Sandalwood-containing compound essential oil composition for soothing nerves and helping sleep as well as preparation method and application thereof

Abstract

The invention discloses a compound essential oil composition containing sandalwood and capable of soothing nerves and helping sleep as well as a preparation method and application of the compound essential oil composition. The compound essential oil composition provided by the invention comprises the following components in parts by weight: 1-11 parts of sweet almond essential oil, 1-5 parts of sandalwood essential oil, 1-8 parts of rhizoma acori graminei essential oil, 1-6 parts of freesia essential oil, 1-4 parts of bitter orange leaf essential oil and 1-5 parts of purslane essential oil. The compound essential oil composition is obtained by compounding specific essential oil components, the components have an obvious synergistic effect, and the compound essential oil composition has the effects of inhibiting gamma-aminobutyric acid (GABA) lytic enzyme activity, promoting activation of a GABA receptor, increasing expression of olfactory dopamine receptor subunit D3, regulating a glutamic acid system, reducing the level of inflammatory factors and the like; the traditional Chinese medicine composition has a mechanism that multiple molecules synergistically act on multiple targets, can play a role in different emotion and physiological states due to the dual regulation effect on part of mechanisms, helps to recover the balance of a nervous system, and has remarkable effects of soothing the nerves and helping sleep.

Description

Technical Field

[0001] The present invention belongs to the technical field of natural plant essential oil combinations, and more specifically relates to a sandalwood-containing compound essential oil combination for calming the nerves and promoting sleep, as well as a preparation method and application thereof. Background Art

[0002] The pace of life for modern people is getting faster and faster, and people are under increasing pressure from work and study. Many people are unable to self-regulate well when facing pressure, and then develop insomnia symptoms, which manifest as tension, fear, dizziness, irritability, etc. Severe cases can cause dizziness, chest tightness, palpitations, shortness of breath, dry mouth, frequent urination, urgency, sweating, tremors and other autonomic nervous system symptoms and motor tension. Anxiety is a chronic, disabling disease that has a great impact on a person's life.

[0003] Medication is undoubtedly an effective treatment for anxiety disorders. Currently, modern medicine primarily focuses on pharmacological intervention and psychological adjustments. While Western medications can achieve some effectiveness, they have drawbacks such as addiction, increased resistance, and numerous side effects. Long-term use can be addictive and detrimental to recovery. Traditional Chinese medicine, however, has a long history of treating anxiety disorders, particularly aromatherapy, which utilizes essential oils through inhalation, massage, and ingestion. Essential oils are lipophilic and easily dissolve in fat. Their relatively short molecular chains allow them to easily penetrate the skin and enter the body through the abundant capillaries beneath subcutaneous fat. Essential oils are composed of very small molecules. These highly volatile substances are absorbed through the nasal mucosa and transmit signals directly to the brain, where they modulate mood and physiological functions through the limbic system. Numerous studies have shown that essential oils can have a wide-ranging impact on the human mind. When essential oils are diffused through a diffuser, the air is filled with aromatic molecules. These molecules are detected by the tiny olfactory hairs in our nasal mucosa, our sense of smell. These molecules are then transported from the nasal mucosa to the olfactory bulb, the olfactory nerves, and the olfactory pathway, ultimately transmitting the olfactory signal to the brain. The aroma of essential oils inhaled through breathing can also influence the functions of the autonomic nervous system, such as heart rate, pulse rate, skin conductivity, skin temperature, and respiratory rate, all of which can be modulated by aroma. Essential oils undoubtedly have activating, sedating, balancing, harmonizing, anti-anxiety, anti-depressant, calming, and arousing effects.

[0004] Currently, many natural plant essential oils have some calming and sleep-inducing effects, but their effectiveness is relatively poor, and multi-target regulation is difficult. Compound essential oils, due to their multiple active components and characteristics, can, to some extent, overcome the shortcomings of Western medicine. However, there is still a lack of more compound essential oil compositions with better active effects and multi-target effects. Identifying more drug candidates for the treatment of insomnia from the treasure trove of plant compounds is of great significance for improving sleep quality. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to solve the defects of existing single essential oils and the shortcomings of compound essential oils for calming the mind and promoting sleep, and to provide a multi-target compound essential oil combination for calming the mind and promoting sleep, as well as a preparation method and application thereof.

[0006] The above-mentioned purpose of the present invention is achieved through the following technical solutions: The invention provides a compound essential oil combination, which comprises the following components in parts by weight: 1-11 parts of sweet almond essential oil, 1-5 parts of sandalwood essential oil, 1-8 parts of calamus essential oil, 1-6 parts of freesia essential oil, 1-4 parts of petitgrain essential oil, and 1-5 parts of purslane essential oil.

[0007] The compound essential oil combination provided by the present invention, by carefully screening and combining multiple plant essential oils, gives full play to its synergistic effect, to achieve the effect of tranquilizing the mind and helping sleep. By the synergistic effect of the multiple essential oils of sweet almond essential oil, sandalwood essential oil, calamus essential oil, freesia essential oil, petitgrain essential oil, purslane essential oil, it is possible to start from multiple targets, inhibit GABA degrading enzyme activity, promote the activation of GABA receptors, increase olfactory bulb dopamine receptor subunit D3 expression, regulate glutamate system, reduce inflammatory factor levels, etc., thereby effectively improving anxiety and depression, tranquilizing the mind and helping sleep. This multi-molecule synergistic mechanism of multiple targets enables the compound essential oil to play an active role under different emotions and physiological states, helps restore the balance of the nervous system, and ultimately achieves the effect of tranquilizing the mind and helping sleep.

[0008] Preferably, the compound essential oil combination comprises the following components in parts by weight: 1-6 parts of sweet almond essential oil, 1-4 parts of sandalwood essential oil, 2-6 parts of calamus essential oil, 1-6 parts of freesia essential oil, 1-4 parts of petitgrain essential oil, and 1-4 parts of purslane essential oil.

[0009] More preferably, the compound essential oil combination comprises the following components in parts by weight: 1-6 parts of sweet almond essential oil, 2-4 parts of sandalwood essential oil, 3-4 parts of calamus essential oil, 4-6 parts of freesia essential oil, 2-4 parts of petitgrain essential oil, and 1-3 parts of purslane essential oil.

[0010] Sweet almond essential oil, a base oil in the formula provided by this invention, is rich in nourishing ingredients such as oleic acid. It acts as a gentle carrier for the essential oil compound, providing deep moisturization, improving dryness and roughness, enhancing skin elasticity, and alleviating allergic symptoms. Its mild properties make the essential oil compound more skin-friendly, providing a good foundation for the active ingredients of other essential oils to exert their efficacy. Santalol, a major component of sandalwood essential oil, has a significant sedative effect, promoting GABA receptor activation and thereby reducing nervous system excitability. Furthermore, α-asarone in calamus essential oil increases GABA levels by inhibiting GABA-degrading enzymes (GABA-T), synergizing with santalol to further enhance the sedative effect of the essential oil compound. This synergistic effect not only quickly relieves nervous tension but also helps improve sleep quality. Linalool, a major component of freesia essential oil, inhibits the binding of glutamate NMDA receptors, thereby exerting a sedative effect. It also increases the expression of vesicular glutamate transporter 1 (VGluT1), regulating the function of the glutamate system. This dual regulatory effect on the glutamate system does not cancel each other out, but rather manifests as subtle adjustments that can be applied across various emotional and physiological states, helping to restore balance to the nervous system. Furthermore, freesia essential oil can also have a calming and hypnotic effect by increasing 5-HT levels in the hypothalamus, amygdala, and hippocampus. Components like limonene in petitgrain essential oil can indirectly activate GABA-A receptors, reduce inflammatory cytokines, and regulate serotonin (5-HT) and norepinephrine, complementing the effects of freesia essential oil and further enhancing the regulatory effects of the essential oil blend on the nervous system. Portulaca oleracea essential oil, rich in components like linoleic acid, possesses potent anti-inflammatory, antioxidant, and repairing properties, helping to promote skin healing, reduce redness and swelling, and combat aging. In essential oil blends, purslane oil not only works synergistically with other essential oils to enhance its calming and sleep-inducing effects, but also provides additional protection and repair for the skin, enhancing the overall efficacy of the blend.

[0011] The present invention provides a preparation method of the compound essential oil combination. The method comprises the following steps: weighing essential oil raw materials of various components by weight, adding sweet almond essential oil, sandalwood essential oil, calamus essential oil, freesia essential oil, petitgrain essential oil and purslane essential oil in this order, and stirring to obtain the compound essential oil combination.

[0012] As a preferred embodiment, the present invention provides a method for preparing each essential oil in the compound essential oil combination, comprising the following steps: (1) Take fresh freesia, calamus, petitgrain, sandalwood, sweet almond, and purslane, wash and dry them, and grind them into 40 mesh powder for later use; (2) Taking the above plant powders respectively, and extracting different plant essential oils using ultrasonic-assisted steam distillation; Among them, the extraction conditions of sweet almond essential oil are: ultrasonic power 150W, extraction time 60min, extraction temperature 90℃; the extraction conditions of sandalwood essential oil are: ultrasonic power 180W, extraction time 70min, extraction temperature 85℃; the extraction conditions of Acorus calamus essential oil are: ultrasonic power 200W, extraction time 60min, extraction temperature 85℃; the extraction conditions of freesia essential oil are: ultrasonic power 150W, extraction time 60min, extraction temperature 85℃; the extraction conditions of freesia essential oil are: ultrasonic power 150W, extraction time 60min, extraction temperature 85℃; the extraction conditions of petitgrain essential oil are: ultrasonic power 170W, extraction time 60min, extraction temperature 90℃; the extraction conditions of purslane essential oil are: ultrasonic power 150W, extraction time 60min, extraction temperature 85℃.

[0013] Preferably, the preparation method of the above-mentioned compound essential oil combination is: weighing the components in the following order and by weight: 1 part of sweet almond essential oil, 2 parts of sandalwood essential oil, 3 parts of calamus essential oil, 6 parts of freesia essential oil, 4 parts of petitgrain essential oil, and 1 part of purslane essential oil; mixing them at 25° C. and stirring them evenly; and dispensing them into clean containers to obtain the compound essential oil.

[0014] The present invention provides application of the compound essential oil combination in calming the mind and promoting sleep.

[0015] The present invention provides use of the compound essential oil combination in preparing a product for calming the mind and promoting sleep.

[0016] The present invention provides use of the compound essential oil combination in preparing a product for improving anxiety and depression.

[0017] The present invention provides use of the compound essential oil combination in improving sleep or in preparing a product for improving sleep.

[0018] The present invention also provides a product containing the compound essential oil combination.

[0019] Preferably, the product is any one of an smear oil, a sachet, a sachet, a scent pillow, an aromatherapy diffuser, an essential oil scent patch or a scent pill.

[0020] In addition, the present invention also provides the use of the above product in calming the mind and promoting sleep.

[0021] The present invention has the following beneficial effects: The present invention provides a compound essential oil for calming the nerves and promoting sleep, comprising sweet almond essential oil, sandalwood essential oil, calamus essential oil, freesia essential oil, petitgrain essential oil, and purslane essential oil. The compound essential oil is obtained by compounding specific essential oil components, and the components have a significant synergistic effect. The compound essential oil has a mechanism of multi-molecule synergistic action on multiple targets, such as inhibiting GABA degrading enzyme activity, promoting GABA receptor activation, increasing olfactory bulb dopamine receptor subunit D3 expression, regulating the glutamate system, and reducing inflammatory factor levels. The dual regulatory effect of the compound essential oil on some of the mechanisms can work under different emotional and physiological conditions, helping to restore the balance of the nervous system, so that the final compound essential oil has the effect of calming the nerves and promoting sleep. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This study investigates the effects of compound essential oils on the cell survival rate of human neuroblastoma cell line (SH-SY5Y).

[0023] Figure 2 This study investigates the effects of compound essential oil on cell survival in a SH-SY5Y cell injury model induced by β-amyloid protein 1-42 (Aβ1-42).

[0024] Figure 3 This is the effect of compound essential oil on acetylcholinesterase activity in the SH-SY5Y cell injury model induced by Aβ1-42.

[0025] Figure 4 The expression of GABAAα1-positive cells and GABAAγ2-positive cells in the hypothalamus and cerebral cortex. DETAILED DESCRIPTION

[0026] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0027] Unless otherwise specified, all reagents and materials used in the following examples were commercially available.

[0028] All raw materials used in the following examples are pure plant essential oils. The preparation method is as follows: fresh freesia, calamus, petitgrain, sandalwood, sweet almond, and purslane are washed, dried, and then ground into a 40-mesh powder for later use. The plant powders are then extracted using ultrasonic-assisted steam distillation to obtain the plant essential oils. Among them, the extraction conditions of sweet almond essential oil are: ultrasonic power 150W, extraction time 60min, extraction temperature 90℃; the extraction conditions of sandalwood essential oil are: ultrasonic power 180W, extraction time 70min, extraction temperature 85℃; the extraction conditions of Acorus calamus essential oil are: ultrasonic power 200W, extraction time 60min, extraction temperature 85℃; the extraction conditions of freesia essential oil are: ultrasonic power 150W, extraction time 60min, extraction temperature 85℃; the extraction conditions of freesia essential oil are: ultrasonic power 150W, extraction time 60min, extraction temperature 85℃; the extraction conditions of petitgrain essential oil are: ultrasonic power 170W, extraction time 60min, extraction temperature 90℃; the extraction conditions of purslane essential oil are: ultrasonic power 150W, extraction time 60min, extraction temperature 85℃.

[0029] Example 1 Weigh the following components in the following order and by weight: 1 part sweet almond essential oil, 2 parts sandalwood essential oil, 3 parts calamus essential oil, 6 parts freesia essential oil, 4 parts petitgrain essential oil, and 1 part purslane essential oil. Mix and stir thoroughly at 25°C. Dispense into clean containers to obtain the compound essential oil.

[0030] Example 2 Weigh the following components in the following order and by weight: 6 parts sweet almond essential oil, 2 parts sandalwood essential oil, 3 parts calamus essential oil, 1 part freesia essential oil, 4 parts petitgrain essential oil, and 1 part purslane essential oil. Mix and stir thoroughly at 25°C. Dispense into clean containers to obtain the compound essential oil.

[0031] Example 3 Weigh the following components in the following order and by weight: 4 parts sweet almond essential oil, 2 parts sandalwood essential oil, 3 parts calamus essential oil, 6 parts freesia essential oil, 1 part petitgrain essential oil, and 1 part purslane essential oil. Mix and stir thoroughly at 25°C. Dispense into clean containers to obtain the compound essential oil.

[0032] Example 4 Weigh the following ingredients in the following order and by weight: 3 parts sweet almond essential oil, 2 parts sandalwood essential oil, 1 part calamus essential oil, 6 parts freesia essential oil, 4 parts petitgrain essential oil, and 1 part purslane essential oil. Mix and stir thoroughly at 25°C. Dispense into clean containers to obtain the compound essential oil.

[0033] Example 5 Weigh the following components in the following order and by weight: 11 parts sweet almond essential oil, 2 parts sandalwood essential oil, 3 parts calamus essential oil, 1 part freesia essential oil, 1 part petitgrain essential oil, and 1 part purslane essential oil. Mix and stir thoroughly at 25°C. Dispense into clean containers to obtain the compound essential oil.

[0034] Example 6 Weigh the following components in the following order and by weight: 6 parts freesia essential oil, 4 parts petitgrain essential oil, 1 part purslane essential oil, 3 parts calamus essential oil, 2 parts sandalwood essential oil, and 1 part sweet almond essential oil. Mix and stir thoroughly at 25°C. Dispense into clean containers to obtain the compound essential oil.

[0035] Example 7 Weigh the following components in the following order and by weight: 1 part sweet almond essential oil, 2 parts sandalwood essential oil, 3 parts calamus essential oil, 6 parts freesia essential oil, 4 parts petitgrain essential oil, and 1 part purslane essential oil. Mix and stir thoroughly at 30°C. Dispense into clean containers to obtain the compound essential oil.

[0036] Comparative Example 1 Weigh the following components in the following order and by weight: 1 part sweet almond essential oil and 1 part purslane essential oil. Mix and stir evenly at 25°C, and dispense into clean containers to obtain the compound essential oil.

[0037] Comparative Example 2 Weigh the following components in the following order and by weight: 1 part sweet almond essential oil, 2 parts sandalwood essential oil, 3 parts calamus essential oil, and 1 part purslane essential oil. Mix and stir evenly at 25°C. Dispense into clean containers to obtain the compound essential oil.

[0038] Comparative Example 3 Weigh the following components in the following order and by weight: 1 part sweet almond essential oil, 2 parts sandalwood essential oil, 4 parts petitgrain essential oil, and 1 part purslane essential oil. Mix and stir thoroughly at 25°C. Dispense into clean containers to obtain the compound essential oil.

[0039] Comparative Example 4 Weigh the following components in the following order and by weight: 1 part sweet almond essential oil, 2 parts sandalwood essential oil, 3 parts calamus essential oil, 6 parts freesia essential oil, and 1 part purslane essential oil. Mix and stir thoroughly at 25°C. Dispense into clean containers to obtain the compound essential oil.

[0040] Test Example 1 Antioxidant Stress Because oxidative stress can lead to mood and sleep disturbances, including insomnia, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging ability of different essential oil compounds was determined. Accurately weigh 4 mg of DPPH and dissolve it in methanol in a 100 mL volumetric flask to create the DPPH solution. Add 0.1, 0.3, and 0.5 mL (make up the remaining volume to 0.5 mL with methanol) of the essential oil compounds from Examples 1-7 and Comparative Examples 1-4, respectively, to a test tube and mix well with 2 mL of the DPPH solution. Incubate in the dark for 30 minutes. Using the corresponding blank solution as a control, measure the absorbance at 517 nm. The DPPH free radical inhibition rate of the different essential oil compounds was calculated using the following formula.

[0041]

[0042] Where: A0 is the absorbance after reaction without adding sample inhibitor; A b is the absorbance after adding sample inhibitor; A * is the absorbance without adding inhibitor and DPPH.

[0043] The measurement results are shown in Table 1, which show that at the same concentration and dosage, the compound essential oils of Examples 1-7 have significantly higher DPPH radical scavenging rates than the compound essential oils of Comparative Examples 1-4, have better antioxidant effects, and can reduce oxidative stress. The essential oil formulas of the embodiments can synergistically enhance the antioxidant effect.

[0044] Table 1 Scavenging effect of compound essential oils on free radicals

[0045] Test Example 2 Antibacterial Experiment The plate spreading method and serial dilution method were used to determine the effects of different essential oil combinations on Staphylococcus aureus ( S. aureus ATCC6538) and Escherichia coli ( E. coli ATCC25922) (purchased from Guangdong Institute of Microbiology) to test the antibacterial activity. Different samples were incubated in 100 μL activated Staphylococcus aureus and Escherichia coli (10 5 The cells were incubated with continuous shaking in a 5% FU / mL (0.1 FU / mL) dilution solution as a control. Antimicrobial activity was determined based on colony counts for Staphylococcus aureus and Escherichia coli. All experiments were performed in triplicate. The antimicrobial rate was calculated using the following formula:

[0046] Where: N control and N experimentalRepresent the bacterial counts of the control and experimental samples, respectively.

[0047] The results are shown in Table 2, which show that the compound essential oils of Examples 1-7 have good antibacterial effects, which are better than those of Comparative Examples 1-4. The compound essential oils have good antibacterial properties, can better meet the needs of special groups, and also provide more opportunities for product market expansion.

[0048] Table 2 Antibacterial test results

[0049] Test Example 3 In vitro efficacy and toxicology experiments of compound essential oils 1. Test materials SH-SY5Y cells (human neuroblastoma cells) were used (purchased from the Cell Bank of the Chinese Academy of Sciences, cell number: SNL-092); culture medium (liquid): DMEM + 10% FBS + 1% P / S. The compound essential oil used was the compound essential oil of Example 1.

[0050] Reagents: 0.25% trypsin digestion solution, phosphate-buffered saline (PBS), dimethyl sulfoxide (DMSO), Aβ1-42 (human amyloid 1-42), and CCK-8 solution.

[0051] 2. Cell culture and solution preparation Cell culture conditions: 37°C, 95% air + 5% carbon dioxide.

[0052] Cell passaging: After disinfecting the operating table, take the SH-SY5Y cell culture medium, pour off the old culture medium and wash with 4 mL of PBS solution, then add 1 mL of trypsin digestion solution. After 1-3 minutes of digestion, add 5 mL of culture medium to terminate the digestion, pipette the cells into a suspension, inoculate the cell suspension into new culture medium and incubate for 24 hours.

[0053] Preparation of Aβ1-42 solution: Dissolve Aβ1-42 powder in culture medium containing 1% dimethyl sulfoxide (DMSO) to prepare an Aβ1-42 solution with a concentration of 200 μM for later use.

[0054] Preparation of compound essential oil solution: Dilute the compound essential oil of formula 1 prepared according to Example 1 with DMSO solution 40 times to prepare a compound essential oil solution with a concentration of 25 μL / mL for later use.

[0055] 3. Effects of compound essential oils on SH-SY5Y cell cytotoxicity SH-SY5Y cells were cultured at 1×10 4 mL -1The cells were inoculated into 96-well plates and cultured until they adhered to the wall. Then, 0 μL, 1.5 μL, 2 μL, 2.5 μL, 3 μL, and 3.5 μL of the compound essential oil solution were taken and co-cultured with the cells for 24 h. Then, 10 μL of CCK-8 solution was added to each well and the cells were cultured at 37°C for 1 h. The optical density (OD) value at a wavelength of 450 nm was detected using a microplate reader, and the cell survival rate was calculated based on the absorbance.

[0056] The results are shown in Table 3 and Figure 1 As shown, when the addition amount of the compound essential oil solution was 2 μL, the compound essential oil had no obvious cytotoxicity to nerve cells and had the greatest protective effect.

[0057] Table 3 Effect of the compound essential oil of Example 1 on the survival rate of SH-SY5Y cells

[0058] 4. Effect of compound essential oil on the survival rate of SH-SY5Y cell injury model induced by Aβ1-42 Blank group: SH-SY5Y cells were cultured at 1×10 4 mL -1 The cells were seeded into 96-well plates and cultured until they adhered to the wall. Then, 20 μL of culture medium containing 1% DMSO was added and cultured for 2 h. After that, 2 μL of DMSO was added and cultured for 12 h. Then, 10 μL of cell counting kit-8 (CCK-8) solution was added and cultured at 37°C for another 1 h.

[0059] Model group: SH-SY5Y cells were cultured at 1×10 4 mL -1 The cells were seeded in a 96-well plate and cultured until they adhered to the wall. 20 μL of Aβ1-42 solution was added and cultured for 2 h. 2 μL of DMSO was added and cultured for 12 h. 10 μL of CCK-8 solution was added and cultured at 37°C for another 1 h.

[0060] Compound essential oil group: SH-SY5Y cells were cultured at 1×10 4 mL -1 The cells were seeded in a 96-well plate and cultured until they adhered to the wall. Then, 20 μL of Aβ1-42 solution was added and cultured for 2 h. Then, 2 μL of compound essential oil solution was added and cultured for 12 h. Finally, 10 μL of CCK-8 solution was added and cultured at 37°C for another 1 h.

[0061] The optical density (OD) values of the above three groups at a wavelength of 450 nm were detected by a microplate reader, and the cell survival rate was calculated based on the absorbance.

[0062] The experimental results are shown in Table 4 and Figure 2As shown, the survival rate of SH-SY5Y cells after treatment with Aβ1-42 was 57.07%, which would damage the cells. However, the survival rate of SH-SY5Y cells treated with the compound essential oil of Example 1 was significantly improved to 93.12%, which could alleviate and restore the damage of SH-SY5Y cells induced by Aβ1-42.

[0063] Table 4 Cell survival rate of the compound essential oil of Example 1 in the SH-SY5Y cell injury model induced by Aβ1-42

[0064] 5. Effect of compound essential oil on acetylcholinesterase activity in Aβ1-42-induced SH-SY5Y cell injury model Blank group: SH-SY5Y cells were cultured at 1×10 5 mL -1 The cells were seeded in a 24-well plate and cultured until they adhered to the wall. Then, 200 μL of culture medium containing 1% DMSO was added and cultured for 2 h. Then, 20 μL of DMSO was added and cultured for 12 h.

[0065] Model group: SH-SY5Y cells were cultured at 1×10 5 mL -1 The cells were seeded in 24-well plates and cultured until they adhered to the wall. Then, 200 μL of Aβ1-42 solution was added and cultured for 2 h, followed by 20 μL of DMSO for 12 h.

[0066] Compound essential oil group: SH-SY5Y cells were cultured at 1×10 5 mL -1 The cells were seeded in a 24-well plate and cultured until they adhered to the wall. Then, 200 μL of Aβ1-42 solution was added and cultured for 2 h. Then, 20 μL of compound essential oil solution was added and cultured for 12 h.

[0067] The cells of the above three groups were disrupted by ultrasonic wave in an ice bath (power 300W, ultrasonic wave 3 seconds, interval 7 seconds, total time 3 minutes). The cell lysate was placed in a centrifuge tube and centrifuged at 8000×g, 4°C for 10 minutes. The supernatant was placed on ice for measurement. Finally, the optical density value at a wavelength of 412 nm was detected by a microplate reader, and the acetylcholinesterase activity was calculated based on the absorbance.

[0068] The experimental results are as follows Figure 3 As shown in Table 5, the acetylcholinesterase activity of cells was significantly increased after treatment with Aβ1-42, and was significantly inhibited after treatment with the compound essential oil, indicating that the compound essential oil can help improve nerve function and have a positive effect on tranquility and sleep by inhibiting acetylcholinesterase activity.

[0069] Table 5 Effect of the compound essential oil of Example 1 on acetylcholinesterase activity in the SH-SY5Y cell injury model induced by Aβ1-42

[0070] Test Example 4 Effect of compound essential oil on sleep latency and sleep time induced by sodium pentobarbital in mice SPF-grade Kunming male mice (Guangdong Medical Laboratory Animal Center) were selected and randomly divided into 10 groups. All groups, except the blank control group, were treated with intraperitoneal injection of p-chlorophenylalanine (PCPA) to induce an insomnia model. Each mouse received a 400 mg / kg dose of PCPA, prepared as a suspension in weakly alkaline saline. The blank control group received aerosolized inhalation of distilled water, while the Example 1-7 groups received aerosolized inhalation of the compound essential oils of Examples 1-7. The Comparative Example 1-4 groups received aerosolized inhalation of the essential oils described in Comparative Examples 1-4. This process was repeated for 10 consecutive days, with each session lasting 40 minutes and a dosage of 80 μL. On the 10th day, all groups received an intraperitoneal injection of 60 mg / kg of pentobarbital solution after aerosolized inhalation of the corresponding substances. The injection time (TR) was recorded. After injection, the mice were placed in a feeding box individually. The time when the righting reflex of each mouse disappeared for 1 minute within 30 minutes was recorded, indicating that the mouse fell asleep (TS). The time when the righting reflex of each mouse was restored was recorded, indicating that the mouse woke up (TW). The sleep latency (SleepLatency) and sleep time (TotalSleepTime) of each mouse were calculated, and the sleep latency time and sleep time were recorded.

[0071] The calculation formula is: SleepLatency = TS-TR, TotalSleepTime = TW-TS As shown in Table 6, compared with the blank control group, Examples 1-7, Comparative Examples 1-4, and the sleep latency were increased and decreased, indicating that they had a certain effect of improving sleep. The effect of the compound essential oil of the two essential oil formulas in Comparative Example 1 on improving sleep was significantly reduced. Overall, at the same essential oil concentration and dosage, the effect of the essential oil compound in Comparative Examples 2-4 was significantly inferior to that of Examples 1-7, indicating that the compound essential oil combined the efficacy of various essential oils, synergized with each other, and harmonized the medicinal properties, so that the entire prescription achieved better results and more effectively promoted sleep.

[0072] Table 6 Effects of different groups of compound essential oils on sleep latency and sleep time in mice

[0073] Test Example 5 Effects of compound essential oil on the expression of GABAAα1 and GABAAγ2 in rat hippocampus and cerebral cortex Establishment of an insomnia model in mice: A 25 mg / kg PCPA weak alkaline saline suspension was injected intraperitoneally for the first time. 28-32 hours after administration, the circadian rhythm of the animals disappeared and the animals remained active day and night, indicating that the model was successfully established.

[0074] Mice were randomly divided into three groups. This experiment included a control group, a model group, and a compound essential oil group. After the PCPA insomnia model was established, the model group did not receive essential oil inhalation intervention, while the compound essential oil group received aromatherapy treatment for 1 hour every day for 7 consecutive days using the compound essential oil of Example 1. The essential oil was diluted with distilled water, and the concentrations of low, medium, and high doses of essential oil were 3×10 -3 g / mL, and the inhalation time was 8:00 am every day.

[0075] Hypothalamus and cerebral cortex from different mice were isolated and heat-removed using sodium citrate buffer for antigen detection. Immunohistochemical staining with SP was performed, developed with 3,3'-diaminobenzidine / hydrogen peroxide (DAB / H₂O₂), and counterstained with hematoxylin. According to the kit instructions, sections with known positive antibodies served as positive controls, and the primary antibody was replaced with PBS as a negative control. Subsequently, sections were placed on a microscope for image acquisition, and the integrated optical density (IOD) of GABAAα1- and GABAAγ2-positive cells was analyzed using Image-Pro Plus 5.1 software.

[0076] The results are as follows Figure 4 As shown in the figure, in each group, the GABAAα1-positive cells and GABAAγ2-positive cells in the hypothalamus and cerebral cortex were purple-gray, and the morphology of the positive cells was round, oval, etc. From the expression of GABAAα1-positive cells and GABAAγ2-positive cells in the hypothalamus and cerebral cortex, the expression of GABAAα1-positive cells and GABAAγ2-positive cells in the model group was significantly reduced compared with the control group ( P <0.01). Compared with the model group, the expression of GABAAα1-positive cells and GABAAγ2-positive cells in the hypothalamus and cerebral cortex in the compound essential oil group was significantly increased ( P <0.01). This shows that aromatherapy with the compound essential oil can significantly increase the expression of GABAAα1 and GABAAγ2 positive cells in the rat hippocampus and cerebral cortex, indicating that the compound essential oil can improve the neural function of mice in the insomnia model by regulating the expression of GABA receptors.

[0077] Test Example 6: Verifying the Effect of Compound Essential Oil on Sleep-Related Gene Expression Using qRT-PCR To validate the multi-target calming and sleep-inducing mechanism of the compound essential oil, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to examine its effects on the expression of genes related to the GABA, dopamine, 5-HT, glutamate, and inflammatory factors in the brain tissue of insomnia model mice. SPF male Kunming mice (weighing 28±2g) were randomly divided into a blank control group, a model group, a compound essential oil group according to Example 1, a positive control group (diazepam), a compound essential oil group according to Comparative Example 1, a compound essential oil group according to Comparative Example 2, and a compound essential oil group according to Comparative Example 3, with 10 mice in each group. Insomnia models were established in the model and treatment groups via intraperitoneal injection of PCPA (400mg / kg). The compound essential oil and single essential oil groups received nebulized inhalation of a 1% compound essential oil or single essential oil solution (80μL / inhalation, 40 minutes / day) for 7 consecutive days. The positive control group received an injection of diazepam (1mg / kg). 24 hours after the last treatment, the hippocampus and hypothalamus tissues of the mice were obtained and quickly frozen in liquid nitrogen for storage.

[0078] Total RNA was extracted from tissues using TRIzol reagent method (TRIzol) and the purity (OD 260 / OD 280 ≥1.8) and integrity, cDNA was synthesized using a reverse transcription kit. Target gene primers were designed and synthesized, including GABAA receptor α1 subunit (GABRA1), dopamine receptor D3 (DRD3), 5-HT1A receptor (HTR1A), glutamate receptor NR2A subunit (GRIN2A), inflammatory factor IL-6, and internal reference gene GAPDH. The qRT-PCR reaction system was 20 μL (containing SYBR Green Master Mix, cDNA, and primers), and the reaction procedure was 95°C pre-denaturation for 5 min, 95°C for 10 s, and 60°C for 30 s for a total of 40 cycles. Melting curve analysis was used to verify amplification specificity. Three technical replicates were set for each sample, and ΔΔ Ct The relative gene expression was calculated by 2^ -ΔΔCt Indicates that GraphPad Prism performed one-way ANOVA and Tukey test (* P <0.05 was considered a significant difference).

[0079] The results, shown in Table 7, show that the compound essential oil group in Example 1 significantly upregulated the expression of GABRA1, DRD3, HTR1A, and BDNF (recovering to 92%, 88%, 95%, and 98% of the blank group, respectively), while simultaneously downregulating the expression of GRIN2A and IL-6 (reduced by 40% and 48% compared to the model group). This suggests that the compound essential oil group synergistically regulates neurotransmitter balance by enhancing GABAergic, dopaminergic, and 5-HTergic signaling, inhibiting glutamate overactivation and inflammatory responses. The diazepam group only improved the GABA system and had no significant effect on dopamine, 5-HT, or inflammatory factors. The compound essential oil groups in Comparative Examples 1-3 showed weaker effects than the compound essential oil group in Example 1, further validating the synergistic mechanism of the compound essential oil group.

[0080] Studies on the individual essential oils in the formula show that while sweet almond essential oil has nourishing and soothing effects, its calming and sleep-inducing effects are relatively limited and lack multiple regulation of neurotransmitters. Although sandalwood essential oil can relax by activating GABA receptors, its effect is relatively limited, making it difficult to comprehensively regulate multiple nervous systems. Acorus calamus essential oil has a significant effect on increasing GABA levels, but it mainly inhibits GABA-T and fails to effectively affect other important neurotransmitters, resulting in a limited range of effects. Freesia essential oil has a certain sedative effect on the glutamate system, but its effect on dopamine and GABA receptors is weaker, and its effect in regulating mood and sleep may be insufficient when used alone. Petitgrain essential oil relieves anxiety by regulating GABA-A receptors and the 5-HT system, but its effect is relatively mild and may not provide significant calming effects in a short period of time. Although purslane essential oil has anti-inflammatory and repairing effects, its direct regulation of the nervous system is limited, making it difficult to exert a strong calming effect. Compound essential oils make up for the shortcomings of single essential oils through the combined effects of multiple ingredients. They can synergistically regulate the nervous system at multiple targets, thereby more effectively achieving the goal of calming the mind and promoting sleep.

[0081] This experiment confirmed the multi-target calming and sleep-inducing effects of the compound essential oil at the gene expression level, which is consistent with previous pharmacodynamic and behavioral results.

[0082] Table 7 qRT-PCR detection results of target genes

[0083] (* in the table indicates a significant difference between the model group and the blank group P <0.05; # indicates significant difference between the experimental group and the model group P <0.05) Test Example 7 Aroma Sensory Evaluation Experiment of Compound Essential Oil 1. Aroma sensory evaluation Aroma sensory testing was conducted on the compound essential oils of Examples 1-7 and Comparative Examples 1-4. Thirty sensory panelists (with normal sense of smell and a 1:1 male-to-female ratio) performed olfactory sensory evaluations. The scoring table for the aroma sensory evaluation is shown in Table 8. The average of each score was taken, and the sensory scores of the different compound essential oils were calculated based on the scoring table.

[0084] Table 8 Sensory evaluation scoring table

[0085] The statistical results are shown in Table 9. According to the sensory evaluation scores of the compound essential oils of each formula, the total scores of the formulas of Examples 1-7 are all higher than 10 points, and the average total score of Comparative Examples 1-4 is 8.5 points, indicating that the compound essential oils of Examples 1-7 have good aroma intensity, coordination and pleasantness, and the aroma is pleasant.

[0086] Table 9 Sensory evaluation scores of compound essential oils of various formulas

[0087] 2. Acceptability Survey Further, the compound plant essential oil of Example 1 and the commercially available lavender-based calming and sleep-inducing compound essential oil were subjected to odor evaluation. 30 volunteers were randomly selected to conduct statistical analysis of the acceptability of different essential oils. The acceptability calculation formula is as follows:

[0088] The statistical results are shown in Table 10, which show that the acceptance rate of the compound plant essential oil of Example 1 with freesia as the main note is 70%, which is much higher than the acceptance rates of 63% and 53% of the control oil with lavender as the main note.

[0089] Table 10 Odor acceptance test results

[0090] In summary, the present invention selects freesia essential oil, petitgrain essential oil, purslane essential oil, Indian sandalwood essential oil, calamus essential oil, and sweet almond essential oil for compounding. In vitro and in vivo sleep experiments show that after these six essential oils are compounded in a specific ratio, they can synergistically regulate the nervous system at multiple targets, and have the effects of inhibiting GABA degrading enzyme activity, promoting the activation of GABA receptors, increasing the expression of olfactory bulb dopamine receptor subunit D3, regulating the glutamate system, and reducing the level of inflammatory factors. The dual regulation of some mechanisms can play a role in different emotions and physiological states, help restore the balance of the nervous system, have a significant calming and sleep-inducing effect, can relieve insomnia, and be used to improve the sleep condition of people with poor sleep. It can subjectively and significantly improve the sleep condition of people with poor sleep, and objectively can increase the deep sleep time and deep sleep ratio of the subjects to a certain extent, reduce the sleep latency, and the odor acceptability has obvious advantages compared with commercially available products, and can be used to prepare more calming and sleep-inducing products.

[0091] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A compound essential oil combination, characterized in that, The invention comprises the following components in parts by weight: 1-11 parts of sweet almond essential oil, 1-5 parts of sandalwood essential oil, 1-8 parts of calamus essential oil, 1-6 parts of freesia essential oil, 1-4 parts of petitgrain essential oil and 1-5 parts of purslane essential oil.

2. The compound essential oil combination according to claim 1, wherein The invention comprises the following components in parts by weight: 1-6 parts of sweet almond essential oil, 1-4 parts of sandalwood essential oil, 2-6 parts of calamus essential oil, 1-6 parts of freesia essential oil, 1-4 parts of petitgrain essential oil and 1-4 parts of purslane essential oil.

3. The compound essential oil combination according to claim 2, wherein The invention comprises the following components in parts by weight: 1-6 parts of sweet almond essential oil, 2-4 parts of sandalwood essential oil, 3-4 parts of calamus essential oil, 4-6 parts of freesia essential oil, 2-4 parts of petitgrain essential oil and 1-3 parts of purslane essential oil.

4. The method for preparing the compound essential oil combination according to any one of claims 1 to 3, wherein Weigh the essential oil raw materials of each component according to weight, and add sweet almond essential oil, sandalwood essential oil, calamus essential oil, freesia essential oil, petitgrain essential oil, and purslane essential oil in this order, and stir and mix evenly to obtain the product.

5. Use of the compound essential oil combination according to any one of claims 1 to 3 in calming the nerves and promoting sleep.

6. Use of the compound essential oil combination according to any one of claims 1 to 3 in preparing a product for calming the nerves and promoting sleep.

7. Use of the compound essential oil combination according to any one of claims 1 to 3 in the preparation of a product for improving anxiety and depression.

8. Use of the compound essential oil combination according to any one of claims 1 to 3 in improving sleep or in preparing a product for improving sleep.

9. A product, characterized in that Contains the compound essential oil combination according to any one of claims 1 to 3.

10. The product according to claim 9, characterized in that: The product is any one of an smear oil, a sachet, a sachet, a scent pillow, an aromatherapy diffuser, an essential oil scent patch or a scent pill.

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