CN113509528A

CN113509528A - Composition and sleep-aiding product containing magnolia sieboldii extract

Info

Publication number: CN113509528A
Application number: CN202110443502.1A
Authority: CN
Inventors: 唐明慧; 宋妮; 艾勇; 何廷刚; 朱思阳; 张炽坚
Original assignee: Hua An Tang Biotech Group Co ltd
Current assignee: Hua An Tang Biotech Group Co ltd
Priority date: 2021-04-23
Filing date: 2021-04-23
Publication date: 2021-10-19

Abstract

The invention relates to the technical field of plant extracts, in particular to a composition containing magnolia sieboldii extract and a sleep-aiding product. The invention discloses a composition, wherein the magnolia sieboldii extract in the composition can be cooperated with vetiver essential oil, frankincense essential oil, real lavender essential oil, sweet marjoram essential oil and Pinus densiflora essential oil to further shorten the sleep latency time of a mouse in a pentobarbital sodium induced sleep experiment and prolong the sleep time; the number of neurons in hippocampal regions of the brain of the mouse is increased; increasing the expression level of 5HT-1A protein, GABAAR alpha 1 protein and mRNA in brain region. The composition has good sleep promoting effect.

Description

Composition and sleep-aiding product containing magnolia sieboldii extract

Technical Field

The invention relates to the technical field of plant extracts, in particular to a composition containing magnolia sieboldii extract and a sleep-aiding product.

Background

With the increase of living pressure, the sleep quality of people can not be guaranteed, and normal work and learning are seriously influenced.

At present, the methods for treating insomnia mainly comprise drug treatment and non-drug treatment. The drug treatment of insomnia is the most effective method, but the drug treatment also has the side effects of drug dependence, withdrawal reaction and the like.

Non-drug therapy mainly comprises aromatherapy, etc., and the medicinal components adopted by aromatherapy are mainly plant extracts. The aromatherapy using the plant extract is not as fast as the drug therapy, but has the advantages of pure nature of the plant extract, high safety, and almost no side effects such as drug dependence and withdrawal reaction.

The Magnolia sieboldii extract has antibacterial, whitening, and wrinkle resisting effects, and is commonly used in daily chemical field. At present, the application of the magnolia sieboldii extract in the sleep aiding field is not found.

Disclosure of Invention

In view of the above, the present invention provides a composition and a sleep-aid product containing magnolia sieboldii extract, wherein the magnolia sieboldii extract in the composition is matched with other components in the composition, such that the sleep latency period can be effectively shortened, the sleep time can be effectively prolonged, the number of hippocampal neurons of the brain, the expression amounts of 5HT-1A protein and GABAAR α 1 protein, and the mRNA level of GABAAR α 1 protein can be effectively increased, and the sleep-aid effect is significant.

The specific technical scheme is as follows:

the present invention provides a composition comprising: magnolia sieboldii extract, vetiver essential oil, frankincense essential oil, true lavender essential oil, sweet marjoram essential oil and Pinus sylvestris essential oil.

The composition provided by the invention has a sleep-aiding effect and an obvious effect. The magnolia sieboldii extract in the composition can be used for enhancing the sleep-aiding effect in cooperation with vetiver essential oil, frankincense essential oil, real lavender essential oil, sweet marjoram essential oil and Pinus sylvestris essential oil.

The Magnolia sieboldii extract is one or more of Magnolia sieboldii alcohol extract, Magnolia sieboldii water extract and Magnolia sieboldii water vapor distillation extract.

Preferably, the alcohol extract, the water extract and the steam distillation extract of the Magnolia sieboldii are Magnolia sieboldii essential oil.

The essential oil is volatile aromatic oil liquid extracted from flowers, leaves, stems, roots or fruits of plants, has the characteristics of strong permeability, various biological activities and the like, and has different effects. The essential oil has the advantages of easy entering into human body to achieve specific effect due to natural characteristics and small molecules. Therefore, the magnolia sieboldii extract of the present invention is preferably magnolia sieboldii essential oil.

In the invention, the raw material of the magnolia sieboldii extract is one or more than two of leaves, roots, flowers, fruits and stems of magnolia sieboldii, and is preferably the magnolia sieboldii leaves. Therefore, the Magnolia sieboldii essential oil in the present invention is preferably Magnolia sieboldii leaf essential oil.

In the invention, the preparation method of the magnolia sieboldii essential oil is preferably as follows:

pulverizing Magnolia sieboldii into 10-20 mesh powder, placing in 3-5 series steam distillation reaction kettles, dispersing uniformly, adding mesh enclosure, introducing steam, opening condenser, reacting for 1-2 hr, collecting extract, and separating oil and water to obtain Magnolia sieboldii essential oil.

In the invention, the vetiver essential oil has the functions of resisting depression, resisting oxidation, calming and helping sleep and the like;

the Olibanum essential oil has antioxidant and antidepressant effects;

the true lavender essential oil has antibacterial, psoriasis resisting, antioxidant, antidepressant, and anxiolytic effects;

the sweet marjoram essential oil has effects of resisting oxidation, protecting liver, protecting heart, resisting bacteria and fungi, resisting tumor, resisting ulcer, and inhibiting cholinesterase;

the Pinus densiflora essential oil has antibacterial and antioxidant effects.

The invention adopts a pentobarbital sodium induced sleep time test, and detects the influence of essential oil on the sleep latency and the sleep time of a pentobarbital sodium sleep experimental mouse through an essential oil aromatherapy. The sleep latency period is the time from the intraperitoneal injection of the pentobarbital sodium to the disappearance of the righting reflex, and the shortening range of the sleep latency period of each group can reflect the good and bad sleep-aiding effect of the essential oil and the sleep time can also intuitively show the good and bad effect of the essential oil on the basis of the numerical values of a PCPA model group and a blank control group. The results show that the composition comprising magnolia sieboldii extract provided by the present invention prolongs the sleep time induced by pentobarbital sodium and shortens the sleep latency time, compared to the composition consisting of vetiver essential oil, boswellia carterii essential oil, true lavender essential oil, sweet marjoram essential oil and pinus sylvestris essential oil.

Further, the invention adopts Nie's staining to detect the number of the mouse neurons. The results show that the composition comprising magnolia sieboldii extract provided by the present invention can increase the normal number of neurons in the hippocampal region of mice compared to the composition consisting of vetiver essential oil, boswellia carterii essential oil, true lavender essential oil, sweet marjoram essential oil and pinus sylvestris essential oil.

The 5HT-1A, 5-hydroxytryptamine 1A receptor (5-hydroxytryptamine receptor 1A), is expressed in neurons of the cerebral cortex, hippocampus and hypothalamus, and is involved in the regulation of sleep, sexual behavior, anxiety and mood. The 5HT-1A receptor is an autoreceptor characterized by expression in presynaptic neurons, limiting the release of 5HT from nerve terminals. The principle of PCPA induced insomnia is that it blocks presynaptic 5HT-1A autoreceptors, depleting 5 HT. GABA, namely gamma-aminobutyric acid (GABA), is a main inhibitory neurotransmitter in the Central Nervous System (CNS), plays an important role in sleep, and is one of important targets of a plurality of drugs for clinically treating insomnia. Related studies have shown that sedatives enhance the GABA (GABA) response through positive modulation of GABAA receptors, i.e., GABA receptors such as GABAAR alpha 1 reflect to some extent the intensity of the hypnotic effect of the drug.

Further, the invention adopts paraffin section immunohistochemical test to detect the expression conditions of 5HT-1A protein and GABAAR alpha 1 protein in the brain tissue of the mouse. The results show that the composition comprising magnolia sieboldii extract provided by the invention can significantly improve the expression of 5HT-1A protein and GABAAR alpha 1 protein in brain tissue, compared to the composition consisting of vetiver essential oil, boswellia carterii essential oil, true lavender essential oil, sweet marjoram essential oil and pinus sylvestris essential oil.

Further, the invention adopts a real-time fluorescent quantitative PCR test to detect the expression condition of GABAAR alpha 1 protein mRNA in the brain tissue of the mouse. The results show that the composition comprising magnolia sieboldii extract provided by the present invention can significantly increase the mRNA level of GABAAR alpha 1 protein in brain tissue compared to a composition consisting of vetiver essential oil, boswellia carterii essential oil, true lavender essential oil, sweet marjoram essential oil, and pinus sylvestris essential oil.

In the invention, the volume ratio of the magnolia sieboldii extract, the vetiver essential oil, the frankincense essential oil, the real lavender essential oil, the sweet marjoram essential oil and the Pinus sylvestris essential oil is (4-8): (1-4): (2-6): (2-5): (1-4): (2-5), preferably 6:2:4:3:2: 3. 8:4:6:5:4:5 or 4:1:2:2:1: 2.

The invention also provides a preparation method of the composition, which comprises the following steps: mixing the Magnolia sieboldii extract, the vetiver essential oil, the Boswellia carterii essential oil, the real lavender essential oil, the sweet marjoram essential oil and the Pinus sylvestris essential oil.

The invention also provides the application of the composition or the composition prepared by the preparation method in preparing sleep-aiding products.

The invention also provides a sleep-aiding product which comprises the composition or the composition prepared by the preparation method.

In the invention, the administration mode of the sleep-aiding product is inhalation, smearing or aromatherapy, and preferably the inhalation is inhalation.

In the invention, the effective dose of the composition in the sleep-aiding product is 625-2500 mu L/day/kg, preferably 2500 mu L/day/kg.

In the invention, the mass content of the composition in the sleep-aiding product is 0.001-10%.

In the invention, the sleep-aiding product is a daily chemical product. The daily chemical product is preferably cosmetics, skin care products or washing products, and specifically comprises toner, lotion, essence, cream, essential oil, shampoo and hair conditioner, aroma, candle and the like.

According to the technical scheme, the invention has the following advantages:

the invention provides a composition, wherein the magnolia sieboldii extract in the composition can cooperate with vetiver essential oil, frankincense essential oil, real lavender essential oil, sweet marjoram essential oil and Pinus sylvestris essential oil, so that the sleep latency of a mouse in a pentobarbital sodium induced sleep experiment is further shortened, and the sleep time is prolonged; the number of neurons in hippocampal regions of the brain of the mouse is increased; increasing levels of 5HT-1A protein and GABAAR alpha 1 protein and mRNA in various brain regions. The composition has good sleep promoting effect.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without inventive exercise.

FIG. 1 is a graph showing the results of testing the sleep latency of various groups of mice according to the present invention;

FIG. 2 is a graph showing the results of testing the sleep time of mice in each group according to the present invention;

FIG. 3 is a microscope photograph of Neisseria staining neurons (scale: 50 μm) of a hippocampal slice of each group of mice provided by an embodiment of the present invention;

FIG. 4 is a statistical chart of the number of normal neurons in the hippocampal region of each group of mice according to the present invention;

FIG. 5 is a microscope photograph (50 μm scale) of the expression of 5HT-1A protein in immunohistochemical sections of the cerebral cortex of each group of mice provided by the example of the present invention;

FIG. 6 is a histogram of 5HT-1A protein expression in immunohistochemical sections of the cerebral cortex of each group of mice provided by the present example;

FIG. 7 shows a microscope image (scale: 50 μm) of GABAAR alpha 1 protein expression from each mouse hippocampal immunohistochemical section provided by example of the present invention;

fig. 8 is a histogram of GABAAR α 1 protein expression in hippocampal immunohistochemical sections of various groups of mice provided by an embodiment of the present invention;

fig. 9 is a histogram of the relative expression level of mRNA of GABAAR α 1 in each mouse brain tissue group according to the embodiment of the present invention.

Detailed Description

In order to make the objects, features and advantages of the present invention more obvious and understandable, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it should be apparent that the embodiments described below are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the examples of the present invention, vetiver essential oil was purchased from Ningbo Cichlon's commerce, Frankincense essential oil was purchased from Yaqi practical (Shanghai) Co., Ltd, real Lavender essential oil was purchased from Chongqing Zhengyuan commercial Co., Ltd, and Murraya koenigii essential oil and Pinus sylvestris essential oil were purchased from Yaqi practical (Shanghai) Co., Ltd.

Example 1

This example is the preparation of essential oil of Magnolia sieboldii leaves

In this embodiment, in the application of the magnolia sieboldii extract disclosed in patent CN 105708760 a as a bacteriostatic agent, the preparation method of example 1 is used for preparing magnolia sieboldii leaf essential oil, and the specific preparation steps are as follows: pulverizing Magnolia sieboldii leaves to 20 meshes, placing in 4 series-connected steam distillation reaction kettles, uniformly dispersing, adding a mesh enclosure, introducing steam, opening a condenser, reacting for 1h, collecting extract, and separating oil and water to obtain Magnolia sieboldii leaf extract essential oil.

Example 2

This example uses chlorphenylalanine (PCPA) to establish a mouse model of insomnia

Male KM mice (30. + -.5 g) at 5-6 weeks of age were acclimatized for one week before molding. When the chlorphenylalanine PCPA suspension is prepared, 600mg of PCPA powder is dissolved in 20mL of physiological saline containing 1% Tween80, and the preparation concentration is 30 mg/mL. The mice except the blank control group are injected into the abdominal cavity according to the dosage of 0.1mL/10g of the weight of the mice, and the blank control group is injected into the abdominal cavity by normal saline containing 1 percent of Tween with the same volume for 2 days continuously. 26-30h after the last intraperitoneal injection of PCPA is finished, the mouse shows behavioral changes (activities in the daytime are ceaseless, excitability is increased, irritability is easy, circadian rhythms disappear, water intake and food intake are increased, and sleeping time is obviously reduced), which indicates that the insomnia model is successfully molded.

Example 3

This example is the preparation of an essential oil composition

The Magnolia sieboldii leaf essential oil, the vetiver essential oil, the frankincense essential oil, the true lavender essential oil, the sweet marjoram essential oil and the Pinus sylvestris essential oil prepared in example 1 were mixed in a mass ratio of 8:4:6:5:4:5, 6:2:4:3:2:3 and 4:1:2:2:1:2, respectively, to obtain essential oil compositions which were respectively marked as test composition 1, test composition 2 and test composition 3.

Mixing vetiver essential oil, frankincense essential oil, real lavender essential oil, sweet marjoram essential oil and Pinus sylvestris essential oil in a mass ratio of 2:4:3:2:3 to obtain an essential oil composition, and recording as a control composition.

Example 4

This example demonstrates the sleep-aiding effect of a composition comprising Magnolia sieboldii leaf essential oil

1. Method for detecting influence of essential oil composition on sleep latency and sleep time of sleeping mice by adopting sodium pentobarbital sleep time test

The effect of the essential oil composition of example 3 on the duration of sleep induction by barbiturates, hypnotics generally prolong the sleep time induced by sodium pentobarbital. Recording the time of falling asleep and the time of sleeping, comparing the difference between the smelling group of the essential oil and the PCPA model control group of each group, and testing the significance of the difference, wherein P <0.05 indicates that the essential oil of the group has significant hypnotic effect, and P <0.01 indicates that the essential oil of the group has very significant hypnotic effect.

1.1 the blank group of mice does not establish a PCPA insomnia model, the PCPA insomnia mice are randomly divided into 4 groups, animals in the experimental group smell the test compositions prepared in the embodiment 3 with the same concentration respectively and are marked as the experimental group 1, the experimental group 2 and the experimental group 3, and the inhalation volume of the mice is 2500 muL/day/kg; essential oil control group sniff the control composition prepared in example 3, mouse inhalation volume was 2500 μ L/day/kg; mice in the blank group and the PCPA model group were inhaled with 1% Tween80 in saline at a volume of 2500. mu.L/kg/day. Each mouse in each group was smelled for 30 minutes daily. Injecting diazepam solution 30min before injecting PCPA into mice of the diazepam positive control group, wherein the dosage is 2 mg/kg;

1.2 after the smelling for 30min, injecting, and injecting pentobarbital sodium into the abdominal cavity of all the mice of all the groups by using a disposable sterile syringe with the specification of 1mL according to the weight of 0.1mL/10g of the mice.

1.3 mice intraperitoneal injection operation: the injection syringe is used to suck up the dose to be injected in advance. The mouse was grasped with the head facing down and the abdomen facing up and the injection needle inserted at a 30 degree angle to the right of the midline of the abdomen. The syringe is withdrawn and reinserted if blood or fluid flows back, indicating improper insertion. The medicine is slowly pushed in.

1.4 record the injection time (T.R). After injection, the mice are independently placed into a breeding box, and index evaluation is carried out as follows: righting reflection disappearance standard: it means that the back of the mouse is kept downwards for more than 30s and the righting reflex does not appear within 1 min. The duration of disappearance of the righting reflex (time of disappearance of righting reflex to time of repeated appearance of righting reflex) and the latency of disappearance of the righting reflex were recorded. If doubting whether the righting reflection is really recovered, the righting reflection is placed on the back immediately after the first righting, if the righting reflection is automatically turned over within 1min, the time of the previous turning is the recovery time, otherwise, the time of the second turning is taken as the standard.

1.4.1 sleep latency: the time from the intraperitoneal injection of the pentobarbital sodium to the disappearance of the righting reflex. The time at which the righting reflex disappeared was recorded for each mouse within 30min, indicating that the mouse was asleep (T.S). The results of the experiment are shown in fig. 1 and table 1.

1.4.2 sleep time: i.e. the time from disappearance of the righting reflex to arousal. The time at which each mouse turned positive reflex was recovered was recorded, indicating that the mice were awake (T.W). The Sleep Latency (Sleep Latency) and Sleep Time (Total Sleep Time) of each mouse were calculated, wherein Sleep Latency is T.S-T.R., and Total Sleep Time is T.W-T.S. The results of the experiment are shown in fig. 2 and table 2.

As can be seen from table 1, the essential oil compositions containing magnolia sieboldii leaf essential oil provided by experimental groups 1 to 3 all have the effect of remarkably shortening the sleep latency of mice, wherein the effect of the experimental group 2 is the best, so that the experimental group 2 is compared with other groups for carrying out subsequent experiments.

Fig. 1 is a graph of the results of the tests on sleep latency of the groups of mice provided in this example (P <0.05 for significance and P <0.01 for extreme significance compared to the model group). As can be seen from fig. 1, the sleep latency was reduced in all mice of the experimental, blank, control and diazepam groups compared to the PCPA model group, with a significant reduction in the diazepam, experimental and blank groups. Compared with the control group (vetiver essential oil + frankincense essential oil + true lavender essential oil + sweet marjoram essential oil + pinus sylvestris essential oil), the experimental group (vetiver magnolia extract + vetiver essential oil + frankincense essential oil + true lavender essential oil + sweet marjoram essential oil + pinus sylvestris essential oil) mice has shorter sleep latency.

As can be seen from table 2, the essential oil compositions containing magnolia sieboldii leaf essential oil provided by the experimental groups 1 to 3 all have the effect of remarkably prolonging the sleep time of mice, wherein the effect of the experimental group 2 is the best, so that the experimental group 2 is compared with other groups for carrying out subsequent experiments.

Fig. 2 is a graph of the test results of sleep time for each group of mice provided by the example of the present invention (P <0.05 for significance and P <0.01 for extreme significance compared to the model group). As can be seen from fig. 2, the sleep time was extended to various degrees in the mice of the experimental group 2, the control group and the diazepam group, compared to the PCPA model group; the control group (vetiver essential oil + frankincense essential oil + true lavender essential oil + sweet marjoram essential oil + pinus sylvestris essential oil) and the experimental group 2 (magnolia sieboldii extract + vetiver essential oil + frankincense essential oil + true lavender essential oil + sweet marjoram essential oil + pinus sylvestris essential oil) have little difference in the degree of prolonging the sleep time of the mice.

TABLE 1

	Experimental group 1	Experimental group 2	Experimental group 3
				Sleep latency period	233.83±10.92	200.75±8.98	230.68±11.23

TABLE 2

	Experimental group 1	Experimental group 2	Experimental group 3
				Sleep time	1288.12±12.23	1325.75±9.04	1302.23±10.12

2. Neisseria staining for detecting the number of mouse neurons in each group

2.1 slicing and baking paraffin: setting the temperature of the constant-temperature drying oven at 60 ℃, and baking slices for 15 min;

2.2 xylene dewaxing: sequentially placing the tissue slices into xylene I and xylene II for 20min respectively;

2.3 gradient alcohol to water: according to the concentration from high to low, putting the slices into 100% ethanol I and 100% ethanol II in turn, each for 10 min; adding 95% ethanol, 90% ethanol, 80% ethanol and 70% ethanol, each for 5 min;

2.4 tap water washing 1 time (2 min/time); washing with double distilled water for 2 times (2 min/time);

2.5 toluidine blue dye liquor: 10 min;

2.6 after the dyeing is finished, water washing is carried out to stop the dyeing: washing with tap water for 1 time (2 min/time); washing with double distilled water for 2 times (2 min/time);

2.7, alcohol dehydration: sequentially adding 70% ethanol, 95% ethanol and 100% ethanol, each for 5 min;

2.8 xylene clarity: 15 min;

2.9 microscopic examination and image acquisition and analysis.

FIG. 3 is a microscope photograph of Neisseria staining neurons of a hippocampal slice of brain tissue of each group of mice in this example;

FIG. 4 is a statistical chart of the number of normal neurons in the hippocampal region of each group of mice in this example. 3-4, the numbers of hippocampal neurons in the mice of experimental group 2 and the control group were increased to different degrees compared to the PCPA model group; the number of neurons in the hippocampal region of the experimental group 2 mice was significantly increased compared to the control group. The effect of the experimental group 2 (the magnolia sieboldii extract, the vetiver essential oil, the frankincense essential oil, the real lavender essential oil, the sweet marjoram essential oil and the Pinus sylvestris essential oil) is better than that of the control group (the vetiver essential oil, the frankincense essential oil, the real lavender essential oil, the sweet marjoram essential oil and the Pinus sylvestris essential oil).

3. Detection of 5HT-1A protein and GABAAR alpha 1 protein expression in brain tissue of mice by paraffin section immunohistochemistry

3.1 Paraffin section dewaxing to Water: placing the slices in xylene I15 min-xylene II 15 min-xylene III 15 min-absolute ethyl alcohol I5 min-absolute ethyl alcohol II 5 min-85% ethyl alcohol 5 min-75% ethyl alcohol 5 min-distilled water washing.

3.2 antigen retrieval: placing the tissue slices in a repairing box filled with citric acid antigen repairing buffer solution (pH6.0) in a microwave oven for antigen repairing, wherein the medium fire is 8min to boil, and the fire is stopped for 7min, wherein excessive evaporation of the buffer solution is prevented, and dry slices are cut. After natural cooling, the slides were washed 3 times for 5min in PBS (pH7.4) with shaking on a destaining shaker.

3.3 blocking endogenous peroxidase: the sections were placed in 3% hydrogen peroxide solution, incubated for 25min at room temperature in the dark, and the slides were washed 3 times 5min each time in PBS (pH7.4) with shaking on a destaining shaker.

3.4 serum blocking, 3% BSA was added dropwise to the assembly circle to cover the tissue evenly, and blocking was carried out at room temperature for 30 min. (Primary antibody was ft sheep-derived blocked with rabbit serum, other sources with BSA)

3.5 plus primary antibody: gently removing the confining liquid, dripping PBS (5HT1AReceptor Antibody, AF5453, dilution ratio IHC 1:50-1: 200; Anti-GABAA Receptor alpha 1Rabbit pAb, GB11716, dilution ratio IHC 1:600-1:1500) on the section, and flatly placing the section in a wet box for incubation at 4 ℃ overnight. (Small amount of water added in wet box to prevent evaporation of antibody)

3.6 adding secondary antibody: slides were washed 3 times in PBS (pH7.4) with shaking on a destaining shaker for 5min each time. After the section is slightly dried, a secondary antibody (HRP-labeled goat anti-rabbit IgG, GB23303, dilution ratio IHC 1:200-1:500) of a primary antibody corresponding species is added dropwise into the ring to cover the tissue, and the tissue is incubated for 50min at room temperature.

3.7DAB color development: slides were washed 3 times for 5min in PBS (pH7.4) with shaking on a destaining shaker. After the section is slightly dried, a DAB color developing solution which is prepared freshly is dripped into the ring, the color developing time is controlled under a microscope, the positive color is brown yellow, and the section is washed by tap water to stop color development.

3.8 counterstaining nuclei: counter-staining with hematoxylin for about 3min, washing with tap water, differentiating with hematoxylin differentiation solution for several seconds, washing with tap water, returning the hematoxylin to blue, and washing with running water.

3.9 dewatering and sealing: placing the slices in 75% alcohol for 5 min-85% alcohol for 5 min-absolute ethanol for 15 min-absolute ethanol II for 5 min-xylene I for 5min, dehydrating, removing the slices from xylene, air drying, and sealing with neutral gum.

3.10 microscopic examination and image acquisition and analysis.

FIG. 5 is a microscope photograph showing the expression of 5HT-1A protein in cerebral cortex of the brain tissue of each group of mice in this example. FIG. 6 is a histogram of 5HT-1A protein expression in the cerebral cortex of the brain tissue of each group of mice in this example. As can be seen from FIGS. 5 and 6, the expression of 5HT-1A protein was significantly reduced in the cerebral cortex region of mice in the PCPA model group, as compared with those of the blank group; compared with the PCPA model group, the 5HT-1A protein expression level of the cerebral cortex of the mice in the experimental group 2 is obviously improved, and the effect is better than that of a control group and a blank group. Fig. 7 is a microscope image of GABAAR α 1 protein expression in hippocampal region of brain tissue of each group of mice in this example. Fig. 8 is a histogram of GABAAR α 1 protein expression in hippocampal regions of brain tissue of mice in each group of this example (P <0.05, indicating significance, and P <0.01, indicating extreme significance, compared to the model group). As can be seen from fig. 7 and 8, GABAAR α 1 protein expression was significantly reduced in hippocampal region of brain tissue in mice of the PCPA model group compared to the blank group; the GABAAR alpha 1 protein expression level of the hippocampal region of the mouse in the experimental group 2 is slightly higher than that of the control group, and the effect is better.

4. Fluorescent quantitative PCR detection of expression quantity of gene mRNA of each group of mice expressing GABAAR alpha 1

3.1 Total RNA extraction (gun head and centrifuge tube are sterilized by moist heat, no RNase)

3.1.1 Take homogenate tube, add 1mL Trizol Reagent, put on ice for precooling.

3.1.2 Add 100mg of tissue to the homogenizer tube.

3.1.3 homogenizer is ground well until no tissue mass is visible.

The supernatant was centrifuged at 3.1.412000 rpm for 10 min.

3.1.5 adding 250 μ l chloroform, inverting the centrifuge tube for 15s, mixing well, standing for 3 min.

3.1.64 ℃ for 10min at 12000 rpm.

3.1.7 transfer the supernatant to a new centrifuge tube, add 0.8 times volume of isopropanol, reverse and mix.

Standing at 3.1.8-20 deg.C for 15 min.

The mixture was centrifuged at 12000rpm at 3.1.94 ℃ for 10min, and the white precipitate at the bottom of the tube was RNA.

3.1.10 the liquid was removed by suction and 1.5mL of 75% ethanol was added to wash the precipitate.

3.1.114 ℃ and then centrifuged at 12000rpm for 5 min.

3.1.12 the liquid was sucked off and the centrifuge tube was placed on a clean bench and blown for 3 min.

3.1.13 Add 15. mu.l RNase-free water to dissolve the RNA.

Incubate at 3.1.1455 ℃ for 5 min.

3.1.15 detection of RNA concentration and purity using Nanodrop 2000: after the blank of the instrument is zeroed, 2.5 mul of RNA solution to be detected is put on a detection base, a sample arm is put down, and light absorption value detection is started by using software on a computer.

3.1.16 the RNA at the excess concentration is diluted in an appropriate ratio to a final concentration of 200 ng/. mu.l.

3.2 reverse transcription (gun head and PCR both sterilized by moist heat, No RNase)

3.2.1A PCR tube was taken and a solution containing 2. mu.g of RNA was added.

3.2.2 Add 1. mu.l oligo (dT) 18.

3.2.3 make up to 12. mu.l with DNase free deionized water.

3.2.4 incubation at 65 ℃ for 5min on a PCR instrument, and rapidly cooling on ice.

3.2.5 mu.l of 5 × Reaction Buffer, 2. mu.l of 10mM dNTP Mix, 1. mu.l of RiboLock RNAase inhibitor (20U/. mu.l)) and 1. mu.l of Revertai M-MuLV reverse transcriptase (200U/. mu.l) were added in this order and mixed by pipetting.

3.2.6 the temperature is kept for 60min at 42 ℃ on a PCR instrument, and the temperature is kept for 5min at 70 ℃ after the completion of the reaction, so that the reverse transcriptase is inactivated.

3.3 quantitative PCR

3.3.1 prepare the following reaction system in 0.2mL PCR tubes, and prepare 3 tubes for each reverse transcription product.

3.3.2 PCR amplification

Pre-denaturation at 95 deg.C for 10min

Cycles (40 times) 95 ℃, 15s → 60 ℃, 60s

Melting curve 60 ℃ → 95 ℃, temperature rise 0.3 ℃ every 15s

3.4 results processing

Δ Δ CT method:

CT (target gene, sample to be tested) -CT (internal standard gene, sample to be tested)

CT (target gene, control sample) -CT (internal standard gene, control sample)

K＝A-B

Expression fold 2^-K

Fig. 9 is a histogram of the relative expression of GABAAR α 1mRNA in brain tissue of each group of mice according to an embodiment of the present invention (P <0.05, indicating significant, and P <0.01, indicating very significant, compared to the model group). As can be seen from fig. 9, the relative expression level of GABAAR α 1mRNA in the PCPA model group was lower than that in the other groups, and the relative expression level of GABAAR α 1mRNA in the experimental group 2 (magnolia sieboldii extract + vetiver essential oil + boswellia essential oil + true lavender essential oil + sweet marjoram essential oil + pinus sylvestris essential oil) was significantly better than that in the PCPA model group and the control group (vetiver essential oil + boswellia essential oil + true lavender essential oil + sweet marjoram essential oil + pinus sylvestris essential oil).

The above-mentioned embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the same; although the present 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 solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Claims

1. A composition, comprising: magnolia sieboldii extract, vetiver essential oil, frankincense essential oil, true lavender essential oil, sweet marjoram essential oil and Pinus sylvestris essential oil.

2. The composition as claimed in claim 1, wherein the mass ratio of the magnolia sieboldii extract, the vetiver essential oil, the boswellia essential oil, the lavender essential oil, the sweet marjoram essential oil and the pinus sylvestris essential oil is (4-8): (1-4): (2-6): (2-5): (1-4): (2-5).

3. The composition of claim 1, wherein the Magnolia sieboldii extract is Magnolia sieboldii essential oil.

4. The composition according to claim 1, wherein the magnolia sieboldii extract, the vetiver essential oil, the boswellia essential oil, the lavender essential oil, the sweet marjoram essential oil and the pinus sylvestris essential oil are in a mass ratio of 8:4:6:5:4:5, 6:2:4:3:2:3 or 4:1:2:2:1: 2.

5. Use of a composition according to any one of claims 1 to 4 in the manufacture of a sleep aid product.

6. A sleep aid product comprising a composition according to any one of claims 1 to 4.

7. A sleep-aid product according to claim 6, wherein said product is administered by inhalation, painting or aromatherapy.

8. A sleep-aid product according to claim 6, wherein the effective amount of the composition in the product is 625-2500 μ L/day/kg.

9. A sleep-aid product according to claim 6, wherein the composition is contained in the sleep-aid product in an amount of 0.001 to 10% by mass.

10. A sleep-aid product according to claim 6, wherein the sleep-aid product is a daily chemical product.