Detailed Description
The term as used herein:
"prepared from … …" is synonymous with "comprising". The terms "comprising," "including," "having," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, step, method, article, or apparatus.
The conjunction "consisting of … …" excludes any unspecified element, step or component. If used in a claim, such phrase will cause the claim to be closed, such that it does not include materials other than those described, except for conventional impurities associated therewith. When the phrase "consisting of … …" appears in a clause of the claim body, rather than immediately following the subject, it is limited to only the elements described in that clause; other elements are not excluded from the stated claims as a whole.
When an equivalent, concentration, or other value or parameter is expressed as a range, preferred range, or a range bounded by a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when ranges of "1 to 5" are disclosed, the described ranges should be construed to include ranges of "1 to 4", "1 to 3", "1 to 2 and 4 to 5", "1 to 3 and 5", and the like. When a numerical range is described herein, unless otherwise indicated, the range is intended to include its endpoints and all integers and fractions within the range.
In these examples, the volume fraction represents a basic unit of measurement of the volume relationship of the components, and 1 part may represent an arbitrary unit volume, for example, 1mL, 1. Mu.L, or 1L, 2.689L, or the like. If we say that the volume fraction of the A component is a part and the volume fraction of the B component is B part, the ratio a of the volume of the A component to the volume of the B component is: b. alternatively, the volume fraction of the A component is aK, and the volume fraction of the B component is bK (K is any number and represents a multiple factor). It is not to be misunderstood that the sum of the parts by volume of all the components is not limited to 100 parts.
"and/or" is used to indicate that one or both of the illustrated cases may occur, e.g., a and/or B include (a and B) and (a or B).
The application provides compound essential oil, which comprises the following raw materials in parts by volume: 5 to 10 parts (for example, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts or any value between 5 and 10 parts), 5 to 20 parts (for example, 5 parts, 7 parts, 9 parts, 11 parts, 13 parts, 15 parts, 17 parts, 19 parts, 20 parts or any value between 5 and 20 parts) of white atractylodes rhizome essential oil, 1 to 5 parts (for example, 1 part, 2 parts, 3 parts, 4 parts, 5 parts or any value between 1 and 5 parts) of rhizoma ligustici wallichii essential oil, 5 to 20 parts (for example, 5 parts, 7 parts, 9 parts, 11 parts, 13 parts, 15 parts, 17 parts, 19 parts, 20 parts or any value between 5 and 20 parts) of dried orange peel essential oil, 2 to 8 parts (for example, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts or any value between 2 and 8 parts), 0.5 to 1 part (for example, 0.5 parts, 6 parts, 4 parts, 5 parts, 5.5 parts or any value between 1.5 parts, 10 parts, 0.5 parts or any value between 5.5 parts, 40 parts of lavender essential oil), 0.5 to 20 parts (for example, 0.5 parts, 40 parts or any value between 0.40 parts).
In a preferred embodiment of the present application, the compound essential oil of the present application comprises the following raw materials in parts by volume: 5 to 8 parts of angelica essential oil, 8 to 15 parts of bighead atractylodes rhizome essential oil, 2 to 4 parts of ligusticum wallichii essential oil, 8 to 15 parts of dried orange peel essential oil, 2 to 5 parts of frankincense essential oil, 0.5 to 0.8 part of grassleaf sweelflag rhizome essential oil and 15 to 30 parts of lavender essential oil.
The application provides a preparation method of the compound essential oil, which comprises the steps of mixing the raw materials. The essential oils of Chinese angelica, largehead atractylodes rhizome, szechuan lovage rhizome, tangerine peel, frankincense, grassleaf sweetflag rhizome and lavender are volatile oils prepared by respectively taking Chinese angelica, largehead atractylodes rhizome, szechuan lovage rhizome, tangerine peel, frankincense, grassleaf sweetflag rhizome and lavender as raw materials and adopting a steam distillation method, an organic solvent extraction method, a carbon dioxide supercritical extraction method or a squeezing method.
The angelica essential oil contains a plurality of components such as ligustilide, n-butenolide, angelone, myrcene and the like, has the functions of improving the learning and memory capacity of rats with vascular cognitive dysfunction models, promoting the expression of the rat Bal-2 protein and inhibiting the expression of the rat Bax protein with cognitive dysfunction (VCI) models. Ligustilide has antiasthmatic effect, and can inhibit central nervous system of animal, and ferulic acid can improve peripheral circulation. The main ingredient of the white atractylodes rhizome essential oil is atractylone, and can be used for resisting oxidation, reducing blood sugar, resisting coagulation, resisting bacteria, dilating blood vessels, inhibiting heart, calming and the like. The rhizoma Ligustici Chuanxiong essential oil has effects of inhibiting animal brain activity, inhibiting spontaneous activity of body, prolonging sleep time, and tranquilizing. The pericarpium Citri Tangerinae essential oil and its main ingredient limonene have effects of relieving pain of central nervous system and promoting nerve center to maintain wakefulness, and limonene may achieve tranquilization by regulating dopamine level and 5-HT receptor function. The Olibanum molecules in the essential oil can help relieve dysphoria, frustration, sadness, etc., calm mind, and help meditation. The rhizoma Acori Graminei essential oil has effects of tranquilizing, improving digestive function, relieving asthma and cough, and has good therapeutic effect on some diseases of nervous system. The lavender essential oil has a sedative effect on the heart, can reduce hypertension and calm palpitation, and is helpful for insomnia. Linalool in lavender essential oil can have beneficial effects on memory loss and behavioral modification caused by sleep deprivation by regulating 5-hydroxytryptamine, cortisol levels.
According to the compatibility principles of the same kind of mutual reinforcement, mutual supplement, mutual opposite and the like, the angelica has the effects of nourishing blood and activating blood, the ligusticum wallichii and the frankincense have the effects of activating blood and removing stasis, and promoting blood circulation and dissipating blood, and the mutual supplement has the effects of enhancing and activating blood and removing stasis, and nourishing blood and activating blood circulation; the bighead atractylodes rhizome is more important than tonifying qi, and the dried orange peel is more important than regulating qi, so that the bighead atractylodes rhizome and the dried orange peel complement each other, and the qi is regulated without hurting the qi; rhizoma Acori Graminei has effects of inducing resuscitation, eliminating phlegm, refreshing mind, improving intelligence, eliminating dampness, and stimulating appetite, and has good central nerve treatment effect; lavender is a flower fragrance type essential oil with pleasant smell, can effectively regulate the smell of the traditional Chinese medicine essential oil, and has the function of pleasurable mind and body; in conclusion, the essential oils complement each other and act together.
Through animal experiment researches, the inventor discovers that the compound essential oil obtained by setting the volume parts of the raw material essential oils in the range has the effects of sedative hypnotic and memory improvement.
However, these compound essential oils are all essential oils extracted from plants, are oily liquids, and have high volatility, so that the stability of the compound essential oils is low. Under the conditions of oxygen, illumination and high temperature, the chemical property of the compound essential oil is unstable, and the medicinal active ingredients can be volatilized, degraded and oxidized rapidly, and can be easily crystallized and separated out under the low temperature condition, so that the drug effect of the essential oil is obviously reduced. Especially, if part of the essential oil is directly used, the essential oil is also easy to generate irritation to skin. Therefore, aiming at the problems that the compound essential oil is not easy to store for a long time and skin irritation is easy to generate, the application also provides a product with sedative hypnotic and memory improving effects, which comprises the compound essential oil. Specifically, the product comprises any one of essential oil balm and essential oil spray, can effectively improve the daily application problem of the essential oil, and also has the capabilities of calming and hypnotizing and improving memory.
In one embodiment of the present application, the preparation method of the essential oil balm specifically includes the following steps:
(1) The base oil and the paste matrix are mixed according to the mass ratio of 9:3, stirring at 50-60 ℃ to obtain a matrix mixture; wherein the base oil comprises at least one of olive oil, sweet almond oil, jojoba oil, wheat germ oil, grape seed oil, coconut oil, sea buckthorn oil, sesame oil, walnut kernel oil and rose hip oil; the ointment matrix comprises at least one of beeswax, vaseline and solid oil, wherein the solid oil comprises benzyl esters of benzoic acid and cinnamic acid, lauryl esters, animal and vegetable fatty glyceride, and their mixture;
(2) The compound essential oil and the matrix mixture in the step (1) are mixed according to the mass ratio of 1: mixing (20-40), stirring at 50-60 deg.c until homogeneous, and cooling to obtain the balsam. Preferably, the mass ratio of the compound essential oil to the matrix mixture in the step (1) is 1:20 to obtain the essential oil balm.
Wherein, the mass ratio of the compound essential oil to the matrix mixture is 1: (20 to 40), for example, may be 1: 20. 1: 22. 1: 25. 1: 28. 1: 30. 1: 35. 1:40 or 1: any value of (20-40).
In one embodiment of the present application, a method for preparing an essential oil spray product specifically includes: the compound essential oil and the emulsifier are mixed according to the mass ratio of 1: uniformly stirring the components in the ratio of (1-3), then continuously dripping water into the components, and obtaining the essential oil spray after the system is clear and transparent.
Preferably, the mass ratio of the compound essential oil to the emulsifier is 1:2 are uniformly stirred according to the proportion.
The emulsifier generally needs to be an essential oil emulsifier, and specifically comprises at least one of polyglycerol fatty acid ester, sucrose fatty acid ester and alkyl polyglucoside.
After the compound essential oil and the emulsifier are uniformly mixed, water is continuously added dropwise for stirring, the stirring speed is 50-700 rpm, the stirring time is 5-60 min, and the mass of the water used is about 1.5-2.5 times of the mass of the mixed system of the compound essential oil and the emulsifier when the system is clarified.
When the essential oil spray is prepared by dripping water, double distilled water, deionized water or pure water is usually selected to ensure that no other ionic impurities exist in the water.
The essential oil balm, the essential oil spray and other products provided by the application are flexible in use mode, easy to absorb, safe, low in toxicity and less in side effect, and are all essential oil extracted from natural plants.
Embodiments of the present invention will be described in detail below with reference to specific examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1: compound essential oil
The embodiment provides a compound essential oil, which comprises the following raw materials in parts by volume:
5 parts of Chinese angelica essential oil, 8 parts of bighead atractylodes rhizome essential oil, 3 parts of ligusticum wallichii essential oil, 10 parts of dried orange peel essential oil, 5 parts of frankincense essential oil, 0.5 part of grassleaf sweelflag rhizome essential oil and 30 parts of lavender essential oil.
The specific preparation method of the compound essential oil comprises the following steps: the preparation method comprises the steps of respectively taking angelica sinensis, bighead atractylodes rhizome, ligusticum wallichii, dried orange peel, frankincense, rhizoma acori graminei and lavender as raw materials, preparing different types of volatile oil by adopting a steam distillation method, wherein the volatile oil is essential oil, and then mixing the different types of essential oil according to the formula amount to obtain the compound essential oil. For example, the compound essential oil can be obtained by uniformly mixing 5mL of angelica essential oil, 8mL of bighead atractylodes rhizome essential oil, 3mL of ligusticum wallichii essential oil, 10mL of dried orange peel essential oil, 5mL of frankincense essential oil, 0.5mL of rhizoma acori graminei essential oil and 30mL of lavender essential oil.
Example 2: compound essential oil
The embodiment provides a compound essential oil, which comprises the following raw materials in parts by volume:
10 parts of Chinese angelica essential oil, 20 parts of bighead atractylodes rhizome essential oil, 5 parts of ligusticum wallichii essential oil, 20 parts of dried orange peel essential oil, 8 parts of frankincense essential oil, 1 part of grassleaf sweelflag rhizome essential oil and 40 parts of lavender essential oil.
The specific preparation method is the same as that of example 1.
Example 3: essential oil balm with sedative hypnotic and memory improving effects
The embodiment provides an essential oil balsam with sedative-hypnotic and memory improving effects, which comprises compound essential oil mixed by the following parts by volume: 7 parts of Chinese angelica essential oil, 15 parts of bighead atractylodes rhizome essential oil, 3 parts of ligusticum wallichii essential oil, 15 parts of dried orange peel essential oil, 8 parts of frankincense essential oil, 0.7 part of grassleaf sweelflag rhizome essential oil and 20 parts of lavender essential oil. Specifically, the compound essential oil can be obtained by uniformly mixing 7L of angelica essential oil, 15L of bighead atractylodes rhizome essential oil, 3L of ligusticum wallichii essential oil, 15L of dried orange peel essential oil, 8L of frankincense essential oil, 0.7L of grassleaf sweelflag rhizome essential oil and 20L of lavender essential oil.
The specific preparation method of the essential oil balm comprises the following steps:
(1) Olive oil and beeswax are mixed according to the mass ratio of 9:3, mixing in proportion, and stirring at 60 ℃ to obtain a matrix mixture;
(2) Weighing the matrix mixture in the step (1), and mixing the matrix mixture with compound essential oil in the implementation according to the mass ratio of 20:1, uniformly stirring at 60 ℃, and cooling to obtain the essential oil balm.
Example 4: essential oil spray with sedative hypnotic and memory improving effects
The embodiment provides an essential oil spray with sedative hypnotic and memory improving effects, which comprises compound essential oil mixed by the following parts by volume: 10 parts of Chinese angelica essential oil, 10 parts of bighead atractylodes rhizome essential oil, 5 parts of ligusticum wallichii essential oil, 10 parts of dried orange peel essential oil, 3 parts of frankincense essential oil, 0.8 part of grassleaf sweelflag rhizome essential oil and 25 parts of lavender essential oil.
The specific preparation method of the essential oil spray in the embodiment comprises the following steps:
the compound essential oil and the polyglycerol fatty acid ester of the embodiment are mixed according to the mass ratio of 1:2, continuously stirring at the stirring speed of 500rpm for 30min to obtain an emulsifying system, then continuously dripping double distilled water into the emulsifying system until the system becomes clear, wherein the mass of the double distilled water is about 2 times that of the emulsifying system, and thus the essential oil spray can be obtained.
Comparative example 1: compound essential oil
The comparative example provides a compound essential oil, which comprises the following raw materials in parts by volume:
5 parts of Chinese angelica essential oil, 10 parts of bighead atractylodes rhizome essential oil, 5 parts of ligusticum wallichii essential oil, 10 parts of dried orange peel essential oil and 10 parts of lavender essential oil.
The specific preparation method is the same as that of example 1.
Comparative example 2: compound essential oil
10 parts of angelica essential oil, 10 parts of bighead atractylodes rhizome essential oil, 10 parts of dried orange peel essential oil, 5 parts of frankincense essential oil and 20 parts of lavender essential oil.
The specific preparation method is the same as that of example 1.
It should be noted that, when researching the technical scheme of the application, the inventor of the application performs safety tests on different essential oils in compound essential oil in advance, and finds that the grassleaf sweelflag rhizome essential oil has certain toxicity, and the specific tests are as follows:
acorus gramineus essential oil sniffing toxicity experiment
Randomly dividing 112 KM mice into 7 groups, each group of 16 mice with half male and female, continuously sniffing in a box body of 30cm×30cm for 45min, wherein a blank control group sniffs 8mL blank water mist, and dose 1 group (sniffing with 2.83mL rhizoma Acori Graminei essential oil+5.17 mL double distilled water, and space concentration of rhizoma Acori Graminei essential oil is 104.81 mL/m) 3 ) Dose 2 groups (3.25 mL essential oil+4.75mL double distilled water, and the spatial concentration of rhizoma Acori Graminei essential oil is 120.37 mL/m) 3 ) Dose 3 groups (3.73 mL essential oil+4.27 mL distilled water, spatial concentration of rhizoma Acori Graminei essential oil is 138.14 mL/m) 3 ) Dose 4 groups (4.28 mL essential oil+3.72 mL distilled water, spatial concentration of rhizoma Acori Graminei essential oil is 158.52 mL/m) 3 ) Dose 5 groups (4.92 mL essential oil+3.08mL distilled water, spatial concentration of rhizoma Acori Graminei essential oil is 182.22 mL/m) 3 ) Dose 6 groups (5.65 mL essential oil+2.35 mL distilled water, spatial concentration of rhizoma Acori Graminei essential oil is 209.26 mL/m) 3 ). The survival of mice after bromine absorption in the different dose groups and the blank group are listed in table 5.
Table 1 survival after mice were dosed table
Grouping
|
Total number of animals (n)
|
Number of deaths (m)
|
Mortality (%)
|
Dose 1 group
|
16
|
0
|
0
|
Dose 2 group
|
16
|
0
|
0
|
Dose 3 group
|
16
|
4
|
25
|
Dose 4 groups
|
16
|
12
|
75
|
Dose 5 group
|
16
|
15
|
93.75
|
Dose 6 group
|
16
|
15
|
93.75
|
Blank group
|
16
|
0
|
0 |
Calculating half-lethal dose LD of rhizoma Acori Graminei essential oil concentration of table 1 on mice according to improved kohlrabi method 50 The spatial concentration of the essential oil of the grassleaf sweelflag rhizome is 151.48mL/m 3 When the amount of the administered 8mL is converted, half of the mice in the bromine-absorbing box of 30cm×30cm can die by absorbing bromine, half of the lethal amount of the grassleaf sweelflag rhizome essential oil accounts for 51.12% of the 8mL of the dosage volume.
This indicates that the high concentration of the grassleaf sweelflag rhizome essential oil is toxic, in order to ensure the safety of the compound essential oil, the mixing proportion of the grassleaf sweelflag rhizome essential oil and other essential oils in the formula should be strictly controlled when the compound essential oil is compounded, for example, the volume content of the grassleaf sweelflag rhizome essential oil in the compound essential oil in the embodiment 1-2 of the present application accounts for 0.81-0.96% of the volume content of the total compound essential oil, and the concentration of the grassleaf sweelflag rhizome essential oil in the subsequent sniffing and use process is far lower than LD 50 Is a concentration of (3). Therefore, the use concentration of the compound essential oil in the technical scheme is within the safe concentration range.
The beneficial effects of the compound essential oil provided by the application are specifically illustrated by animal experiments.
1. Experimental animal
The number of male KM mice is 108, the weight is 20+/-2 g, the mice are randomly divided into 6 groups according to the weight, 18 mice in each group are subjected to experiments, and a blank group, a model group, a positive group, a low-dose group, a medium-dose group and a high-dose group are arranged.
2. Experimental medicine
Diazepam tablets; compound essential oil prepared in example 1.
3. Experimental method
The temperature of the feeding environment is 24-26 ℃, the humidity is 50-60%, the brightness and the darkness are alternated for 12 hours, free drinking and feeding are performed during the period, and the experiment is started after one week of adaptive feeding. Except for blank groups, all other groups adopt an intraperitoneal injection of paracetamol animal insomnia model. P-chlorophenylalanine (PCPA) is precisely weighed and prepared into suspension by using normal saline for standby, each mouse is injected with the PCPA suspension according to the dosage of 300mg/kg, the injection is continuously carried out for two days, after the first injection is carried out for 28-32h, the animals have obvious behavioral changes of continuous activities, enhanced excitability, enhanced aggressiveness, grey stool and the like in the day and night, which indicates that the insomnia model of the mice is successfully prepared, and the blank group is treated by injecting the normal saline with the same volume.
After successful modeling, mice in the positive group are given an equal volume of diazepam aqueous solution every day, diazepam is the first choice drug for treating insomnia, and the administration dosage of the mice is 1.3mg/kg according to the surface area conversion formula of the animal administration dosage body. The blank group and the model group irrigate the stomach with equal volume of distilled water, the low dose group, the medium dose group and the high dose group perform the inhalation intervention every day, each dose group inhales the compound essential oil mixed solution mixed by compound essential oil and water with different volume percentage concentrations every day, the volume percentage concentrations are respectively 0.25%, 0.5% and 1%, and the continuous administration is carried out for 7 days.
The animal aromatherapy chamber is a 50X 40cm big box structure made of organic glass, 4 small box structures which are 20X 20cm and can be filled with air are arranged in the animal aromatherapy chamber, an ultrasonic aromatherapy machine can be placed in the middle of the box, mice are sequentially placed in the aromatherapy box, the prepared compound essential oil mixed solution is added into the aromatherapy machine, the box cover is covered, and the mice are inhaled for 60 minutes. The positive group is filled with equal volume of 1.3mg/kg concentration diazepam water solution, and the blank group, the model group and the compound essential oil administration group are filled with equal volume of distilled water.
3.1 sodium pentobarbital-induced sleep latency and sleep duration
9 mice in each group were randomly labeled, and after 30min from the end of the last dose or aromatherapy, the labeled 9 mice in each group were intraperitoneally injected with a threshold dose of 55mg/kg of pentobarbital sodium (pre-experimental results indicated that the experimental animals were 100% asleep at this concentration). After injection, mice were placed on a warm pad and the time to fall asleep was noted, with the disappearance of the eversion of the mice for 1 minute as an indicator of falling asleep. The time from injection of the drug to disappearance of the flip-flop is the latency of the sleep time, the time from disappearance of the flip-flop until restoration is the sleep time, and the latency of the sleep time and the sleep time are recorded.
As shown in fig. 1 and 2, the sleep latency is significantly increased and the sleep duration is significantly reduced in the model group compared to the blank group; the sleep latency is significantly reduced and the sleep duration is significantly enhanced in the group 3 mice with different doses of the positive group and the compound essential oil compared to the model group mice. This shows that the compound essential oil can obviously reduce the sleep latency of mice through the way of sniffing, prolongs the sleep time and is dose-dependent.
3.2 weight changes in mice
Weighing the weight of each group of mice before molding, and recording; after successful molding, the mice in the positive group, the model group and the compound essential oil administration group with different doses are filled with distilled water with the same volume as the positive group, the model group and the compound essential oil administration group, and the mice in the low dose group, the medium dose group and the high dose group are subjected to sniffing intervention every day, and after 7 days of continuous administration, the weights of the mice are weighed before sacrifice, and the weight increment of the mice is calculated before and after the continuous administration.
As shown in fig. 3, the mice in the model group had significantly reduced body weight compared to the blank group; compared with the model group, the body weight of the mice in the positive group and the 3 groups with different doses of compound essential oil is obviously increased.
3.3 open field experiments with mice
Open field experiments were performed using unlabeled mice in experiment 3.1, each group of mice was subjected to an autonomous activity test experiment for each group of mice after 30min of last administration, mice were acclimatized in the environment for 3min, the moving distance, average speed, resting time behavioural parameters were counted for 5min, and differences between the behavioural parameters of each group of mice were compared.
As shown in (a), (b), and (c) of fig. 4, the maximum speed, average speed, and moving distance of the model group are significantly increased compared to the blank group; compared with the model group, the maximum speed, average speed and moving distance of the mice in the positive group and the 3 groups with different doses of compound essential oil are obviously reduced.
3.4 mouse diving experiments
The test of diving platform is carried out on mice in different groups, when the administration is carried out on day 4, the mice are firstly placed on the copper grid, when the copper grid is electrified, the mice which jump on the copper grid receive electric shock, the normal response is to avoid the electric shock jumping on the platform, most of the mice possibly jump down the platform again or repeatedly to receive electric shock, and the mice jump back to the platform quickly when receiving the electric shock. The training was performed for 5 minutes, and the number of electric shocks (number of errors) received by each mouse was recorded as the learning score. And (3) re-testing after 24 hours, wherein the mice are firstly placed on a diving platform during the test, and the time of first jumping (latency period) and the number of errors within 5 minutes are recorded, so that the memory retention condition is reflected.
As shown in fig. 5, the latency of mice in the model group was extremely significantly reduced compared to the blank group; compared with the model group, the latency of the mice in the positive group and the 3 groups with different doses of compound essential oil is obviously increased and dose dependency exists.
3.5 experiments on the Effect of Compound essential oil on the level of mouse Hippocampus 5-HT, GABA, glu, ach
The experiment was performed using the mice in experiment 3.2, and after 30min of last administration on day 7, hippocampal tissue was rapidly isolated. Washing off brain tissue blood and impurities in ice PBS buffer solution, placing brain tissue into a centrifuge tube, adding 9 times of PBS buffer solution with brain tissue weight, homogenizing, centrifuging (6000 r/min,20 min), and collecting supernatant. After the supernatant samples of the brain tissues of each group of mice are treated according to the specification of an enzyme-linked immunosorbent assay (ELISA) kit of the mice 5-HT, GABA, glu, ach, the content of 5-HT, GABA, glu, ach in the sea horse is detected at 450nm by adopting an enzyme-labeled instrument.
5-HT, GABA, glu, ach are all neurotransmitters in the brain, and specifically, 5-HT neurons have an effect of modulating the sleep-wake cycle, and are one of the hinges of sleep and wake. GABA is the most important and abundant inhibitory neurotransmitter in the central nervous system, and hypofunction may lead to insufficient inhibitory function in the brain, etc. Glu is a major excitatory neurotransmitter that maintains normal functions of the nervous system, and is involved in regulating a variety of cellular activities and physiological functions, such as survival and apoptosis, learning, memory, cognition, emotion, and movement of cells. Ach is a very common neurotransmitter, and is an important neurotransmitter involved in learning and memory in cholinergic systems, and damage to cholinergic systems can cause defects in learning and memory functions, and is related to senile hypomnesis, age-related memory damage and the like.
As shown in fig. 6-9, the concentration content of hippocampus 5-HT, GABA, ach was significantly reduced in the model group mice and the concentration content of GLU was significantly increased compared to the blank group; compared with mice in a model group, the concentration content of hippocampus 5-HT, GABA, ach of mice in 3 groups with different doses of positive group and compound essential oil is obviously increased, and the concentration content of GLU is obviously reduced, which indicates that the compound essential oil can achieve sedative hypnotic effect through an inhalation mode.
3.6 experiments on the Effect of Compound essential oil on the hypothalamic 5-HT, GABA, glu level in mice
Experiments were performed using the mice in experiment 3.2, and hypothalamic tissues were rapidly isolated 30min after the last dose on day 7. Washing off brain tissue blood and impurities in ice PBS buffer solution, placing brain tissue into a centrifuge tube, adding 9 times of PBS buffer solution with brain tissue weight, homogenizing, centrifuging (6000 r/min,20 min), and collecting supernatant. Samples of brain tissue supernatant from each group of mice were treated according to the instructions of the enzyme-linked immunosorbent assay (ELISA) kit for mice 5-HT, GABA, glu and the hypothalamus content 5-HT, GABA, glu was detected at 450nm using an ELISA apparatus.
As shown in fig. 10-12, the concentration levels of 5-HT and GABA were significantly reduced in the hypothalamus mice of the model group compared to the blank group, while the concentration level of GLU was significantly increased; compared with the model group, the concentration content of hypothalamus 5-HT and GABA of the 3 groups of mice with the positive group and the compound essential oil with different dosages is obviously increased, and the concentration content of GLU is obviously reduced. This indicates that the compound essential oil can achieve sedative hypnotic effect by inhalation.
3.7 Compound essential oil vs. Abeta in mouse serum and Hippocampus 1-42 Influence of content experiment
Experiments were performed using the mice in experiment 3.2, and after 30min of last administration on day 7, the serum and hippocampal tissues of the mice were removed and were prepared according to aβ 1-42 Kit operations for detection of Abeta in serum and hippocampus 1-42 The content is as follows.
Beta-amyloid protein (Abeta) 1-42 ) Can inhibit proliferation of neural stem cells, destroy neuron membrane, penetrate water and ions and cause cell death,thereby damaging neurons and a large amount of Abeta 1-42 Is easy to reduce the memory and learning and cognition functions of animals.
As shown in fig. 13-14, the serum of mice in the model group and aβ in the hippocampus were compared with the blank group 1-42 The concentration content of (2) is significantly increased; compared with the model group, the serum of 3 groups of mice with different dosages of positive group and compound essential oil and Abeta in sea horse 1-42 The concentration content of (2) is obviously reduced, which indicates that the compound essential oil can obviously reduce Abeta in serum and hippocampus by an olfactory inhalation mode 1-42 Is never indirectly improved in memory function.
3.8 experiment of influence of Compound essential oil on MT content in Hippocampus of mice
Experiments were performed using the mice in experiment 3.2, and after 30min of last administration on day 7, the hippocampal tissues of the mice were removed and operated according to MT kit for detecting MT content in hippocampus.
Melatonin (MT) is an indole hormone secreted by the pineal gland of mammals and humans and has a variety of physiological activities. It can induce natural physiological sleep, correct disordered sleep-wake cycle, and research finds that MT has various pharmacological actions similar to benzodiazepines, such as sedative hypnotic, antiepileptic, etc.; MT can increase GABA content of hypothalamus, brainstem and cerebral cortex, and can enhance central inhibition of GABA by facilitating GABA binding with its receptor in brain.
As shown in fig. 15, the MT content in the hippocampus was significantly reduced in the mice of the model group compared to the blank group; compared with mice in a model group, the MT content in the hippocampus of 3 groups of mice with positive groups and compound essential oil at different doses is obviously increased, which indicates that the compound essential oil can obviously increase the MT content in the hippocampus by an inhalation mode so as to achieve sedative-hypnotic effect.
The experimental results are all statistically analyzed by mean+ -SD and SPSS 25.0 software, the comparison among groups is analyzed by a single-factor ANOVA method, the variance is even by an LSD method, the variance is uneven by a gas-Howell method, in the statistical results, delta P is less than 0.05 and delta P is less than 0.01 compared with a blank group, and P is less than 0.05 and P is less than 0.01 compared with a model group.
The animal experiment results show that the above-mentioned materials,the compound essential oil of the example 1 provided by the application can reduce the sleep latency induced by pentobarbital sodium, prolong the sleep duration, increase the weight, increase the 5-HT and GABA levels in the hippocampus and hypothalamus in the brain and simultaneously reduce the level of hippocampal GLU by sniffing administration so as to achieve the sedative hypnotic effect. Increase learning and memory capacity by increasing diving experiment learning latency, and decrease Abeta in serum and sea horse 1-42 Further improving learning memory and cognitive function.
Animal experiments were also performed in this application on the compound essential oil of example 2, selected animals and experimental methods refer to example 1 above, and finally the sodium pentobarbital-induced sleep latency and sleep duration of mice were tested. Tables 2 and 3 record the data for sleep latency and sleep duration for the different test groups of example 1 and example 2, respectively.
TABLE 2 sleep latency results
Table 3 sleep duration results
Group of
|
Example 1
|
Example 2
|
Blank group
|
31.45±17.55
|
31.45±18.77
|
Model group
|
20.03±10.31
|
11.22±4.54
|
Positive group
|
49.45±16.19
|
32.00±17.34
|
Low dose group
|
37.35±11.39
|
27.01±16.95
|
Medium dose group
|
38.52±16.58
|
28.81±16.16
|
High dose group
|
41.49±20.33
|
29.30±12.90 |
From tables 2 and 3, the model group in example 2 showed a significant increase in sleep latency and a significant decrease in sleep duration compared to the blank group; the sleep latency was significantly reduced and sleep duration was significantly enhanced in the positive group, the compound essential oil low, medium, and high dose group 3 mice of example 2 compared to the model group mice. This shows that the experimental rule of the compound essential oil in the embodiment 2 of the application in the sleep latency period and the experimental rule of the sleep duration are the same as the experimental rule of the compound essential oil in the embodiment 1, which further indicates that the compound essential oils in the embodiment 1 and the embodiment 2 of the application can obviously reduce the sleep latency period of mice and prolong the sleep time through the sniffing way.
Further analysis found that: the sleep latency and sleep duration of example 1 are dose dependent, while the sleep latency differences between the different dose groups of example 2 are larger, and the sleep duration is shorter than that of example 1, which indicates that example 1 of the present application has better effect in improving sleep disorders, insomnia than that of example 2.
The application also selects some volunteers with sleep disorder for testing, and the specific characteristic analysis is as follows:
40 adults 20-50 years old are selected, each subject is a brain worker, the working time per week is not less than 5 days, the working time per day is not less than 8 hours, the subjects face electronic products (including but not limited to mobile phones and computers) for a long time, the sleep time is >30 minutes, the night wakefulness is equal to or greater than 2 times, the total sleep time is less than 6 hours, the sleep quality is reduced, and the daily dysfunction is accompanied.
The above test volunteers did not participate in any other or adjuvant treatment during the test period, the above group was randomly divided into 4 groups, and subjects in different groups were subjected to the experiment of falling asleep with the prescriptions of example 1, example 2, comparative example 1, comparative example 2, respectively, with "fragrance" before sleeping, and continuously conducted for 2 days, i.e., 1-3 drops of compound essential oil (theoretical 0.05-0.15 mL) were dropped to the fragrance-expanded woods before sleeping every day, and placed at the bedside after absorption. The questionnaire is then completed as if it were filled in according to the test index of table 4 below.
Table 4 test index
Table 5 test results
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Table 5 shows the test results of different test groups after 2 days of test, and basically all the test groups subjectively think that after using the compound essential oil, the sleep time is shortened, the sleep time is increased, the dream times are reduced, the night starting times are reduced, the daytime working state is improved, and the like. But the fragrance and comfort level of example 1 and example 2 are more acceptable to people than those of the formulations of comparative example 1 and comparative example 2, and the dream condition, the second day mental state and the night awakening condition, example 1 and example 2 are also superior to comparative example 1 and comparative example 2. After the average calculation of the values of all the test indexes, the average values of the test indexes of the example 1 and the example 2 are also higher than those of the comparative examples 1 and 2. It can be seen that the compound essential oils of example 1 and example 2 of the present application have better sedative-hypnotic effects.
After that, 10 subjects using the compound essential oil of example 1 were counted, and the change of sleep characteristics before and after the test was performed, and the results of the related tests are shown in fig. 16.
The results of fig. 16 show that: the compound essential oil disclosed by the embodiment 1 of the application can be used for remarkably improving the sleep balance index of people with difficult sleep; the sleep latency can be obviously reduced, the average sleep latency after one day is 31.4 minutes, and the average sleep latency after two days is 17.7 minutes; the total sleeping time and sleeping efficiency of the tested person are also improved to a certain extent. In addition, the compound essential oil of example 1 was used without changing the sleep structure ratio of the human subject.
In addition, the application also performs skin irritation and storage stability investigation on the product prepared from the compound essential oil.
Skin irritation investigation of essential oil balm products
SD rats, male and female halves, weigh 180+ -20 g. After separating male and female SD rats, the rats were randomly separated into 6 groups of female rats and 6 groups of male rats according to body weight, and each group was uniformly applied to the backs of rats with 17.5% stock solution (33/40 matrix mixture+7/40 compound essential oil), 25% stock solution (3/4 matrix mixture+1/4 compound essential oil), 35% stock solution (13/20 matrix mixture+7/20 compound essential oil), 50% stock solution (1/2 matrix mixture+1/2 compound essential oil), 70% stock solution (3/10 matrix mixture+7/10 compound essential oil), and 100% stock solution (100% compound essential oil).
Taking 0.5mL of a test medicament to be directly smeared on one side of the back skin of a rat, smearing blank base oil on the other side of the back skin of the rat as a control, then covering with two layers of gauze and one layer of cellophane, fixing by using an adhesive tape, adhering for at least 4 hours, removing the smears after adhering, cleaning by warm water, observing and recording whether the coated parts have erythema and edema by naked eyes respectively at 1 hour, 2 hours, 4 hours, 24 hours, 48 hours and 72 hours, and inspecting whether the parts of the blood vessel, the muscle, the skin, the mucous membrane and the like of the animal cause local reactions such as reddish swelling, hyperemia, exudation, denaturation or necrosis according to the scoring principle specified in the technical guidelines of local irritation and hemolysis research of traditional Chinese medicines and natural medicaments.
TABLE 6 skin changes at various stages in rats
According to the results of the above Table 6, it is shown that the male rats of 100% stock solution group were slightly reddish after 2 hours of administration, and the female rats of 50% stock solution or more were slightly reddish; after 24 hours of administration, 70% of the stock solution groups of the male rats have slight reddening; after 72 hours of administration, the skin of the administered rats was restored to the previous state. Therefore, the essential oil balm prepared by the method is in a reasonable range, and the essential oil balm can reduce the irritation to skin and is safe and effective.
Stability investigation of essential oil spray product
(1) Centrifugal stability
The essential oil spray prepared in example 4 was centrifuged at 8000r/min for 15min, and the appearance after centrifugation was observed for the presence of a delamination demulsification phenomenon. The results show that the spray is still clear and transparent after centrifugation, and is not broken by layering, so that the spray has good centrifugal stability.
(2) Dilution stability
The essential oil spray and double distilled water are diluted according to the proportion of 100, 200, 400, 800 and 1200 times, and whether layering and demulsification of appearance are observed. The results show that no layering phenomenon is observed after the spray is uniformly mixed with double distilled water in any proportion, and the solution uniformity is good, so that the spray has good dilution stability.
(3) Temperature stability
Spraying essential oil in a closed container, standing at 4deg.C, 25deg.C and 37deg.C, and observing appearance change. The results show that the appearance of the oil spray has no layering demulsification phenomenon, and the essential oil spray has temperature stability.
(4) Long term stability
Spraying essential oil into a sealed sample bottle, storing at normal temperature for 60d, and observing the appearance and testing the change of the particle size of the essential oil with the 0d, 7d, 15d, 30d and 60d respectively. The results show that the appearance and the particle size of the oil spray are good, the layering demulsification phenomenon is not seen, and the essential oil spray has long-term stability, as shown in Table 7.
TABLE 7 Long term stability particle size and PDI results
Days/d
|
Particle size (nm)
|
PDI
|
0
|
26.21±2.73
|
0.30±0.06
|
7
|
23.49±0.68
|
0.27±0.02
|
15
|
23.90±0.55
|
0.26±0.01
|
30
|
22.52±0.41
|
0.23±0.02
|
60
|
23.17±0.50
|
0.30±0.01 |
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Furthermore, those skilled in the art will appreciate that while some embodiments herein include some features but not others included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention and form different embodiments. For example, in the claims below, any of the claimed embodiments may be used in any combination. The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.