Refreshing compound essential oil as well as preparation method and application thereof

Abstract

The invention provides refreshing compound essential oil as well as a preparation method and application thereof, and belongs to the technical field of application of natural products. The refreshing compound essential oil is prepared from the following raw materials in parts by volume: 7 to 10 parts of agilawood essential oil, 10 to 15 parts of peppermint essential oil, 9 to 15 parts of ravenalka essential oil, 6 to 10 parts of sweet basil essential oil, 5 to 8 parts of cypress essential oil, 8 to 14 parts of eucalyptol rosemary essential oil and 10 to 16 parts of lemon essential oil. Experiments prove that the compound essential oil has the effects of refreshing and restoring consciousness, and is good in in-vivo safety.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of natural product application, and particularly relates to a refreshing compound essential oil and a preparation method and application thereof. BACKGROUND

[0002] Plant essential oils are outstanding in mood regulation and physical and mental health. Through aroma therapy, their aromatic molecules can regulate mood, relieve anxiety and stress, and help improve sleep quality, becoming a natural choice for modern people to balance body and mind. In the field of physical health care, essential oils can be absorbed through massage or bathing, which can relieve muscle pain, promote blood circulation, and assist in the treatment of chronic diseases such as arthritis. Plant essential oils are also used for household cleaning and air purification, and have natural antibacterial properties. Modern pharmacological studies have shown that aromatic plant essential oils have pharmacological activities such as antioxidant, antibacterial, and sedative, and can regulate the autonomic nervous system, central nervous system and / or endocrine system through aromatic molecules, thereby affecting mood, physiological state and behavior. Therefore, modern aromatherapy is widely used in the auxiliary treatment of chronic pain, depression, anxiety, insomnia and other diseases related to the nervous system.

[0003] However, existing essential oil products generally have the problems of single function, lack of scientific verification, and unknown safety. A compound essential oil with refreshing and brain-awakening function is disclosed, which contains 14 plant ingredients, and the excessive types of ingredients can easily cause many problems; high concentration of natural borneol may cause neurotoxicity, and Hypericum perforatum is a photosensitive ingredient, which needs to be used in the dark; jasmine, magnolia and clove are common allergens, and sensitive skin or pollen allergy sufferers are prone to redness and itching when using them; meanwhile, the compound essential oil contains lavender (sedative), rosemary (refreshing) and mint (stimulating) which are contradictory ingredients, and may weaken the effect of single function. In addition, clinical studies have reported that essential oils may cause various adverse reactions, and the disclosed compound essential oil has rarely been subjected to safety experiments. Therefore, it is particularly important to develop a high-efficiency and safe refreshing and brain-awakening product. SUMMARY

[0004] Therefore, the purpose of the present application is to provide a refreshing and brain-awakening compound essential oil and a preparation method and application thereof, which can significantly improve the refreshing and brain-awakening synergistic effect of smellers by skillfully matching various aromatic compounds, and has good safety.

[0005] To solve the above technical problems, the present application provides the following technical solutions:

[0006] The present application provides a refreshing and brain-awakening compound essential oil, which comprises the following raw materials by volume: 7-10 parts of linaloe essential oil, 10-15 parts of peppermint essential oil, 9-15 parts of rosemary leaf essential oil, 6-10 parts of sweet basil essential oil, 5-8 parts of silk cypress essential oil, 8-14 parts of eucalyptol rosemary essential oil and 10-16 parts of lemon essential oil.

[0007] Preferably, it further comprises jojoba oil 45-55 parts.

[0008] Preferably, it comprises the following ingredients by volume: agarwood essential oil 9 parts, peppermint essential oil 12 parts, rosemary leaf essential oil 10 parts, sweet basil essential oil 8 parts, silk cypress essential oil 6 parts, eucalyptol rosemary essential oil 10 parts, and lemon essential oil 12 parts.

[0009] Preferably, it further comprises jojoba oil 50 parts.

[0010] The application provides a preparation method of the invigorating compound essential oil, comprising the following steps: adding agarwood essential oil, peppermint essential oil, rosemary leaf essential oil, sweet basil essential oil, silk cypress essential oil, eucalyptol rosemary essential oil, and lemon essential oil into jojoba oil, mixing uniformly, and standing.

[0011] Preferably, after adding each essential oil, the mixture is mixed uniformly.

[0012] Preferably, the standing time is 12-36 hours.

[0013] The application provides an application of the invigorating compound essential oil or the invigorating compound essential oil obtained by the preparation method in the preparation of an invigorating product.

[0014] Preferably, the invigorating compound essential oil is suitable for improving mental state, enhancing attention, improving cognitive function, and relieving mental fatigue.

[0015] Preferably, the product comprises one or more of the following: fragrance clasp, rattan incense, sachet, body spray, essential oil cream, and scented candle.

[0016] Compared with the prior art, the application has the following beneficial effects:

[0017] The application is guided by aromatherapy theory, and selects agarwood essential oil, peppermint essential oil, rosemary leaf essential oil, sweet basil essential oil, silk cypress essential oil, eucalyptol rosemary essential oil and lemon essential oil as the base oil of jojoba oil. The above essential oils are combined to achieve the synergistic effect of significantly improving the refreshing effect of smellers. The agarwood essential oil can calm the nervous system and relieve anxiety, so that the user is in a positive psychological state. The peppermint essential oil can stimulate the cerebral cortex through menthol to increase attention and alertness. The combination of the two can strengthen the refreshing effect on the basis of soothing emotions, reduce the mental consumption caused by anxiety and tension, and directly stimulate the nerves to improve the degree of wakefulness. The rosemary leaf essential oil can stabilize the psychology, and the eucalyptol rosemary essential oil can activate brain cells and remove drowsiness. The combination of the two can act on the psychology and physiology, stabilize the emotions first, then activate the brain, make people get rid of drowsiness and psychological confusion, achieve the effect of refreshing and improving memory. The combination of the agarwood essential oil for calming the nervous system and the sweet basil essential oil for refreshing and relieving headache can relieve headache and other physical discomforts, so that people can keep awake and positive in a comfortable state of mind and body. The silk cypress essential oil contains cedrol, which can inhibit the violent fluctuation of emotions. The lemon essential oil is rich in limonene and citral, which can stimulate the secretion of dopamine and serotonin to bring a fresh and pleasant feeling. The combination of the silk cypress essential oil and the lemon essential oil can not only soothe the nervous emotions, but also boost the spirit. The above essential oils of the application are combined to achieve the effect of significantly refreshing and waking up. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The resting time of the compound essential oil after the mice inhaled for 5 minutes in the open field experiment.

[0019] Figure 2 The average speed of movement of the compound essential oil after the mice inhaled for 5 minutes in the open field experiment.

[0020] Figure 3 The total distance of movement of the compound essential oil after the mice inhaled for 5 minutes in the open field experiment.

[0021] Figure 4 The sleep time of the refreshing and waking up 1 after the mice inhaled for 5 minutes in the sleep induction experiment. The left graph is the experimental result of male mice, and the right graph is the experimental result of female mice.

[0022] Figure 5 The sleep latency of the refreshing and waking up 1 after the mice inhaled for 5 minutes in the sleep induction experiment. The left graph is the experimental result of male mice, and the right graph is the experimental result of female mice.

[0023] Figure 6 The activity frequency of the refreshing and waking up 1 after the mice inhaled for 5 minutes in the spontaneous movement experiment. The left graph is the experimental result of male mice, and the right graph is the experimental result of female mice.

[0024] Figure 7 The length of time of autonomous movement activity after 1 minute of mouse sniffing of refreshing brain 1. The left graph is the experimental result of male mice, and the right graph is the experimental result of female mice.

[0025] Figure 8 The results of neurotransmitter determination after 1 minute of mouse sniffing of refreshing brain 1.

[0026] Figure 9 Blood biochemical ALT changes after 1 minute of mouse sniffing of refreshing brain 1.

[0027] Figure 10 Blood biochemical AST changes after 1 minute of mouse sniffing of refreshing brain 1.

[0028] Figure 11 Blood biochemical UREA changes after 1 minute of mouse sniffing of refreshing brain 1.

[0029] Figure 12 Blood biochemical CREA changes after 1 minute of mouse sniffing of refreshing brain 1.

[0030] Figure 13 Changes in organ index after 1 minute of mouse sniffing of refreshing brain 1.

[0031] Figure 14 H&E staining of heart, liver, spleen, lung, and kidney after sniffing of compound essential oil.

[0032] Figure 15 Brain region distribution map.

[0033] Figure 16 Channel β / α value before and after sniffing refreshing brain 1.

[0034] Figure 17 Channel △β / α value before and after sniffing refreshing brain 1.

[0035] Figure 18 Channel △β / α value change rate before and after sniffing refreshing brain 1. DETAILED DESCRIPTION

[0036] The application provides a refreshing compound essential oil, which comprises the following ingredients by volume: 7-10 parts of agarwood essential oil, 10-15 parts of peppermint essential oil, 9-15 parts of rosemary leaf essential oil, 6-10 parts of sweet basil essential oil, 5-8 parts of cypress essential oil, 8-14 parts of eucalyptol rosemary essential oil and 10-16 parts of lemon essential oil; preferably, the refreshing compound essential oil comprises the following ingredients by volume: 8-9.5 parts of agarwood essential oil, 11-14 parts of peppermint essential oil, 10-14 parts of rosemary leaf essential oil, 7-9 parts of sweet basil essential oil, 5.5-7.5 parts of cypress essential oil, 9-13 parts of eucalyptol rosemary essential oil and 11-15 parts of lemon essential oil; the refreshing compound essential oil of the application further comprises 45-55 parts of jojoba oil, preferably 46-54 parts. As an implementable mode, the refreshing compound essential oil preferably comprises the following ingredients by volume: 9 parts of agarwood essential oil, 12 parts of peppermint essential oil, 10 parts of rosemary leaf essential oil, 8 parts of sweet basil essential oil, 6 parts of cypress essential oil, 10 parts of eucalyptol rosemary essential oil and 12 parts of lemon essential oil; the relaxing compound essential oil further comprises 50 parts of jojoba oil. The compound essential oil of the application can significantly improve the refreshing effect of the sniffer by skillfully matching various aromatic compounds. The essential oil of the application is commercially available and well known in the art. The compound essential oil of the application is not only suitable for driving scenes, but also can be widely used in offices, learning places and various environments, thereby providing a safe and long-acting solution for relieving the fatigue of modern people.

[0037] In the application, the warm woody aroma of agarwood essential oil can pacify the nervous system, relieve anxiety and tension, and keep people in an active state; the peppermint essential oil has a cooling smell and can effectively refresh the mind; the main component menthol in the peppermint essential oil can stimulate the cerebral cortex, increase attention and vigilance, and make people more awake; the smell of mint can improve cognitive function, improve learning efficiency and reduce fatigue; the rosemary leaf essential oil can restore chaotic and disordered psychological state, stimulate refreshing and keep self-confidence; the sweet basil essential oil has the effects of refreshing the mind and stimulating the sense of smell, and has good curing effect on headache and migraine; the cypress essential oil has the effects of relaxing tension and improving self-confidence, and can refresh the mind, improve attention and concentration when used with lemon essential oil and peppermint essential oil; the eucalyptol rosemary essential oil can remove drowsiness, activate brain cells and help improve overall memory quality, making people more alert; the lemon essential oil has the characteristics of refreshing and refreshing, can effectively stimulate the nervous system of the brain, enhance concentration and memory, the limonene component in the lemon essential oil can promote the secretion of neurotransmitters, improve learning motivation and learning efficiency, and the aroma of lemon can improve mood and relieve tension and stress.

[0038] The application further provides a preparation method of the compound essential oil for refreshing and invigorating the brain, comprising the following steps: dropping agarwood essential oil, peppermint essential oil, rosemary leaf essential oil, sweet basil essential oil, silk cypress essential oil, eucalyptol rosemary essential oil and lemon essential oil into jojoba oil, uniformly mixing, and standing. The application uniformly mixes after adding one kind of essential oil. The uniformly mixing method of the application is gently shaking. The standing time of the application is 12-36 h, preferably 18-32 h, and more preferably 24 h. The purpose of standing in the application is to fully integrate all raw materials.

[0039] The application further provides application of the compound essential oil for refreshing and invigorating the brain or the compound essential oil for refreshing and invigorating the brain obtained by the preparation method in preparation of a product for refreshing and invigorating the brain. The compound essential oil for refreshing and invigorating the brain of the application is suitable for improving mental state, enhancing attention, improving cognitive function and relieving mental fatigue.

[0040] In the application, the product comprises one or more of fragrance buckles, rattan incense, sachets, body sprays, essential oil creams and scented candles.

[0041] In the application, the compound essential oil is evaluated for refreshing and invigorating the brain by using open field experiments, sleep induction experiments and autonomous movement experiments. It is verified by experiments that, under the condition of sniffing for 5 min, the compound essential oil can improve the average speed and total distance of movement of mice in the open field experiment, shorten the rest time, shorten the sleep time in the sleep induction experiment, and improve the activity frequency of mice in the autonomous movement experiment, thus exhibiting a relatively significant effect of refreshing and invigorating the brain. The autonomous activity instrument model is a method for reflecting the functional state of the central nervous system, observing and researching the neuro-psychological changes of experimental animals, and evaluating the autonomous behavior, exploratory behavior and tension of animals in a novel environment. The open field model and the autonomous activity instrument have similarities, and both are used to observe the neuro-psychological changes of animals, mainly observing the movement distance of animals, the number of grooming behaviors, and the time of staying in the corner and the middle area. The sleep induction experiment is a common animal behavior research method, mainly used to explore the mechanism of mouse sleep, evaluate sleep induction factors and the influence of related test products on the sleep state of mice. By inducing mice into a sleep state through a specific method, behavior data related to sleep can be obtained.

[0042] In the present application, the invigorating compound essential oil reduces the content of 3-Methoxytyramine, Succinic acid and Serotonin, and increases the content of L-Aspartic acid by using neurotransmitter assay. The 3-Methoxytyramine is a metabolite of dopamine, and the generation of 3-Methoxytyramine is reduced due to the slow metabolism of dopamine. Since the metabolism of dopamine is inhibited, the extracellular dopamine level is increased, continuously stimulating dopamine receptors, which enhances dopaminergic neurotransmission, and improves mental state and invigorates the brain. Serotonin is closely related to mood regulation and sleep, and when the content of Serotonin decreases, the sleep regulation mechanism of mice is also affected, resulting in shorter sleep time. Succinic acid is involved in the energy metabolism process of cells, especially in the tricarboxylic acid cycle, and its decrease may affect the energy supply of nerve cells; when the content of succinic acid decreases and affects the energy supply of the tricarboxylic acid cycle, the body may activate other energy metabolism pathways to compensate for the function of nerve cells, and even increase the excitability of the brain to a certain extent, thereby producing an invigorating effect. L-Aspartic acid is an excitatory neurotransmitter, and its increase means that the excitability of the mouse brain is enhanced, and the improvement of brain excitability can improve the alertness of the mouse, making it more rapid in response to external stimuli.

[0043] In the present application, the invigorating compound essential oil is subjected to brain wave test for invigorating effect, and the experimental results show that the invigorating compound essential oil increases the β / α value in the left frontal lobe F3 and right frontal lobe F4 channels of the brain. The frequency of human brain waves varies between 1-30 times per second, which can be divided into four wave bands, namely δ (1-3 Hz), θ (4-7 Hz), α (8-13 Hz) and β (14-30 Hz). Among them, the α wave, with a frequency of 8-13 times per second and an average of about 10 times, is the basic rhythm of normal brain waves, and its frequency is quite constant without external stimulation; this rhythm is most obvious when a person is awake, quiet and closed eyes, and the α wave disappears immediately when the eyes are opened or other stimuli are received. The β wave has a frequency of 14-30 times per second, and appears when the spirit is nervous and the emotion is excited or hyperactive, and the original slow wave rhythm can be replaced by this rhythm when a person is suddenly awakened from sleep. Therefore, it is generally believed that the β wave is the main wave form when the cerebral cortex is excited. △β / α value > 0 has invigorating effect; △β / α value ≤ 0 has no invigorating effect.

[0044] In the present application, all components or reagents are commercially available unless otherwise specified.

[0045] The technical solutions in the present application will be clearly and completely described below in combination with the embodiments in the present application. Apparently, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative effort belong to the protection scope of the present application.

[0046] Example 1

[0047] The refreshing mind A is prepared according to the following volume ratio raw material formula: 10 parts of agarwood essential oil, 15 parts of peppermint essential oil, 12 parts of rosemary leaf essential oil, 10 parts of sweet basil essential oil, 8 parts of silk cypress essential oil, 12 parts of eucalyptol rosemary essential oil, 13 parts of lemon essential oil and 50 parts of jojoba oil.

[0048] The preparation steps of the compound essential oil are as follows:

[0049] a. Pour 50 milliliters of jojoba oil into a mixing container.

[0050] b. Drop the agarwood essential oil, peppermint essential oil, rosemary leaf essential oil, sweet basil essential oil, silk cypress essential oil, eucalyptol rosemary essential oil and lemon essential oil into the jojoba oil in turn, and shake the container gently after adding each raw material to ensure uniform mixing.

[0051] c. After all the raw materials are added, tightly cover the container cover and shake gently for 3 min until all the ingredients are completely mixed evenly.

[0052] d. Let the mixed essential oil stand for 24 h to ensure that all the ingredients are fully integrated to achieve the best effect. Store in a dark place at room temperature.

[0053] Example 2

[0054] The refreshing mind 1 is prepared according to the following volume ratio raw material formula: 9 parts of agarwood essential oil, 12 parts of peppermint essential oil, 10 parts of rosemary leaf essential oil, 8 parts of sweet basil essential oil, 6 parts of silk cypress essential oil, 10 parts of eucalyptol rosemary essential oil, 12 parts of lemon essential oil and 50 parts of jojoba oil.

[0055] The preparation steps are the same as those in Example 1.

[0056] Example 3

[0057] The refreshing mind 2 is prepared according to the following volume ratio raw material formula: 8 parts of agarwood essential oil, 15 parts of peppermint essential oil, 10 parts of rosemary leaf essential oil, 8 parts of sweet basil essential oil, 6 parts of silk cypress essential oil, 12 parts of eucalyptol rosemary essential oil, 16 parts of lemon essential oil and 50 parts of jojoba oil.

[0058] The preparation steps are the same as those in Example 1.

[0059] Example 4

[0060] Awakening 3 was prepared according to the following volume parts of raw material formula: Agarwood essential oil 7 parts, peppermint essential oil 10 parts, rosemary leaf essential oil 15 parts, sweet basil essential oil 10 parts, silk cypress essential oil 8 parts, eucalyptol rosemary essential oil 12 parts, lemon essential oil 12 parts and jojoba oil 50 parts.

[0061] Preparation steps are the same as Example 1.

[0062] Experimental Example 1

[0063] 1. Behavioral experiment

[0064] Experimental institution: Chiral Drug Research Center, Innovative Traditional Chinese Medicine Research Institute, Shanghai University of Traditional Chinese Medicine, After C57 / 6J mice sniffed essential oil, a series of behavioral experiments were conducted to verify the refreshing effect of essential oil. The test setting is as follows:

[0065] Test sample information: refreshing A prepared in Example 1, refreshing 1 prepared in Example 2, refreshing 2 prepared in Example 3, refreshing 3 prepared in Example 4.

[0066] Animal welfare: During the experiment, animal management and use comply with relevant requirements of national and local regulations (Regulations on the Administration of Laboratory Animals (3rd revised edition), Shanghai Municipal Administration of Laboratory Animals), as well as Guide for the Care and Use of Laboratory Animals (2011) issued by the National Research Council of the United States. The animal management and use of this experiment has been approved by the Institutional Animal Care and Use Committee (IACUC) of the experimental institution. The approved ethics number is: 2411107.

[0067] Experimental animals: C57 / 6J mice, SPF level, about 22-25g, animal source and license Animal source: Shanghai Jihui Experimental Animal Technology Co., Ltd.

[0068] Method of giving compound essential oil and experimental design: Compound essential oil was given to mice by sniffing, 30 μL of compound essential oil was dropped on a cotton ball and placed in the mouse cage to volatilize the essential oil, with a frequency of 5 min / time. To ensure that the Control group and each essential oil group do not interfere with each other, the process of giving essential oil was carried out in airtight fresh air cage box. Open-field test, sleep induction test and voluntary movement test were conducted, with n=6 for open-field test, n=10 for sleep induction test and voluntary movement test.

[0069] Animal breeding: quarantine and adaptation: all experimental animals have quality certificate, and directly into the SPF level animal room; before the experiment, need to adapt for at least 3 days, and the experimental personnel in the adaptation period and experimental period every day to observe the health of the cage side. Animal breeding follows the following standards: male and female cages, no more than 5 in each cage, the feeding environment requires temperature 20~26℃, relative humidity 40~70%, air change frequency≥15 times per hour, 12h light and dark automatic light (7:00PM light off, the next day 7:00M light on). The feed is cobalt-free feed, and the drinking water is self-made deionized water. 60 Irradiation sterilization of SPF mice is maintained by Jiangsu Cooperation Pharmaceutical Biological Engineering Co., Ltd. Animals are free to drink self-made deionized water. Corn cob bedding is provided by Texas Valley Agricultural Technology Co., Ltd.

[0070] 1.1 Open field test

[0071] The normal mice were randomly divided into 5 groups, Control group, Tishenxingshen A group, Tishenxingshen 1 group, Tishenxingshen 2 group and Tishenxingshen 3 group, 6 in each group. 30 μL compound essential oil was dropped on the cotton ball and placed in the mouse cage to volatilize the essential oil. The mice in the essential oil group were given essential oil by sniffing for 5 min. The Control group was not treated by sniffing. The laboratory was quiet and ventilated, and the light was dim. After the experiment started, the animals were placed in the center of the open box. After 2 min of adaptation, the camera and timer were started, and the movement data of the mice in the open field for 5 min were recorded, including rest time, average speed and total distance.

[0072] The results showed that compared with the Control group, the rest time of mice given Tishenxingshen A, Tishenxingshen 1 and Tishenxingshen 2 was significantly reduced Figure 1 )(*P<0.05, **P<0.01), the average speed of mice given Tishenxingshen 1, Tishenxingshen 2 and Tishenxingshen 3 was significantly increased Figure 2 )(*P<0.05, **P<0.01), and the total distance of mice given Tishenxingshen 1 was significantly increased Figure 3 )(*P<0.05). The above data showed that Tishenxingshen 1 had obvious refreshing effect, and was selected for subsequent experiments.

[0073] 1.2 Sleep induction experiment

[0074] The normal mice were randomly divided into 2 groups, namely Control group and Vigor 1 group, 10 in each group. 30 μL of compound essential oil was dropped on a cotton ball and placed in the mouse cage to let the essential oil volatilize. The mice in the Vigor 1 group were given Vigor 1 by sniffing for a total of 5 min, and the mice in the Control group were not given sniffing. Before induction, the mice needed to adapt to the environment for 1-2 hours, and then were induced by intraperitoneal injection of 300 mg / kg chloral hydrate. During the experiment, the sleep latency and sleep duration were recorded. Short latency and long sleep duration indicated poor mouse energy.

[0075] The results showed that, compared with the Control group, Vigor 1 could significantly reduce the sleep duration of the mice Figure 4 (*P<0.05), and had a wake-up effect. Vigor 1 could significantly increase the sleep latency of female mice Figure 5 (*P<0.05), but had no significant effect on the sleep latency of male mice Figure 5 .

[0076] 1.3 Spontaneous Motor Activity Test in Mice

[0077] The normal mice were randomly divided into 2 groups, namely Control group and Vigor 1 group, 10 in each group. 30 μL of compound essential oil was dropped on a cotton ball and placed in the mouse cage to let the essential oil volatilize. The mice in the Vigor 1 group were given Vigor 1 by sniffing for a total of 5 min, and the mice in the Control group were not given sniffing. The mice were placed in the lower left corner of the self-activity box with their heads facing the corner, and the number of activities and the activity duration of the mice within 5 min were recorded. After each mouse test, the excrement in the box was cleaned, and the instrument was wiped with 60% alcohol to prepare for the next mouse test.

[0078] The results showed that, compared with the Control group, Vigor 1 could significantly increase the number of activities of the mice Figure 6 (*P<0.05), indicating that Vigor 1 had a significant invigorating effect under the condition of giving essential oil for 5 min. However, it had no significant effect on the self-activity duration of the mice Figure 7 .

[0079] 1.4 Statistical Analysis

[0080] Statistical analysis of post-experiment data was performed using GraphPad Prism 8.0 software. Statistical differences were analyzed by One-way analysis of variance (ANOVA), Two-way ANOVA and T-test. ****P<0.0001, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 indicate significant differences. * indicates compared with the control group. Ns: P>0.05, indicating no significant difference.

[0081] 2. Neurotransmitter assay

[0082] Experimental institution: Chiral Drug Research Center, Innovative Traditional Chinese Medicine Research Institute, Shanghai University of Traditional Chinese Medicine. After the C57 / 6J mice inhaled the compound essential oil, the brain tissue was detected for neurotransmitters to obtain the changes of neurotransmitters in the brain of mice after inhaling the essential oil.

[0083] Test sample: refreshing 1 prepared in Example 2.

[0084] Animal welfare is the same as Step 1 of this example.

[0085] Experimental animals: male, C57 / 6J mice, SPF level, about 22-25 g. The grouping is as follows: 7 in the Control group and 7 in the refreshing 1 group. Animal source: Shanghai Jihui Experimental Animal Technology Co., Ltd.

[0086] Selection of compound essential oil administration method: the compound essential oil was administered to the mice by sniffing, and the refreshing essential oil was administered at a frequency of 5 min / time. To ensure that the Control group and the essential oil group do not interfere with each other, the administration of the essential oil was carried out in airtight fresh air cage boxes.

[0087] Animal feeding is the same as Step 1 of this example.

[0088] 2.1 Animal sampling: C57 / 6J mice were sacrificed under isoflurane anesthesia, the mouse head skin and skull were carefully peeled off, the brain was immediately frozen in liquid nitrogen after being taken out, and then transferred to a-80℃ refrigerator. When the sample is sent, the brain tissue should be stored in dry ice. The neurotransmitter assay was completed by Shanghai Oulei Biomedical Technology Co., Ltd.

[0089] 2.2 Sample pretreatment

[0090] Take appropriate amount of sample, accurately weigh and record, then load into 1.5 mL EP tube (EP tube is pre-cooled at -20℃); add two small steel balls, add 400 μL 85% acetonitrile (V / V, containing succinic acid-2,2,3,3-d4 and tryptophan-[2H5], 1 mM BHT, containing 0.15% formic acid); put into the grinder (45HZ, 2min); ice water bath ultrasonic 10min, centrifugation 10min (4℃, 12000rpm), take 300 μL supernatant dry; with 300 μL water (containing L-2-chlorophenylalanine) reconstituted, vortex 30s, ice water bath ultrasonic 5min; centrifugation 10min (4℃, 13000rpm), take the supernatant and transfer to brown sample vial, LC-MS analysis.

[0091] 2.3 Chromatography method: injection amount: 5 μL; flow rate: 0.3 mL / min; mobile phase: A (0.1% formic acid-water solution), B (0.1% formic acid-methanol); gradient elution method: 0 min A / B (99:1, V / V), 1 min A / B (99:1, V / V), 6 min A / B (5:95, V / V), 7 min A / B (5:95, V / V), 7.01 min A / B (99:1, V / V), 8 min A / B (99:1, V / V).

[0092] 2.4 Mass spectrometry method: gas curtain gas: 30 (psi); collision-induced ionization (collision-activated dissociation, CAD) parameters: medium; positive ion spray voltage: 5500V; negative ion spray voltage: -4500V; ion source temperature: 450℃; column temperature: 40℃; spray gas (Gas1): 50 (psi); auxiliary heating gas (Gas2): 50 (psi).

[0093] 2.5 Neurotransmitter assay: The content of 24 neurotransmitters was detected. The 24 neurotransmitters were: 3,4-Dihydroxyphenylacetic acid, 3-Methoxytyramine, 4-Aminobutyric acid, L-5-hydroxytryptophan, 5-Hydroxyindole-3-acetic acid, Serotonin, L-Glycine, L-Glutamic acid, L-Glutamine, L-Tyrosine, L-Tryptophan, L-Aspartic acid, L-Histidine, Dopamine, Homovanillic acid, Succinic acid, Taurine, Picolinic acid, Kynurenine, Nicotinic acid, Indolelactic acid, Indoleacetic acid, L-Levadopa, Acetylcholine chloride.

[0094] 2.6 Statistical analysis: The data of each evaluation index after the experiment was statistically analyzed using GraphPad Prism 8.0 software, and the statistical difference of the data was analyzed by One-way analysis of variance (ANOVA), Two-way ANOVA and T-test. *P < 0.05, **P < 0.01, ***P < 0.001 indicate significant difference. * indicates compared with the normal group. ns: P > 0.05, indicating no significant difference.

[0095] 2.7 Neurotransmitter assay experimental results of the brain refreshing group 1

[0096] The results showed that compared with the normal group, the contents of 3-Methoxytyramine, Succinic acid and Serotonin in the brain refreshing group 1 were significantly decreased (*P < 0.05), the content of L-Aspartic acid was significantly increased (*P < 0.05), and there was no significant difference in the other indicators. Figure 8). 3-Methoxytyramine is a metabolite of dopamine. Although the slowing of dopamine metabolism theoretically weakens dopaminergic nerve activity, it actually stimulates receptors due to the elevated extracellular dopamine levels, which can enhance neurotransmission to refresh the mind; the decrease in serotonin content affects sleep regulation in mice, resulting in shorter sleep time; the decrease in succinic acid content affects the energy supply of nerve cells, and the body compensates by increasing brain excitability to refresh the mind; L-Aspartic acid as an excitatory neurotransmitter enhances brain excitability and alertness in mice. Thus, the refreshing and invigorating 1 formula achieves the effect of refreshing and invigorating the mind.

[0097] 3. Animal safety evaluation

[0098] Experimental institution: Chiral Drug Research Center, Innovative Chinese Medicine Research Institute, Shanghai University of Traditional Chinese Medicine. Safety evaluation of compound essential oil after sniffing by C57 / 6J mice.

[0099] Test sample: refreshing and invigorating 1 prepared in Example 2.

[0100] Animal welfare: same as Step 1.

[0101] Experimental system: male C57 / 6J mice, SPF level, about 22-25 g. Grouping: 11 in the Control group and 11 in the refreshing and invigorating 1 group. Animal source: Shanghai Jihui Experimental Animal Technology Co., Ltd.

[0102] Animal feeding: same as Step 1 of this example.

[0103] 3.1 Animal sampling: after the experiment was completed, the C57 / 6J mice were weighed and blood was collected from the eye socket, then they were sacrificed under isoflurane anesthesia, and the mouse heart, liver, spleen, lung, and kidney were removed and weighed. Then they were fixed in 4% paraformaldehyde, blood biochemistry and blood routine were determined by Seville Biological Technology Co., Ltd., and H&E staining was completed by Shanghai Yingpaina Biological Technology Co., Ltd.

[0104] 3.2 Statistical analysis: each evaluation index was analyzed using GraphPad Prism 8.0 software after the experiment, and the statistical difference of the data was analyzed by One-way analysis of variance (ANOVA), Two-way ANOVA, and T-test. *P<0.05, **P<0.01, ***P<0.001 indicate significant differences. * indicates compared with the control group. Ns: P>0.05, indicating no significant difference.

[0105] 3.3 Experimental procedures and results

[0106] 3.3.1 Blood biochemistry experimental procedures and results

[0107] The experiment aimed at four key indicators of mice blood: alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen (UREA) and creatinine (CREA). The mouse blood was centrifuged at 4℃, 3000r / min for 15min after overnight at 4℃, and the supernatant was immediately detected. The reagent kit used in the experiment was from Changchun Hui Li Biotechnology Co., Ltd., and the working reagent was prepared according to the instruction. After setting the corresponding parameters on the automatic biochemical instrument, the sample was loaded, and the automatic biochemical instrument was used for automatic determination. Since there was a sample in the Control group that was significantly different from other data in the group, considering that the mouse itself had some diseases or other reasons to interfere with the experimental data, the data of this sample was excluded from the statistics.

[0108] In terms of liver function related indicators, ALT and AST are sensitive markers of liver cell damage. Statistical analysis showed that there was no significant difference between the ALT and AST indicators of the experimental group and the control group (ns: P>0.05). This indicates that after inhaling the refreshing 1 compound essential oil, the liver cells of mice did not appear obvious damage, and the liver maintained normal metabolism and detoxification function. Figure 9 , Figure 10 In terms of kidney function related indicators, UREA and CREA are important indicators reflecting the excretion and metabolism function of kidney. The statistical results showed that there was no significant difference between the UREA and CREA indicators of each experimental group and the control group (ns: P>0.05). This shows that under the conditions of this experiment, the refreshing 1 compound essential oil has no adverse effects on the excretion and metabolism function of mouse kidney, and the kidney can maintain normal physiological state.

[0109] In summary, after the mice inhaled the refreshing 1 compound essential oil, the four indicators of blood biochemical detection ALT, AST, UREA and CREA did not appear obvious abnormal changes. This indicates that the refreshing 1 compound essential oil has good safety in vivo, and has no adverse effects on the liver and kidney function of mice. Figure 11 , Figure 12 In summary, after the mice inhaled the refreshing 1 compound essential oil, the four indicators of blood biochemical detection ALT, AST, UREA and CREA did not appear obvious abnormal changes. This indicates that the refreshing 1 compound essential oil has good safety in vivo, and has no adverse effects on the liver and kidney function of mice.

[0110] In summary, after the mice inhaled the refreshing 1 compound essential oil, the four indicators of blood biochemical detection ALT, AST, UREA and CREA did not appear obvious abnormal changes. This indicates that the refreshing 1 compound essential oil has good safety in vivo, and has no adverse effects on the liver and kidney function of mice.

[0111] 3.3.2 Blood routine experiment steps and results

[0112] After blood routine test of each group of mice using full-automatic animal blood cell analyzer, the blood routine individual data of the group of refreshing the mind 1 is shown in Table 1. The results show that, compared with the control group, the mice in the experimental group treated with compound essential oil sniffing, the blood routine indexes, including white blood cell count and classification (percentage and absolute value of neutrophils, lymphocytes, mononuclear cells, eosinophils, basophils), red blood cell count, hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin content, mean corpuscular hemoglobin concentration, platelet count, etc., are within the normal range. It is proved that the compound essential oil of refreshing the mind 1 does not show obvious blood toxicity in vivo, which preliminarily proves the blood safety of the compound essential oil of refreshing the mind 1 in the experimental model.

[0113] Table 1 Blood routine test results

[0114]

[0115]

[0116] 3.3.3 Experimental results of organ index

[0117] The heart, liver, spleen, lung and kidney of mice were weighed and the organ index was calculated, and the calculation formula was: organ index = organ weight (g) / body weight (g) x 100%.

[0118] Statistical analysis of organ index of mice in experimental group (refreshing the mind 1 group) and normal group was carried out, and the results showed that, compared with the normal group, the heart, liver, spleen, lung and kidney organ index of mice in the group of refreshing the mind 1 had no significant difference. Figure 13 It is shown that under the conditions set in this experiment, after sniffing compound essential oil, no obvious effect on the relative weight of heart, liver, spleen, lung and kidney of mice was produced, which suggests that the compound essential oil does not significantly interfere with the normal growth and development of these organs in vivo, and proves the safety of the compound essential oil of refreshing the mind 1 to the organs in this experiment.

[0119] 3.3.4 H&E staining steps and results

[0120] The mouse heart, liver, spleen, lung, kidney tissues were placed in 4% paraformaldehyde for more than 24h, and the fixed specimens were prepared into paraffin sections as follows: (1) Paraffin section deparaffinization to water: first immerse the section in environmentally friendly deparaffinization liquid, twice, 20min each time; then transfer to anhydrous ethanol for immersion twice, 5min each time; then put into 75% alcohol for 5min; finally use tap water to clean the section, complete the deparaffinization process. (2) Rewarming fixation of frozen section: take the frozen section from the-20℃ refrigerator, restore to room temperature, fix with tissue fixative for 15min, then rinse with running water. (3) Pretreatment: immerse the section in high-definition constant dye pretreatment liquid for 1min. (4) Hematoxylin staining: transfer the section to hematoxylin dyeing liquid for 5min, then rinse with tap water, differentiate, rinse with tap water, then return to blue, finally rinse with running water. (5) Eosin staining: immerse the section in 95% alcohol for 1min, then put into eosin dyeing liquid for 15 seconds. (6) Dehydration and mounting: the section is immersed in anhydrous ethanol for three times, 2min each time; then in n-butanol for two times, 2min each time; then in xylene for two times, 2min each time, finally mount with neutral balsam.

[0121] After H&E staining of the mouse heart, liver, spleen, lung, kidney tissues, the pathological morphological changes of each tissue were observed under an optical microscope. In the tissue sections of the control group mice, the myocardial fibers of the heart tissue were arranged in order, the cross striations were clear, the cell nuclei were normal, and no inflammatory cell infiltration or necrotic area was observed. The hepatocytes in the liver tissue were regular in shape, the cytoplasm was abundant, the nucleolus was clear, the liver lobule structure was complete, and there was no abnormal change in the hepatic portal area. The white pulp and red pulp of the spleen had clear boundaries, the lymphocytes were evenly distributed, and there was no abnormal condition such as atrophy or hyperplasia of the spleen nodule. The alveolar structure in the lung tissue was complete, the alveolar wall was thin and not thickened, there was no exudate in the alveolar cavity, and the bronchial epithelial cells were normal in shape. The glomerular structure in the kidney tissue was normal, the renal tubular epithelial cells were regular in shape, the lumen was unobstructed, and there was no protein cast or cell shedding phenomenon. Figure 14 ) After the mice were given Tiaogan 1 compound essential oil for sniffing treatment, the H&E staining results of the heart, liver, spleen, lung, and kidney tissues of the mice showed no significant difference compared with the normal group.

[0122] Based on the safety evaluation and efficacy research results of this experiment, the Tiaogan 1 compound essential oil has good safety in vivo, and has no obvious adverse effects on the liver and kidney function, blood system, and tissue structure of the main organs of the mice.

[0123] 4. Brain wave test

[0124] Experimental institution: Guangzhou Baiyun Meiguan Shangyan International Cosmetics Research Institute, brain wave test of Tiaogan 1 compound essential oil for invigorating the brain.

[0125] Tested product: invigorating 1 prepared in Example 2.

[0126] Fragrance preparation: 150 μL of compound essential oil was dropped on the fragrance pad, and put into the fragrance buckle. The fragrance buckle was fixed on the mask with a magnet near the nose.

[0127] Subjects: A total of 34 people, 4 men and 30 women, aged 24 to 45, with an average age of 36.5 ± 5.9, who met the subject's voluntary selection criteria.

[0128] 4.1 Test method: The qualified subjects were monitored for 2 min of EEG waves without wearing the fragrance buckle (T0); the subjects were monitored for 1 min of EEG waves with deep inhalation (T1) while wearing the product fragrance buckle; the subjects were monitored for 3 min of EEG waves with continuous inhalation (T2) while wearing the product fragrance buckle; the subjects were monitored for 5 min of EEG waves after inhalation (T3) after removing the product fragrance buckle. The β / α values of the EEG waves in the F3 (left frontal lobe) and F4 (right frontal lobe) channels were taken before product inhalation (T0), deep inhalation (T1), continuous inhalation (T2), and after inhalation (T3) for statistical analysis. The channel corresponding to the brain part is shown in Table 2 and Figure 15 .

[0129] Table 2 Test channel and site control table

[0130] Channel Fp1 Fp2 F3 F4 C3 C4 P3 P4 Region Left frontal lobe Right frontal lobe Left frontal lobe Right frontal lobe Left central region Right central region Left parietal lobe Right parietal lobe Channel O1 O2 F7 F8 T3 T4 T5 T6 Region Left occipital lobe Right occipital lobe Left frontal lobe Right frontal lobe Left temporal lobe Right temporal lobe Left posterior temporal lobe Right posterior temporal lobe

[0131] 4.2 Test equipment: Digital electroencephalogram topographic instrument, XL-998C, 16 channels, Jiangsu Mutai Medical Technology Co., Ltd.

[0132] 4.3 Environmental conditions: Temperature 21-26℃, humidity 40-60% RH.

[0133] 4.4 Test procedure: The subjects' information was registered, and the informed consent form was understood and signed, and the inclusion and exclusion criteria were completed to enter the group; rest for 30 min in an environment of 21-26℃; wear the EEG monitoring device and monitor the EEG for 2 min; wear the product fragrance buckle and take a deep breath, monitor the EEG for 1 min; continuously sniff normally while monitoring the EEG for 3 min; remove the fragrance buckle and monitor the EEG for 5 min.

[0134] 4.5 Data statistics

[0135] The β / α values of the EEG waves in the F3 (left frontal lobe) and F4 (right frontal lobe) channels were taken for descriptive statistics.

[0136] The β / α values of the brain waves in the F3 (left frontal lobe) and F4 (right frontal lobe) channels were subjected to normal distribution test. If the distribution was normal, paired T test was used for statistical analysis. If the test data was not normally distributed, rank sum test method was used for statistical analysis. The above statistical analysis was a two-tailed test, and the significance level α = 0.05.

[0137] 4.6 Result judgment: P≤0.05, there is significant difference; P≤0.01, there is extremely significant difference; P>0.05, there is no significant difference. △β / α value>0, there is refreshing effect; △β / α value≤0, there is no refreshing effect.

[0138] 4.7 The test results are shown in Table 3 and Figures 16-18 The results show that, compared with the pre-usage of the refreshing 1 compound essential oil, the β / α values in the F3 (left frontal lobe) and F4 (right frontal lobe) channels of the subjects during the deep inhalation (T1), continuous inhalation (T2) and post-inhalation (T3) periods have significant differences, and the β / α values show a significant upward trend. The test results can be used to support the product's refreshing effect. During the entire test period, no adverse reaction events were found in the subjects due to the use of the product.

[0139] Table 3 β / α values of the subjects in the F3 (left frontal lobe) and F4 (right frontal lobe) channels of the refreshing 1 product

[0140]

[0141]

[0142] Experimental Example 2

[0143] The tables used in this experimental example are the Positive and Negative Affect Schedule (PANAS) and Table 4 Emotional Aroma Evaluation Table. The Positive and Negative Affect Schedule (PANAS) is referenced in the literature (Yao, et al. Applicability of the Positive and Negative Affect Schedule in Hospitalized Patients [J]. China Health Quality Management, 2019, 26(04): 64-66.).

[0144] The Positive and Negative Affect Schedule (PANAS) includes two emotional dimensions of positive (positive emotion) and negative (negative emotion), each dimension contains 10 items, and each item is ① very few or none, ② very few, ③ moderate, ④ quite a lot, ⑤ very much, corresponding to 1 point, 2 points, 3 points, 4 points, and 5 points, respectively. According to the filling information of the volunteers, the total scores of positive emotion and negative emotion were calculated; the total score of positive emotion is the total score of items 1-10, and the total score of negative emotion is the total score of items 11-20.

[0145] Table 4 Emotional evaluation of fragrance table ① little or no, ② very little, ③ moderate, ④ quite a lot, ⑤ very much corresponding score of 1 point, 2 points, 3 points, 4 points, 5 points. According to the information of volunteers filling out the table, the comprehensive score of emotional evaluation of fragrance table is calculated. The comprehensive score of emotional evaluation of fragrance table is the average score of items A-D.

[0146] Table 4 Emotional evaluation of fragrance table

[0147] Item ① Very little or none ② Very little ③ Moderate ④ Quite a lot ⑤ Very much A. Intensity of aroma □ □ □ □ □ B. Degree of pleasantness of aroma □ □ □ □ □ C. Degree of physical and mental relaxation □ □ □ □ □ D. Degree of calm and comfort □ □ □ □ □

[0148] Twelve kinds of essential oils including agarwood essential oil, bergamot essential oil, peppermint essential oil, lemon essential oil, green orange essential oil, rosemary leaf essential oil, sweet basil essential oil, silk cypress essential oil, atlantic cedar essential oil, eucalyptol rosemary essential oil, eucalyptus essential oil and tea tree essential oil are combined to prepare four kinds of compound essential oils according to different formulations.

[0149] Combination 1: 7:15:10:8:5:5:5:10:5, respectively, in volume ratio, mix evenly, and the total amount of mixture is 15 mL.

[0150] Combination 2: 10:15:15:10:9:5:5:5:5, respectively, in volume ratio, mix evenly, and the total amount of mixture is 15 mL.

[0151] Combination 3: 9:20:10:5:10:5:5:5:10, respectively, in volume ratio, mix evenly, and the total amount of mixture is 15 mL.

[0152] Combination 4: 10:15:15:10:5:10:10, respectively, in volume ratio, mix evenly, and the total amount of mixture is 15 mL.

[0153] Ten volunteers with mild anxiety (using the Generalized Anxiety Disorder Scale (GAD-7) score, volunteers with scores in the 5-9 score interval are volunteers with mild anxiety) were selected to go to the sensory laboratory for evaluation and testing. Equal amounts (15 mL) of essential oils were taken and placed directly into the same aromatherapy machine in a 25 square independent space, with a release set every 2 min. After half an hour, one person entered every 10 min, and the cycle was repeated until all 10 volunteers were finished. The 10 volunteers respectively smelled four kinds of compound essential oils, and needed to complete the evaluation and testing in the sensory laboratory. After smelling the aroma of each group of essential oil samples, fill in the positive and negative emotion scale (PANAS) and Table 4 emotional evaluation table. After the test, the scores of each group were counted, and the results are shown in Table 5. According to the data score and discussion, agarwood essential oil, peppermint essential oil, rosemary leaf essential oil, sweet basil essential oil, silk cypress essential oil, eucalyptol rosemary essential oil and lemon essential oil were selected as raw materials.

[0154] Table 5 Positive / Negative Emotion Scale Score (M ± SD)

[0155]

[0156] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled persons in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered within the protection scope of the present application.

Claims

1. A stimulating and invigorating compound essential oil, characterized in that, The ingredients include the following parts by volume: 7-10 parts agarwood essential oil, 10-15 parts peppermint essential oil, 9-15 parts ravensa leaf essential oil, 6-10 parts sweet basil essential oil, 5-8 parts cypress essential oil, 8-14 parts eucalyptol rosemary essential oil, and 10-16 parts lemon essential oil.

2. The invigorating and mind-refreshing compound essential oil as described in claim 1, characterized in that, It also includes 45-55 parts of jojoba oil.

3. The invigorating and mind-refreshing compound essential oil as described in claim 1, characterized in that, The ingredients include the following parts by volume: 9 parts agarwood essential oil, 12 parts peppermint essential oil, 10 parts ravensa leaf essential oil, 8 parts sweet basil essential oil, 6 parts cypress essential oil, 10 parts eucalyptol rosemary essential oil, and 12 parts lemon essential oil.

4. The relaxing and soothing compound essential oil as described in claim 3, characterized in that, It also includes 50 parts jojoba oil.

5. The method for preparing the invigorating and refreshing compound essential oil according to any one of claims 1-4, characterized in that, The steps include: adding agarwood essential oil, peppermint essential oil, ravensa leaf essential oil, sweet basil essential oil, cypress essential oil, eucalyptol rosemary essential oil, and lemon essential oil to jojoba oil, mixing well, and letting it stand.

6. The preparation method according to claim 5, characterized in that, Mix well after each essential oil is added.

7. The preparation method according to claim 5, characterized in that, The settling time is 12-36 hours.

8. The use of the invigorating compound essential oil as described in any one of claims 1-4 or the invigorating compound essential oil obtained by the preparation method described in claim 5 or 6 in the preparation of invigorating products.

9. The application as described in claim 8, characterized in that, The invigorating and refreshing compound essential oil is suitable for improving mental state, enhancing attention, improving cognitive function, and relieving mental fatigue.

10. The application as described in claim 8, characterized in that, The products include one or more of the following: fragrance clips, reed diffusers, sachets, body sprays, essential oil balms, and scented candles.

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