A multi-target synergistic anti-fatigue refreshing compound essential oil and a use method thereof

Abstract

The application discloses a kind of multi-target synergistic anti-fatigue refreshing compound essential oil and its using method, it is related to anti-fatigue refreshing product technical field, the essential oil is composed of active ingredient and carrier component;The active ingredient is composed of the following weight percentage: lemon essential oil 5%~20%, rosemary essential oil 3%~15%, suhe fragrant essential oil 2%~10%, mint essential oil 3%~12%, and auxiliary essential oil 15%~25%;The carrier component is isopropyl myristate, and the weight percentage is 25%~55%;The sum of the weight percentage of active ingredient group and carrier component is 100%;The application is directed to the mental depression state of mouse induced by chronic restraint stress (CRS), can significantly improve its spontaneous motor activity and exploration behavior, relieve the problem of activity reduction and power loss caused by fatigue, help body to restore normal behavior performance, simulate to improve the fatigue symptoms of human caused by long-term stress, professional body position fixation or movement deprivation.

Description

Technical Field

[0001] This invention relates to the field of anti-fatigue and energizing products, specifically to a multi-target synergistic anti-fatigue and energizing compound essential oil and its application method. Background Technology

[0002] With the accelerating pace of modern life, chronic fatigue has become a common health problem. Its pathogenesis is characterized by a complex interplay of multiple factors. It involves not only the imbalance of neurotransmitters (such as dopamine, γ-aminobutyric acid, and serotonin) in the central nervous system, but also stress-induced endocrine disorders caused by overactivation of the hypothalamus-pituitary-adrenal axis (HPA axis), cell membrane damage caused by oxidative stress, and inhibition of neuronal activity in central excitatory brain regions such as the hippocampus and cortex. Current interventions for chronic fatigue mainly fall into two categories: one is chemically synthesized anti-fatigue drugs, which can achieve short-term energizing effects by rapidly regulating a single neurotransmitter (such as increasing dopamine levels), but long-term use can easily lead to side effects such as drug dependence and increased burden on liver and kidney function, and cannot improve the root causes of chronic fatigue caused by oxidative stress or HPA axis disorders; the other is single-ingredient plant essential oils or traditional fragrance products, such as common peppermint essential oil and lemon essential oil, which mostly only achieve superficial energizing effects by activating nasal sensory nerves or briefly stimulating the sympathetic nervous system, and cannot synergistically act on multiple pathological links such as neurotransmitter imbalance, HPA axis regulation, oxidative damage repair, and central neuron activation that cause fatigue, resulting in limited anti-fatigue effects and short duration of action, making it difficult to meet the needs of people with long-term or deep fatigue.

[0003] Traditional research often focuses on the analysis of single energy metabolism pathways or single active ingredients, failing to fully utilize the synergistic effects of multiple active ingredients in plant essential oils. It also lacks systematic correlation analysis of the "active ingredient-molecular target-multi-pathological link of fatigue," making it difficult for existing essential oil-based anti-fatigue products to overcome the bottleneck of single-mechanism intervention and address the complex pathological problems of chronic fatigue from multiple dimensions. Therefore, there is an urgent need to develop an anti-fatigue and energizing product that can simultaneously regulate neurotransmitter balance, HPA axis function, oxidative stress damage, and central neuronal activity through multi-target synergistic effects, and is safe, convenient, and suitable for daily use. This would solve the problem of limited efficacy and short-lasting effects of existing anti-fatigue products due to their reliance on single mechanisms of action and inability to cover multiple pathological links of fatigue.

[0004] In response to this, this application proposes a multi-target synergistic anti-fatigue and refreshing compound essential oil and its application method to solve the above problems. Summary of the Invention

[0005] The purpose of this invention is to provide a multi-target synergistic anti-fatigue and refreshing compound essential oil and its usage method, so as to solve the problem that existing anti-fatigue products rely on a single mechanism of action and cannot cover multiple pathological aspects of fatigue, resulting in limited effects and short-lasting effects.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A multi-target synergistic anti-fatigue and refreshing compound essential oil, wherein the essential oil is composed of active ingredients and carrier components;

[0008] The active ingredient comprises the following weight percentages:

[0009] Lemon essential oil 5%–20%, rosemary essential oil 3%–15%, styrax essential oil 2%–10%, peppermint essential oil 3%–12%, and auxiliary essential oils 15%–25%;

[0010] The carrier component is isopropyl myristate, with a weight percentage of 25% to 55%; the sum of the weight percentages of the active ingredient group and the carrier component is 100%.

[0011] Furthermore, the auxiliary essential oil is at least one of the following: grapefruit essential oil (2%–5%), calamus essential oil (1%–4%), benzoin essential oil (2%–5%), eucalyptus essential oil (1%–3%), angelica essential oil (1%–3%), neroli essential oil (2%–4%), vetiver essential oil (3%–6%), borneol essential oil (1%–3%), and sweet marjoram essential oil (1%–3%).

[0012] A method for using a multi-target synergistic anti-fatigue and refreshing compound essential oil, wherein the essential oil is administered via inhalation. The compound essential oil is diluted to a concentration of 0.1% to 0.5% and then nebulized, and inhaled through the respiratory tract to exert its anti-fatigue and refreshing effects.

[0013] Furthermore, the concentration of the compound essential oil is 0.3%, the diameter of the atomized particles is 1-5 μm, and the duration of a single inhalation is 5-15 minutes.

[0014] Furthermore, the inhaled drug delivery is achieved via an electronic nebulizer, aromatherapy device, or nasal inhaler, and is used no more than three times daily.

[0015] Furthermore, the compound essential oil was obtained through network pharmacology screening and targets at least 65 key anti-fatigue targets of HSP90AA1 and MAPK1.

[0016] Furthermore, the method is used to improve anxiety-like states and regulate the levels of central neurotransmitters DA, GABA, and 5-HT.

[0017] Compared with existing technologies, this invention provides a multi-target synergistic anti-fatigue and invigorating compound essential oil and its application method. Targeting the lethargic state induced by chronic restraint stress (CRS) in mice, it can significantly improve spontaneous motor activity and exploratory behavior, alleviate the problems of reduced activity and lack of motivation caused by fatigue, help the body restore normal behavioral performance, and simulate and improve fatigue symptoms caused by long-term stress, occupational fixed posture, or exercise deprivation in humans. Compared with traditional invigorating ingredients such as single peppermint essential oil, its compound system has a more comprehensive mechanism of action, and can simultaneously act on multiple dimensions such as neurotransmitter regulation, stress endocrine regulation, oxidative damage repair, and central neuron activation, avoiding the limitations of single-mechanism intervention and achieving a more lasting and thorough anti-fatigue effect. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0019] Figure 1 A graph showing the weight change of mice provided in an embodiment of the present invention;

[0020] Figure 2 This is a diagram illustrating the performance of mice in an open field test according to an embodiment of the present invention.

[0021] Figure 3 This is a diagram illustrating the performance of mice in the elevated cross maze experiment provided in an embodiment of the present invention.

[0022] Figure 4 This is a graph showing the serum HPA hormone levels in mice provided in an embodiment of the present invention.

[0023] Figure 5 Mouse serum neurotransmitter content provided in the embodiments of the present invention Figure 1 ;

[0024] Figure 6 Mouse serum neurotransmitter content provided in the embodiments of the present invention Figure 2 ;

[0025] Figure 7 The graph shows the levels of oxidative stress indicators in mouse serum provided in this embodiment of the invention. Detailed Implementation

[0026] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0027] As attached Figure 1 To be continued Figure 7 As shown:

[0028] A multi-target synergistic anti-fatigue and invigorating compound essential oil, using:

[0029] Raw material and instrument specifications:

[0030] 1. Raw material parameters are shown in Table 1 below;

[0031] Table 1

[0032]

[0033] 2. Instruments and equipment:

[0034] Gas chromatography-mass spectrometry: Agilent 7890A-5975C (DB-5MS column, 30m×0.25mm×0.25μm);

[0035] Electronic atomizer: RELX 4th generation (adjustable atomized particle diameter 1-5μm);

[0036] Aroma diffuser: MUJI ultrasonic aroma diffuser (capacity 200mL, atomization rate 30mL / h);

[0037] Microplate reader: Thermo Multiskan FC (wavelength range 405-630nm);

[0038] Pathology microtome: Leica RM2235 (section thickness 3-5μm);

[0039] Heart rate variability analyzer: Schiller AT-104 (sampling frequency 1000Hz);

[0040] Compound essential oil formulation and preparation:

[0041] Example 1:

[0042] The precise formula (1000g scale) is shown in Table 2 below;

[0043] Table 2

[0044]

[0045] Preparation process:

[0046] S1. Pretreatment: The single essential oil was left to stand at 4℃ for 24 hours and then filtered through a 0.22μm organic filter membrane;

[0047] S2, Gradient Mixing: Preheat the carrier to 30℃, add the core essential oil (stir for 8 minutes each) and auxiliary essential oil (add in groups, stir for 5 minutes each) sequentially while stirring at 300r / min.

[0048] S3. Homogenization and ripening: Homogenize at 1500 r / min for 15 min, ripen at 25℃ in the dark for 72 h, and then dispense after confirming the stability of the components by GC-MS detection.

[0049] Example 2:

[0050] Formula composition: 5% lemon essential oil, 2% styrax essential oil, 10% total auxiliary essential oils, and 55% carrier;

[0051] Core ingredients (100g): 5g lemon, 3g rosemary, 2g styrax, 3g peppermint, 10g auxiliary essential oils, 55g carrier;

[0052] Preparation process: Same as in Example 1.

[0053] Example 3:

[0054] Formula characteristics: 20% lemon essential oil, 15% rosemary essential oil, 30% total auxiliary essential oils, and 25% carrier;

[0055] Core ingredients (100g): 20g lemon, 15g rosemary, 10g styrax, 12g peppermint, 30g auxiliary essential oils, 25g carrier;

[0056] Preparation process: Same as in Example 1.

[0057] Comparative example:

[0058] Comparative Example 1 (lacking styrax):

[0059] Formula: Remove the styrax essential oil from Example 1, replenish the carrier to 460g, and keep the other ingredients unchanged;

[0060] Objective: To verify the necessity of styrax for HPA axis adjustment.

[0061] Comparative Example 2 (Single Peppermint Essential Oil Control Group):

[0062] Recipe: 200g peppermint essential oil, 800g isopropyl myristate;

[0063] Comparative Example 3:

[0064] Formula: 3% lemon essential oil, 1% styrax essential oil, 5% auxiliary essential oils, 85% carrier.

[0065] Comparative Example 4 (Control group of commercially available similar products):

[0066] Sample: A certain brand of "energizing essential oil" (main ingredients: peppermint, lemon, vetiver, no styrax);

[0067] Core characterization detection:

[0068] 1. GC-MS component analysis, the results are shown in Table 3 below;

[0069] Table 3

[0070] Total number of volatile components 82 kinds 80 kinds 83 kinds ≥80 kinds Isopropyl myristate percentage 14.7% 12.3% 17.6% 12%—18% Key active ingredient (limonene) 8.2% 3.1% 13.5% ≥3.0% Peroxide value 3.2 meq / kg 2.8 meq / kg 4.1 meq / kg ≤10meq / kg

[0071] 2. Physicochemical stability test (refer to GB / T 14454.2), the results are shown in Table 4 below;

[0072] Table 4

[0073] Heat resistant (40℃×24h) Clear and transparent, without layering Slightly cloudy with trace amounts of sediment. No abnormalities Cold-resistant (-5℃×24h) Clear and transparent, without crystallization Needle-like crystals appeared, and the layers separated after recovery. No abnormalities Accelerated aging (50℃×30d) Limonene retention rate: 96.2% Limonene retention rate: 78.5% Principal component retention rate ≥95%

[0074] Multi-dimensional efficacy and safety verification

[0075] 1. Animal experiments (SPF-grade Kunming mice, n=80, grouped as follows: blank group / model group / Example 1-3 / Comparative Example 1-4)

[0076] (1) Fatigue model construction: Chronic restraint stress (CRS) - 6 hours of restraint per day for 7 consecutive days

[0077] (2) Dosage regimen: Nebulized inhalation, concentration 0.3%, twice daily for 10 minutes each time, for 7 consecutive days.

[0078] (3) The core detection results are shown in Table 5 below;

[0079] Table 5

[0080] Behavioral indicators Open field movement distance (m) 4.5±0.3（↑55%） 3.8±0.2（↑31%） 4.2±0.2（↑45%） 3.2±0.2（↑10%） 3.0±0.2（↑3%） 2.9±0.2 Opening time of the elevated cross-shaped maze (s) 38±4（↑81%） 30±3（↑43%） 35±3（↑67%） 22±2（↑5%） 20±2（↓4%） 21±3 Sodium pentobarbital sleep duration (min) 35±3（↓42%） 42±4（↓31%） 38±3（↓38%） 55±4（↓12%） 60±5（↓3%） 60±4 Biochemical indicators Brain DA content (ng / mL) 192±11（↑37%） 165±9（↑18%） 185±10（↑32%） 150±8（↑7%） 142±7（↑1%） 140±10 Serum cortisol (μg / dL) 8.2±0.6（↓45%） 10.5±0.8（↓30%） 9.1±0.7（↓40%） 13.8±1.0（↓12%） 15.2±1.1（↓3%） 15.0±1.2 Liver tissue MDA (nmol / mg) 3.0±0.2（↓43%） 3.8±0.3（↓28%） 3.3±0.2（↓38%） 4.6±0.3（↓17%） 5.2±0.4（↓5%） 5.3±0.4 Molecular mechanism D1R protein expression in the hippocampus (grayscale value) 0.62±0.04（↑55%） 0.55±0.03（↑38%） 0.59±0.03（↑48%） 0.45±0.02（↑13%） 0.42±0.02（↑5%） 0.40±0.02

[0081] Note: Compared with the model group, ↑ indicates an increase, ↓ indicates a decrease, P<0.05; D1R protein was detected using Western blot.

[0082] 2. Concentration gradient verification experiment (essential oil in Example 1), the results are shown in Table 6 below;

[0083] Table 6

[0084] 0.1% 3 3.2±0.2 18% No abnormalities Weak effect 0.3% 3 4.5±0.3 37% No abnormalities Optimal and safe 0.5% 3 4.6±0.3 39% Some mice exhibited agitation. Limited improvement in effectiveness, decreased safety.

[0085] 3. Multi-device compatibility test (Essential oil in Example 1, 0.3% concentration), the results are shown in Table 7 below;

[0086] Table 7

[0087] Electronic atomizer 42.3% 55% No malfunctions after 2 hours of continuous operation Ultrasonic Aroma Diffuser 38.6% 48% Uniform atomization, no liquid accumulation Nasal inhaler 45.1% 58% Single dose controllable

[0088] As can be seen above, formula effectiveness: Examples 1 to 3 can all achieve the effects of anti-fatigue + anti-anxiety through multi-target regulation (65 targets such as HSP90AA1, MAPK1, etc.). Among them, Example 1 (intermediate value formula) has the best effect, significantly better than the comparative example (P<0.05);

[0089] Concentration and equipment compatibility: A dilution concentration of 0.3% is the optimal dose, and equipment such as electronic atomizers and aromatherapy diffusers can all stably exert their effects.

[0090] Example 4:

[0091] Experiment on the construction of a mental depression model in chronic restraint stress (CRS) mice and weight changes:

[0092] I. Experimental purpose:

[0093] Verify whether chronic restraint stress (CRS) can successfully construct a mental depression model in mice, and evaluate the improvement effect of the project essential oil on the weight metabolic status of CRS mice.

[0094] II. Experimental materials:

[0095] Experimental animals: SPF-grade healthy male ICR mice, with a body weight of about 25 g, provided by Shanghai Jiesijie Experimental Animal Co., Ltd., animal production license number: SCXK (Shanghai) 2023-0004.

[0096] Experimental reagents: Project essential oil (Fragrant and Refreshing Fragrance and Gentle Breeze Vitality Essential Oil), 0.3% peppermint essential oil (positive control), distilled water (blank / model control).

[0097] Experimental equipment: Special mouse restraint tubes, electronic balance (accuracy 0.001 g).

[0098] III. Experimental methods:

[0099] Animal grouping: The mice were divided into 4 groups, with 10 mice in each group, namely the blank group, the model group, the positive control group (peppermint essential oil group), and the essential oil group (project essential oil group).

[0100] Model construction: The mice in the model group, the positive control group, and the essential oil group received CRS treatment for 19 days. From 9:00 AM to 5:00 PM every day, their physical activities were continuously restricted for 8 hours using special restraint tubes (the head could breathe freely); the blank group had no restraint treatment. ]>

[0101] Administration method: After the restraint period, the blank group and the model group inhaled distilled water nebulizer for 2 hours, the positive control group inhaled 0.3% peppermint essential oil nebulizer for 2 hours, and the essential oil group inhaled 0.3% project essential oil nebulizer for 2 hours, once a day for 19 days.

[0102] Indicator detection: The weight of mice in each group was weighed and recorded every 2 days, and the range of weight change was calculated.

[0103] IV. Experimental Results:

[0104] like Figure 1 As shown, the weight change trend of mice in the blank group was as follows: the weight of mice in the blank group continued to increase from day 1 to day 13; the weight of mice in the model group, positive control group and essential oil group decreased from day 1 to day 4, with the model group showing the most significant decrease, from 34.394g to 32.577g (a decrease of about 5.3%).

[0105] Weight recovery: Starting from day 7, the weight of each model group began to rise and stabilized after day 10; the weight of the essential oil group and the positive control group fluctuated less after day 7, while the weight of the model group still fluctuated more significantly.

[0106] V. Experimental Conclusions:

[0107] CRS treatment resulted in a significant decrease in mouse body weight, successfully establishing a mouse model of lethargy (simulating human stress-induced fatigue).

[0108] Both the project essential oil and peppermint essential oil can stabilize the weight gain trajectory of CRS mice, and the project essential oil has a more prominent effect on regulating weight metabolism, suggesting that it can improve stress-induced metabolic disorders.

[0109] Example 5:

[0110] Open field (OFT) assay to assess the effects of essential oils on the movement and exploratory behavior of CRS mice:

[0111] I. Experimental Objective:

[0112] The effects of the essential oil on spontaneous movement activity and exploratory behavior in CRS mice were quantified using an open field experiment, and its anti-fatigue and energizing effects were verified.

[0113] II. Experimental Materials:

[0114] Experimental animals: Same as in Example 4 (CRS model mice).

[0115] Experimental equipment: 4 rectangular open field boxes (40cm×40cm×31cm), spontaneous activity video analysis system (Shanghai Jiliang Software Technology Co., Ltd., model JLBchv-LAM-4).

[0116] III. Experimental Methods:

[0117] Experimental environment preparation: Keep the experimental environment quiet and dimly lit, with a temperature of 20°C-24°C and a relative humidity of 40%-50%. Move the mice to the test room 2 hours before the experiment to adapt to the environment.

[0118] Experimental procedure: Four groups of mice were gently placed in the center of an open field box (facing away from the experimenter). Activity data for 12 minutes was recorded using a video analysis system, and the last 10 minutes of data were selected for analysis. The open field box was cleaned with 75% ethanol after the experiment.

[0119] Detection metrics: Central Residency Time (CRT), Number of Wall Climbs (CT), Average Speed ​​(AS), Total Distance Traveled (TDT).

[0120] IV. Experimental Results

[0121] like Figure 2 As shown, compared with the blank control group:

[0122] The CRS model group mice showed a significant decrease in total movement distance of 20.03%, a 21.69% reduction in time spent in the central region, a 7.02% reduction in the number of times they climbed and stood up, and a 17.05% decrease in average movement speed.

[0123] The frequency of climbing and standing, the time spent in the central area, the movement speed and the total movement distance of mice in the essential oil group were significantly higher than those in the model group. Moreover, the frequency of climbing and standing and the movement speed were better than those in the positive control group, and the behavioral performance was close to or even exceeded that of the blank group.

[0124] V. Experimental Conclusions:

[0125] CRS treatment significantly suppressed spontaneous motor activity and exploratory behavior in mice, successfully inducing a lethargic-like state consistent with reduced activity characteristics during human fatigue.

[0126] The essential oils in this project, administered via inhalation, can effectively reverse CRS-induced loss of behavioral motivation, exhibit significant anti-fatigue pharmacological activity, and show better improvement in some behaviors than peppermint essential oil alone.

[0127] Example 6:

[0128] The elevated cross maze test (EPM) was used to assess the effects of essential oils on anxiety-like states in CRS mice.

[0129] I. Experimental Objective:

[0130] Using the "approach-avoidance conflict" principle of the elevated cross maze, the study evaluated the alleviating effect of the essential oils on anxiety-like behavior in CRS mice, supplementing their anti-fatigue-related mood-regulating effects.

[0131] II. Experimental Materials:

[0132] Experimental animals: Same as in Example 4 (CRS model mice).

[0133] Experimental equipment: Elevated cross maze device (arm width 5cm, arm length 35cm, central area 5cm×5cm, closed arm height 15cm, 40-55cm above the ground), elevated cross maze video analysis system (Shanghai Jiliang Software Technology Co., Ltd., model JLBchv-EPMM).

[0134] III. Experimental Methods:

[0135] Experimental environment preparation: Keep the environment quiet, free from strong light and noise, and the temperature between 20°C and 24°C.

[0136] Experimental procedure: The mouse was gently placed in the center of the maze (facing the open arm), and its movement path was recorded over 6 minutes using a video system. The percentage of times the mouse entered the open arm (OE%) and the percentage of time it entered the open arm (OT%) were calculated. The maze was cleaned with 75% ethanol after the experiment.

[0137] IV. Experimental Results:

[0138] like Figure 3 As shown, the OE% and OT% of mice in the CRS model group were significantly lower than those in the blank control group, suggesting that chronic restraint stress significantly amplifies the anxiety level of mice.

[0139] The OE% and OT% of mice in the essential oil group were significantly higher than those in the model group, recovering to levels close to those of the blank control group. The positive control group also showed some improvement, but the effect was weaker than that of the essential oil group.

[0140] V. Experimental Conclusions:

[0141] CRS not only induces fatigue in mice, but also accompanies significant anxiety-like behavior, consistent with the synergistic occurrence of "fatigue + anxiety" under long-term stress in humans.

[0142] The essential oils used in the project can enhance mice's exploration motivation and reduce environmental fear, producing a significant anti-anxiety effect. This indicates that its anti-fatigue efficacy covers both "behavioral activation" and "emotional regulation" dimensions, resulting in a more comprehensive intervention.

[0143] Example 7:

[0144] The effect of essential oils on HPA axis hormone levels in CRS mice was detected by ELISA.

[0145] I. Experimental Objective:

[0146] By detecting key hormones (CRH, ACTH, CORT) in the serum hypothalamic-pituitary-adrenal (HPA) axis, the study revealed the regulatory mechanism of the essential oils on the stress endocrine system of CRS mice.

[0147] II. Experimental Materials:

[0148] Laboratory animals: Same as in Example 4, samples were collected 1 day after the behavioral experiment.

[0149] Experimental reagents: Mouse CRH ELISA kit, ACTH ELISA kit, CORT ELISA kit (all from Jiangxi Jianglan Pure Biological Reagent Co., Ltd., 96T specification), PBS buffer (Wuhan Saiweier Biotechnology Co., Ltd., catalog number G0002).

[0150] Experimental equipment: Microplate reader (TECAN, model Tecan sunrise F50), plate washer (Shenzhen Raydu Life Science Co., Ltd., model RY-3000), small refrigerated centrifuge (Eppendorf, model Centrifuge 5427 R).

[0151] III. Experimental Methods:

[0152] Sample collection: After mice were anesthetized with ether, blood was collected from the orbital venous plexus and the serum was separated by centrifugation (4°C, 3000 rpm, 10 minutes).

[0153] ELISA assay: After diluting the serum sample according to the kit instructions, add it to the antibody-coated ELISA plate, incubate, add the enzyme-labeled detection antibody, wash, add the chromogenic substrate, and measure the absorbance (450nm) using an ELISA reader. Calculate the hormone concentration using a standard curve.

[0154] IV. Experimental Results:

[0155] like Figure 4 As shown, compared with the blank group, the serum CORT level of mice in the model group increased significantly, and the CRH and ACTH levels increased synchronously, proving that CRS successfully activated the HPA axis function.

[0156] The serum CORT level in mice in the essential oil group was significantly lower than that in the model group; the levels of CRH, ACTH, and CORT in the positive control group were all significantly lower; the levels of CRH and ACTH in the essential oil group did not decrease synchronously with CORT, which is speculated to be related to the compensatory inhibition of ACTH release by arginine vasopressin.

[0157] V. Experimental Conclusions:

[0158] CRS-induced fatigue is closely related to overactivation of the HPA axis, consistent with the endocrine disorder mechanism of stress-induced fatigue in humans (imbalance of excitation in the HPA axis driving center).

[0159] The essential oil in the project can effectively inhibit the overactivation of the HPA axis in CRS mice (core downregulation of CORT) and relieve fatigue by regulating the neuroendocrine pathway. Its regulatory mechanism is different from that of peppermint essential oil alone (it may involve more complex neuromodulatory compensation).

[0160] Example 8:

[0161] The effect of essential oils on serum neurotransmitter levels in CRS mice was detected by ELISA.

[0162] I. Experimental Objective:

[0163] The study measured serum levels of excitatory neurotransmitters (DA), inhibitory neurotransmitters (GABA), and serotonin (5-HT) to elucidate the mechanism by which the essential oils improve fatigue by regulating neurotransmitter balance.

[0164] II. Experimental Materials:

[0165] Experimental animals and samples: Same as in Example 7 (serum samples).

[0166] Experimental reagents: Mouse DA ELISA kit, GABA ELISA kit, 5-HT ELISA kit (all from Jiangxi Jianglan Pure Biological Reagent Co., Ltd., 96T specification).

[0167] Experimental equipment: Same as in Example 7 (ELISA reader, plate washer, centrifuge).

[0168] III. Experimental Methods:

[0169] The ELISA detection procedure is the same as in Example 7, with a detection wavelength of 450nm. The concentrations of DA, GABA, and 5-HT are calculated respectively.

[0170] IV. Experimental Results:

[0171] like Figure 5 and Figure 6 The serum GABA (4.60±0.52mol / L) and 5-HT (3.59±0.46ng / mL) levels in the model group mice were 29.23%–38.53% lower than those in the blank group (GABA 6.50±0.55mol / L, 5-HT 5.84±0.59ng / mL), while the DA level was significantly increased.

[0172] In the essential oil group, GABA, 5-HT, and DA levels in mice were restored to levels close to those in the blank group. Among them, 5-HT was restored to 5.0±0.29 ng / mL (an increase of 39.3% compared with the model group), and the regulatory effect of DA level was better than that of the positive control group.

[0173] V. Experimental Conclusions:

[0174] CRS causes neurotransmitter imbalance in mice (reduction of inhibitory neurotransmitters and abnormal increase of excitatory neurotransmitters), consistent with the neural mechanism of "central inhibition-excitation imbalance" during human fatigue.

[0175] The essential oils in this project can effectively rebuild the neurotransmitter balance in CRS mice. By increasing GABA (regulating central inhibition), restoring 5-HT (improving emotional fatigue), and optimizing DA (enhancing central excitation), it alleviates central inhibition through multiple targets and exerts an invigorating effect.

[0176] Example 9:

[0177] The effect of essential oils on serum oxidative stress markers (MDA) in CRS mice was detected by ELISA.

[0178] I. Experimental Objective:

[0179] Serum malondialdehyde (MDA, a marker of oxidative stress) levels were measured to verify that the essential oils used in the project alleviate CRS-induced fatigue damage through an antioxidant mechanism.

[0180] II. Experimental Materials:

[0181] Experimental animals and samples: Same as in Example 7 (serum samples).

[0182] Experimental reagents: Mouse MDA ELISA kit (Jiangxi Jianglan Pure Biological Reagent Co., Ltd., 96T specification).

[0183] Experimental equipment: Same as in Example 7 (ELISA reader, plate washer, centrifuge).

[0184] III. Experimental Methods:

[0185] The serum MDA concentration was calculated using the same ELISA detection procedure as in Example 7, through the MDA standard curve.

[0186] IV. Experimental Results:

[0187] like Figure 7 As shown, the serum MDA content in the blank group mice was 5.56±0.70 nmol / mL, while that in the model group it increased to 6.33±0.43 nmol / mL (an increase of 13.85%), suggesting that CRS leads to oxidative stress damage and aggravated cell membrane lipid peroxidation.

[0188] The serum MDA level in the essential oil group mice recovered to 6.08±0.76 nmol / mL, which was significantly lower than that in the model group. The decrease in MDA level in the positive control group was smaller than that in the essential oil group.

[0189] V. Experimental Conclusions:

[0190] Oxidative stress injury (elevated MDA) is one of the important pathological mechanisms of CRS-induced fatigue, consistent with the "imbalance between the oxidative and antioxidant systems" during human fatigue.

[0191] The essential oils in this project can alleviate oxidative stress damage caused by CRS through antioxidant mechanisms, restore cell membrane stability and antioxidant system function, and provide mechanistic support for their anti-fatigue effects through the "oxidative protection" dimension.

[0192] Example 10:

[0193] Immunohistochemical assay of mouse brain c-fos to evaluate the effect of essential oils on neuronal activation

[0194] I. Experimental Objective:

[0195] By detecting the expression of c-fos protein (a marker of neuronal excitability) in the hippocampus and cortical regions of the brain, the activation effect of the essential oil on the central nervous system excitatory pathways in CRS mice was clarified.

[0196] II. Experimental Materials:

[0197] Experimental animals and samples: Same as in Example 4. Brain tissue was taken one day after the behavioral experiment (the left hemisphere was fixed in 4% paraformaldehyde).

[0198] Experimental reagents: Mouse c-fos ELISA kit (Jiangxi Jianglan Pure Biological Reagent Co., Ltd., 96T), environmentally friendly dewaxing and clearing solution (Wuhan Saiweier Biotechnology Co., Ltd., catalog number G1128), DAB chromogenic reagent (Wuhan Saiweier Biotechnology Co., Ltd., catalog number G1212), hematoxylin staining solution (Wuhan Saiweier Biotechnology Co., Ltd., catalog number G1004).

[0199] Experimental equipment: pathological slide machine (Shanghai Leica Instruments Co., Ltd., model RM2016), upright optical microscope (Nikon, Japan, model NIKON ECLIPSE E100), imaging system (Nikon, Japan, model NIKON DS-U3).

[0200] III. Experimental Methods:

[0201] Brain tissue processing: The fixed brain tissue was dehydrated by gradient alcohol, cleared with xylene, embedded in paraffin, sectioned (5 μm thick), and baked (60°C).

[0202] Immunohistochemical procedure: Dewaxing → Antigen retrieval → Blocking endogenous enzymes with 3% hydrogen peroxide → BSA blocking → Incubation with primary antibody (anti-c-fos antibody) at 4°C overnight → Incubation with secondary antibody (HRP labeling) at room temperature → DAB staining → Hematoxylin counterstaining → Dehydration and clearing → Mounting → Microscopic observation and imaging.

[0203] Indicator determination: c-fos positive expression is brown plaques, and the number of positive plaques in the hippocampus and cortex is counted.

[0204] IV. Experimental Results:

[0205] Compared with the control group, the number of c-fos positive plaques in the hippocampus and cortex of mice in the model group was significantly reduced, suggesting that CRS inhibits the excitability of central neurons.

[0206] The number of c-fos positive plaques in the hippocampus and cortex of mice in the essential oil group was significantly increased compared with that in the model group, and the expression effect was better than that in the positive control group, especially in the CA1 area (excitatory brain region) where the activation was more obvious.

[0207] V. Experimental Conclusions:

[0208] CRS leads to fatigue by inhibiting the excitability of central neurons (reduced c-fos expression), which is consistent with the core mechanism of "central nervous system inhibition" during human fatigue.

[0209] The essential oils in this project can specifically activate the central nervous system excitation pathways in CRS mice (especially the CA1 region of the hippocampus), enhance neuronal excitability by increasing c-fos expression, and directly exert an energizing and anti-fatigue effect from the "central activation" level, with an activation effect superior to that of peppermint essential oil alone.

[0210] As can be seen from the above, chronic restraint stress (CRS)-induced lethargy in mice can significantly improve their spontaneous motor activity and exploratory behavior, alleviate the problem of reduced activity and lack of motivation caused by fatigue, help the body restore normal behavioral performance, and simulate and improve the fatigue symptoms caused by long-term stress, occupational fixed posture or exercise deprivation in humans.

[0211] Compared to traditional energizing ingredients such as peppermint essential oil alone, its compound system has a more comprehensive mechanism of action, which can simultaneously act on multiple dimensions such as neurotransmitter regulation, stress endocrine regulation, oxidative damage repair and central neuron activation, avoiding the limitations of single-mechanism intervention and achieving a more lasting and thorough anti-fatigue effect.

[0212] Based on the precise blend of 12 single essential oils, combined with gas chromatography-mass spectrometry (GC-MS) technology, the composition of its volatile components is clearly identified. The types and contents of active ingredients are controllable, avoiding the problem of unstable efficacy caused by component fluctuations, and ensuring that different batches of products can exert a stable anti-fatigue effect.

[0213] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A multi-target synergistic anti-fatigue and invigorating compound essential oil, characterized in that, The essential oil is composed of active ingredients and carrier components; The active ingredient comprises the following weight percentages: Lemon essential oil 5%–20%, rosemary essential oil 3%–15%, styrax essential oil 2%–10%, peppermint essential oil 3%–12%, and auxiliary essential oils 15%–25%; The carrier component is isopropyl myristate, with a weight percentage of 25% to 55%; the sum of the weight percentages of the active ingredient group and the carrier component is 100%.

2. The multi-target synergistic anti-fatigue and invigorating compound essential oil according to claim 1, characterized in that, The auxiliary essential oil is at least one of the following: grapefruit essential oil 2%–5%, calamus essential oil 1%–4%, benzoin essential oil 2%–5%, eucalyptus essential oil 1%–3%, angelica essential oil 1%–3%, neroli essential oil 2%–4%, vetiver essential oil 3%–6%, and borneol essential oil 1%–3%.

3. A method of using a multi-target synergistic anti-fatigue and invigorating compound essential oil, characterized in that, The essential oil is administered via inhalation. The compound essential oil is diluted to a concentration of 0.1% to 0.5% and then nebulized, allowing it to be inhaled through the respiratory tract to exert its anti-fatigue and refreshing effects.

4. The method of using a multi-target synergistic anti-fatigue and invigorating compound essential oil according to claim 3, characterized in that, The compound essential oil is diluted to a concentration of 0.3%, the atomized particle diameter is 1-5 μm, and the duration of a single inhalation is 5-15 minutes.

5. The method of using a multi-target synergistic anti-fatigue and invigorating compound essential oil according to claim 3, characterized in that, The inhaled drug delivery is achieved through an electronic nebulizer, aromatherapy device, or nasal inhaler, and is used no more than 3 times per day.

6. The method of using a multi-target synergistic anti-fatigue and invigorating compound essential oil according to claim 3, characterized in that, The compound essential oil was obtained through network pharmacology screening and is used to target the key anti-fatigue targets of HSP90AA1 and MAPK1.

7. The method of using a multi-target synergistic anti-fatigue and invigorating compound essential oil according to claim 3, characterized in that, The method is used to improve anxiety-like states and regulate the levels of central neurotransmitters DA, GABA, and 5-HT.

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