the Platycladus orientalis essential oil.

7. The use according to claim 1, characterized in that: The arborvitae essential oil is used for reducing protein carbonyl levels, increasing antioxidant enzyme activity and improving glutathione content.

8. The use according to claim 1, characterized in that: The oriental arborvitae essential oil is used for promoting delayed-type allergic reaction and improving the activity of natural killer cells.

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