CN113288834A

CN113288834A - New application of Magnolia sieboldii extract

Info

Publication number: CN113288834A
Application number: CN202110443501.7A
Authority: CN
Inventors: 艾勇; 唐明慧; 宋妮; 何廷刚; 朱思阳; 蔡少纯
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: 2021-08-24

Abstract

The invention relates to the technical field of plant extracts, in particular to a new application of Magnolia sieboldii. The invention discloses application of Magnolia sieboldii in preparation of sleep-aiding products, and the Magnolia sieboldii extract has the sleep-aiding effect for the first time, and can shorten the sleep latency time of a mouse in a pentobarbital sodium induced sleep experiment and prolong the sleep time; increasing body weight in insomnia mice; increasing the number of neurons in each region of the brain of the mouse; increase the expression level of 5HT-1A protein in hippocampal region of brain.

Description

New application of Magnolia sieboldii extract

Technical Field

The invention relates to the technical field of plant extracts, in particular to a new application of a magnolia sieboldii extract.

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.

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 invention provides the application of the magnolia sieboldii extract in the preparation of sleep-aiding products, and the magnolia sieboldii extract can effectively shorten the sleep latency, prolong the sleep time, improve the number of neurons in each area of the brain and the expression level of 5HT-1A protein, and increase the weight of an insomnia mouse.

The specific technical scheme is as follows:

the invention provides an application of magnolia sieboldii extract in preparing a sleep-aiding product.

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 leaf 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 disclosed by the 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.

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-assisting effect of the essential oil and the good and bad curative effect of the magnolia sieboldii essential oil by taking the numerical values of a PCPA model group and a blank control group as the reference, and the sleep time can also intuitively show the good and bad curative effect of the magnolia sieboldii essential oil. The result shows that the magnolia sieboldii leaf essential oil provided by the invention can prolong the sleep time induced by the sodium pentobarbital and shorten the sleep latency time.

Further, the invention adopts Nie's staining to detect the number of the mouse neurons. The result shows that the magnolia sieboldii leaf essential oil provided by the invention can improve the normal neuron number of the cerebral cortex region and the hypothalamus region of a mouse.

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.

Furthermore, the invention adopts paraffin section immunohistochemical test to detect the expression of hippocampal 5HT-1A protein in brain tissue of mouse. The result shows that the magnolia sieboldii leaf essential oil provided by the invention can obviously improve the expression of the hippocampus 5HT-1A protein in brain tissues.

Furthermore, the invention also tests the body weight of the mice before and after the model building and before and after the treatment. The result shows that the weight of the insomnia mouse remarkably increases after the treatment by the magnolia sieboldii leaf essential oil provided by the invention.

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

The invention also provides a sleep-aiding product, which comprises the magnolia sieboldii extract.

In the invention, the Magnolia sieboldii extract is Magnolia sieboldii essential oil.

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 magnolia sieboldii extract in the sleep-aiding product is 625 mu L/day/kg-2500 mu L/kg/day, preferably 1250 mu L/day/kg or 2500 mu L/day/kg.

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 discovers that the magnolia sieboldii extract has the sleep-aiding effect for the first time, and the magnolia sieboldii extract can shorten the sleep latency of a mouse in a pentobarbital sodium induced sleep experiment and prolong the sleep time; increasing body weight in insomnia mice; increasing the number of neurons in each region of the brain of the mouse; improve the expression level of 5HT-1A protein in hippocampal region of mouse brain.

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 image of Neisseria staining neurons of brain tissue sections of various groups of mice provided by the embodiment of the present invention (scale is 50 μm);

FIG. 4 is a statistical plot of the number of normal neurons in the cerebral cortex of each group of mice provided by the present invention;

FIG. 5 is a statistical graph of the number of normal neurons in the hypothalamic region of each group of mice provided by the present invention;

FIG. 6 is a microscope photograph (50 μm scale) of the expression of 5HT-1A protein in immunohistochemical sections of hippocampal regions of various groups of mice as provided in the examples of the present invention;

FIG. 7 is a bar graph of 5HT-1A protein expression in immunohistochemical sections of hippocampal regions of various groups of mice provided by the examples of the present invention;

fig. 8 is a line graph showing the change of body weight of each group of mice provided by 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.

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. After the last intraperitoneal injection of PCPA is finished, the behavior of the mouse changes (the activity is ceaseless in the daytime, the excitability is increased, the irritability is easy, the circadian rhythm disappears, the water intake and the food intake are both increased, and the sleeping time is obviously reduced) 26-30 hours later, which indicates that the insomnia molding is successful.

Example 3

This example verifies the sleep-aiding effect of Magnolia sieboldii leaf essential oil

1. Method for detecting influence of Magnolia sieboldii leaf essential oil with different concentrations on sleep latency and sleep time of sleeping mice by adopting sodium pentobarbital animal sleep time test

The influence of the magnolia sieboldii leaf essential oil on the sleep induction time of the barbiturate medicaments is tested, and the hypnotic medicaments can prolong the sleep induction time of the sodium pentobarbital generally. Recording the time of falling asleep and the time of sleeping, comparing the difference between the smelling group and the PCPA model control group of the essential oil at each concentration, and testing the significance of the difference, wherein the sleep time is prolonged, P <0.05 shows that the essential oil at the concentration has significant hypnotic effect, and P <0.01 shows that the essential oil at the concentration has extremely 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 5 groups, and animals smell the Magnolia sieboldii leaf essential oil with different gradient concentrations respectively: the inhalation volume of the mice in the low-concentration group is 625 mu L/kg/day, the inhalation volume of the mice in the medium-concentration group is 1250 mu L/kg/day, the inhalation volume of the mice in the high-concentration group is 2500 mu L/day/kg, the mice in the blank group are inhaled with physiological saline containing 1% Tween80, the volumes of the mice in the blank group are 2500 mu L/day/kg, and each mouse in each group smells for 30 minutes every day;

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.

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.

Fig. 1 is a graph of the results of the tests on sleep latency in the groups of mice of 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, compared to the PCPA model group, the sleep latency of mice in the low and medium-high concentration magnolia sieboldii leaf essential oil group is significantly shortened and dose-dependent, wherein the high concentration magnolia sieboldii leaf essential oil group has a better treatment effect.

Fig. 2 is a graph of the test results of the sleep time of the mice in each group of 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. 2, the sleep time of the low and medium concentration Magnolia sieboldii leaf essential oil group is prolonged compared with the PCPA model group, wherein the sleep time of the sleep experiment mice can be better prolonged by the low concentration Magnolia sieboldii leaf essential oil.

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 brain tissue of each group of mice in the present example; FIG. 4 is a statistical chart of the number of normal neurons in the cerebral cortex of the mice in each group of the present example; FIG. 5 is a statistical chart showing the number of normal neurons in the hypothalamic region of each group of mice in this example. As can be seen from FIGS. 3 to 5, the number of neuronal cells in the hypothalamic region of the brain cortical region of mice in the PCPA model group was significantly reduced compared to the blank group. The essential oil of the leaf of Magnolia sieboldii with low and medium concentration shows the phenomenon of the number of neurons in the cerebral cortex area and the hypothalamus area which are increased in different degrees and are in a dose-dependent manner. Of these, the Magnolia sieboldii leaf essential oil-high concentration group showed the most significant increase in the number of neurons in both the cerebral cortical area and the hypothalamus area.

3. Immunohistochemical detection of expression of 5HT-1A protein in hippocampal region of mice in each group by paraffin section

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 in PBS (pH7.4) on a destaining shaker for 5min each.

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 blocking solution, adding PBS (5HT1A Receptor Antibody, AF5453, dilution ratio IHC 1:50-1:200) in a certain proportion, and incubating the slices in a wet box at 4 deg.C 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. 6 is a microscope photograph showing the expression of 5HT-1A protein in immunohistochemical sections of hippocampal regions of mice in each group of the present example; figure 7 is a histogram of 5HT-1A protein expression in hippocampal immunohistochemical sections of mice in each group of this example (P <0.05 for significance and P <0.01 for extreme significance compared to model group). As can be seen from FIGS. 6 and 7, the expression of 5HT-1A protein in hippocampal region of brain tissue was significantly reduced in mice of PCPA model group compared to mice of blank group; compared with a PCPA model group, the expression level of the 5HT-1A protein in the hippocampus of the brain tissue of a mouse in the low-concentration and medium-concentration Magnolia sieboldii leaf essential oil group is obviously improved, wherein the expression level of the high-concentration group is most obvious.

4. The body weights of the mice in each group were measured

Before modeling, after modeling and after treatment, the change of the body mass of each group of mice is recorded to reflect the change of the physiological condition of the mice before modeling, after modeling and after treatment. The collected data were plotted as a line graph of the rate of change of body mass, which is shown in fig. 8.

FIG. 8 is a line graph showing the change in body weight of the mice in each group of this example. As can be seen from fig. 8, the body weight was significantly reduced after PCPA molding in mice compared to the blank group; compared with the PCPA model group, the mice treated by diazepam and Magnolia sieboldii leaf essential oil have different degrees of weight regain along the fold line. The weight gain of the high-concentration group is most obvious.

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. Application of Magnolia sieboldii extract in preparing sleep-aiding product is provided.

2. The use of claim 1, wherein the Magnolia sieboldii extract is one or more of an alcohol extract, an aqueous extract and a steam distillation extract of Magnolia sieboldii.

3. The use as claimed in claim 1, wherein the source of the Magnolia sieboldii extract is one or more selected from the group consisting of leaves, roots, flowers, fruits and stems of Magnolia sieboldii.

4. The use according to claim 1, wherein the magnolia sieboldii extract is present in the sleep-aid product in an amount of 0.001% to 10% by weight.

5. Use according to claim 1, wherein the sleep aid product is a daily chemical product.

6. The use according to any one of claims 1 to 5, wherein the Magnolia sieboldii extract is Magnolia sieboldii essential oil.

7. A sleep-aiding product is characterized by comprising an extract of Magnolia sieboldii.

8. A sleep aid product according to claim 7, wherein the Magnolia sieboldii extract is Magnolia sieboldii essential oil.

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

10. A sleep-aid product according to any one of claims 7 to 9, wherein the effective dose of magnolia sieboldii extract in the sleep-aid product is 625 μ L/day/kg to 2500 μ L/day/kg.