Composition for improving sleep

Abstract

The invention discloses a composition for improving sleep, which is characterized in that the composition comprises panax notoginseng stem and leaf total saponins, a spina date seed extract, a lavender extract and lavender essential oil; wherein the mass ratio of the panax notoginseng stem and leaf total saponins to the semen ziziphi spinosae extract to the lavender extract to the lavender essential oil is (2.50 to 3.50) to (45.00 to 55.00) to (55.00 to 70.00) to (4.00 to 20.00). The composition has the effect of exciting a 5-HT1A receptor, can improve the sleep condition of a mouse suffering from insomnia caused by p-chlorophenylalanine (PCPA), and has sedative and hypnotic effects on the mouse, and the action mechanism may be related to the improvement of the levels of 5-hydroxytryptamine (5-HT) and gamma-aminobutyric acid (GABA) in the brain and the reduction of the level of glutamic acid (Glu) in the brain.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a composition for improving sleep. Background Art

[0002] Insomnia refers to a sleep disorder syndrome in which the initiation and maintenance of sleep are impaired, resulting in the inability of sleep quality or quantity to meet individual physiological needs, which has a significant impact on the patient's daily activities. It is often accompanied by fatigue, headaches, mood disorders (such as depression, anxiety), and impaired cognitive function. At the same time, insomnia increases the risk of chronic diseases such as cardiovascular and cerebrovascular diseases and diabetes, and accelerates the aging process of the human body, seriously threatening people's quality of life and physical and mental health. According to a WHO survey, the incidence of insomnia is about 27%, the prevalence of insomnia in the general population in China is about 15%, and the incidence rate in the adolescent group is between 16.57% and 38.20%, which is a high-risk group for insomnia, showing that insomnia is becoming younger and younger. Therefore, how to improve sleep quality has become one of the most concerned health issues. At this stage, the methods to improve insomnia mainly include drug therapy, cognitive behavioral therapy, physical therapy, etc. Drug therapy is mainly based on chemical synthetic sedatives. Commonly used drugs in clinical practice are barbiturates and benzodiazepines. Non-benzodiazepines Chemical synthetic drugs such as GABAA receptor agonists and melatonin have the advantages of fast onset and clear effects. However, these sedatives have side effects such as low safety, drowsiness, addiction and tolerance. Cognitive behavioral therapy has high limitations on its treatment targets, has high requirements for the patient's psychological cognitive level and a relatively long treatment cycle. Physical therapy has disadvantages such as high equipment cost and expensive treatment costs. Traditional Chinese medicine rehabilitation technology has great advantages in the treatment of this disease, mainly including acupuncture, massage, moxibustion, medicinal bath, emotional therapy, ear acupoint pressure beans, Chinese medicine atomization, etc. Chinese medicine atomization is a kind of Chinese medicine fumigation. The gas after Chinese medicine atomization contains drug ions such as aromatic turbidity and spicy fragrance. After acting on the skin mucosa and acupoints, it regulates the higher nerve center, endocrine and immune system through the neurohumoral and meridian systems, and quickly adjusts the qi and blood functions of the human body's internal organs. Traditional Chinese medicine treatment can effectively improve the symptoms of insomnia, with small side effects and no addiction, which can effectively make up for the shortcomings of Western medicine in improving insomnia.

[0003] A composition for improving sleep as claimed in the present invention is not found in the prior art. Summary of the invention

[0004] Based on this, the present invention provides a composition for improving sleep, which composition comprises total saponins from Panax notoginseng stems and leaves, semen ziziphi spinosae extract, lavender extract and lavender essential oil; wherein, the mass ratio between the total saponins from Panax notoginseng stems and leaves, the semen ziziphi spinosae extract, the lavender extract and the lavender essential oil is (2.50 - 3.50):(45.00 - 55.00):(55.00 - 70.00):(4.00 - 20.00).

[0005] Further, the composition consists of total saponins from Panax notoginseng stems and leaves, semen ziziphi spinosae extract, lavender extract and lavender essential oil. Further, the mass ratio between the total saponins from Panax notoginseng stems and leaves, the semen ziziphi spinosae extract, the lavender extract and the lavender essential oil is (2.80 - 3.30):(47.00 - 53.00):(59.00 - 66.00):(4.00 - 20.00). Further, the mass ratio between the total saponins from Panax notoginseng stems and leaves, the semen ziziphi spinosae extract, the lavender extract and the lavender essential oil is approximately 3.12: approximately 50.00: approximately 62.50:(4.00 - 20.00). Further, the mass ratio between the total saponins from Panax notoginseng stems and leaves, the semen ziziphi spinosae extract, the lavender extract and the lavender essential oil is approximately 3.12: approximately 50.00: approximately 62.50: approximately 4.69. Further, the mass ratio between the total saponins from Panax notoginseng stems and leaves, the semen ziziphi spinosae extract, the lavender extract and the lavender essential oil is approximately 3.12: approximately 50.00: approximately 62.50: approximately 18.75. Further, the mass ratio between the total saponins from Panax notoginseng stems and leaves, the semen ziziphi spinosae extract, the lavender extract and the lavender essential oil is not 12.5:25:31.25:4.69. Further, the mass ratio between the total saponins from Panax notoginseng stems and leaves, the semen ziziphi spinosae extract, the lavender extract and the lavender essential oil is not 12.5:25:15.62:18.75. Further, the mass ratio between the total saponins from Panax notoginseng stems and leaves, the semen ziziphi spinosae extract, the lavender extract and the lavender essential oil is not 3.12:25:15.62:4.69. Further, the in vitro administration concentration of the total saponins from Panax notoginseng stems and leaves in the composition is 2.5 - 3.5 μg / ml, such as approximately 3.12 μg / ml. Further, the in vitro administration concentration of the semen ziziphi spinosae extract in the composition is 45 - 55 μg / ml, such as approximately 50 μg / ml. Further, the in vitro administration concentration of the lavender extract in the composition is 55 - 70 μg / ml, such as approximately 62.5 μg / ml. Further, the in vitro administration concentration of the lavender essential oil in the composition is 4 - 20 μg / ml, such as approximately 4.69 μg / ml or 18.75 μg / ml.

[0006] Furthermore, the preparation method of the total saponins from Panax notoginseng stems and leaves comprises the following steps: (1) Weigh an appropriate amount of Panax notoginseng stems and leaves, extract with a solvent, filter, and combine the filtrates; (2) Load the combined filtrate onto macroporous resin and perform alcohol elution; and (3) Concentrate, dry, and sieve the eluate to obtain the total saponins from Panax notoginseng stems and leaves.

[0007] Furthermore, in step (1), the solvent is water. Further, in step (1), the number of extractions is 1 to 5 times, such as 3 times. Further, in step (2), the macroporous resin is D101 macroporous resin. Further, in step (2), the concentration of the alcohol is 0.1% to 100%, such as 1% to 99%, such as 5% to 95%, such as 10% to 90%, such as 20% to 80%, such as 30% to 80%, such as about 20%, such as about 30%, such as about 40%, such as about 50%, such as about 60%, such as about 70%, such as about 80%, such as about 90%, such as about 95%, such as about 99%. Further, in step (3), the drying is freeze-drying.

[0008] Furthermore, the preparation method of the semen ziziphi spinosae extract comprises the following steps: (1) Weigh an appropriate amount of semen ziziphi spinosae, crush and sieve, add a first solvent to soak, heat under reflux for extraction, and filter to obtain filter residues; (2) Evaporate the solvent from the filter residues to obtain defatted medicinal residues, add a second solvent, reflux for extraction, and filter to obtain a filtrate; and (3) Rotavapor-concentrate the filtrate to obtain a concentrated solution, dry to obtain the semen ziziphi spinosae extract.

[0009] Further, in step (1), the sieve is a No. 4 sieve. Further, in step (1), the first solvent is petroleum ether. Further, in step (1), the volume / mass (ml / g) ratio of the first solvent to the semen ziziphi spinosae is 1 to 5, such as about 2.5. Further, in step (1), the mass of the semen ziziphi spinosae is 50 to 100 g, such as about 80 g. Further, in step (1), the number of times of heating under reflux is 1 to 3 times, such as 2 times. Further, in step (1), the temperature of heating under reflux is 50 to 70 °C, such as about 60 °C. Further, in step (1), the time of heating under reflux is 1 to 3 h each time, such as about 2 h.

[0010] Further, in step (2), the evaporation of the first solvent is carried out in a fume hood. Further, in step (2), the second solvent is ethanol. Further, the volume percentage concentration of the ethanol is 60% - 80%, for example, about 70%. Further, in step (2), the volume / mass (ml / g) ratio of the second solvent to the defatted medicinal residues is 5 - 10, for example, about 8. Further, in step (2), the number of times of reflux extraction is 1 - 3 times, for example, 2 times. Further, in step (2), the time of each reflux extraction is 1 - 3 h, for example, about 2 h.

[0011] Further, in step (3), the temperature of rotary evaporation and concentration is 50 - 70°C, for example, about 60°C. Further, in step (3), the end point of rotary evaporation and concentration is that the concentrated solution has no alcohol smell. Further, in step (3), the drying is freeze-drying. Further, the yield of the wild jujube seed extract is 10% - 20%, for example, about 13.1%.

[0012] Further, the preparation method of the lavender extract comprises the following steps: (a) Weigh an appropriate amount of lavender, crush it, add a third solvent for the first soaking, filter to obtain a first filtrate and residues; (b) Add the third solvent to the residues for the second soaking, filter to obtain a second filtrate; and (c) Combine the first filtrate and the second filtrate, carry out rotary evaporation and concentration to obtain a concentrated solution, and dry the concentrated solution to obtain the lavender extract.

[0013] Further, the third solvent is ethanol. Further, the volume percentage concentration of the ethanol is 70% - 90%, for example, about 80%.

[0014] Further, in step (a), the volume / mass (ml / g) ratio of the third solvent to the lavender is 5 - 15, for example, about 10. Further, in step (a), the mass of the lavender is 80 - 120 g, for example, about 100 g. Further, in step (a), the volume of the third solvent is 800 - 1200 ml, for example, about 1000 ml. Further, in step (a), the time of the first soaking is 48 - 96 h, for example, about 72 h.

[0015] Further, in step (b), the time of the second soaking is 48 - 96 h, for example, about 72 h. Further, in step (b), the volume of the third solvent is 800 - 1200 ml, for example, about 1000 ml.

[0016] Further, in step (c), the drying is freeze-drying.

[0017] Further, the yield of the lavender extract is 10% - 20%, for example, about 16.6%.

[0018] Furthermore, the composition further comprises one or more pharmaceutically acceptable excipients. Further, the excipient is selected from one or more of the following: diluent, wetting agent, binder, disintegrant, inclusion agent, flavoring agent, sustained release agent, glidant, lubricant, dispersant, plasticizer, light shielding agent, and antioxidant. Further, the dosage form of the composition is powder, tablet, dripping pill, capsule, film, lozenge, granule, injection, or oral liquid.

[0019] Furthermore, the composition further comprises one or more sleep-improving drugs and / or extracts. Further, the drug is selected from one or more of the following: benzodiazepine receptor agonists, antidepressant drugs, and melatonin receptor agonists. Further, the benzodiazepine receptor agonists include benzodiazepine -type drugs and non-benzodiazepine -type drugs. Further, the benzodiazepine -type drugs are estazolam, triazolam, and diazepam. Further, the non-benzodiazepine -type drugs are zolpidem, zopiclone, and eszopiclone. Further, the antidepressant drugs include noradrenergic and specific 5-HTergic antidepressant drugs, as well as 5-HT receptor blockers and reuptake inhibitors. Further, the noradrenergic and specific 5-HTergic antidepressant drug is mirtazapine. Further, the 5-HT receptor blocker and reuptake inhibitor is trazodone. Further, the melatonin receptor agonists are agomelatine and ramelteon.

[0020] According to another aspect of the present invention, there is provided a method for preparing the above-mentioned composition, the method comprising mixing total saponins from Panax notoginseng stems and leaves, Ziziphus jujuba seed extract, lavender extract and lavender essential oil according to the following mass ratio: (2.50 - 3.50):(45.00 - 55.00):(55.00 - 70.00):(4.00 - 20.00). Further, the mass ratio among the total saponins from Panax notoginseng stems and leaves, the Ziziphus jujuba seed extract, the lavender extract and the lavender essential oil is (2.80 - 3.30):(47.00 - 53.00):(59.00 - 66.00):(4.00 - 20.00). Further, the mass ratio among the total saponins from Panax notoginseng stems and leaves, the Ziziphus jujuba seed extract, the lavender extract and the lavender essential oil is approximately 3.12: approximately 50.00: approximately 62.50:(4.00 - 20.00). Further, the mass ratio among the total saponins from Panax notoginseng stems and leaves, the Ziziphus jujuba seed extract, the lavender extract and the lavender essential oil is approximately 3.12: approximately 50.00: approximately 62.50: approximately 4.69. Further, the mass ratio among the total saponins from Panax notoginseng stems and leaves, the Ziziphus jujuba seed extract, the lavender extract and the lavender essential oil is approximately 3.12: approximately 50.00: approximately 62.50: approximately 18.75. Further, the mass ratio among the total saponins from Panax notoginseng stems and leaves, the Ziziphus jujuba seed extract, the lavender extract and the lavender essential oil is not 12.5:25:31.25:4.69. Further, the mass ratio among the total saponins from Panax notoginseng stems and leaves, the Ziziphus jujuba seed extract, the lavender extract and the lavender essential oil is not 12.5:25:15.62:18.75. Further, the mass ratio among the total saponins from Panax notoginseng stems and leaves, the Ziziphus jujuba seed extract, the lavender extract and the lavender essential oil is not 3.12:25:15.62:4.69.

[0021] According to another aspect of the present invention, there is provided the use of the above-mentioned composition in the preparation of medicaments, foods and / or health products for improving sleep.

[0022] Further, the effect of the composition in improving sleep is achieved through one or more of the following ways: increasing the intracellular calcium ion concentration, improving general behavioral abnormalities, relieving anxiety behaviors, improving sleep quality and improving neurotransmitter metabolism disorders. Further, the general behavioral abnormalities include one or more of the following: weight loss, dull hair and irritable state. Further, the improvement of sleep quality includes shortening the sleep latency and prolonging the sleep duration. Further, the improvement of neurotransmitter metabolism disorders includes one or more of the following: increasing the content of 5-hydroxytryptamine in the hypothalamus, increasing the content of 5-hydroxytryptamine in the hippocampus, increasing the content of γ-aminobutyric acid in the hypothalamus, increasing the content of γ-aminobutyric acid in the hippocampus and decreasing the content of glutamate in the hypothalamus.

[0023] Advantages of the present invention:

[0024] This composition has the effect of activating 5-HT1A receptors, can improve the sleep of mice with insomnia induced by parachlorophenylalanine (PCPA), has a sedative and hypnotic effect on mice, and its mechanism of action may be related to increasing the levels of 5-hydroxytryptamine (5-HT) and γ-aminobutyric acid (GABA) in the brain and decreasing the level of glutamate (Glu) in the brain. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without exceeding the scope of protection required by the present invention.

[0026] Figure 1 Schematic diagram of the effects of three different traditional Chinese medicine extracts (total saponins from Panax notoginseng stems and leaves, Ziziphus jujuba seed extract, lavender extract) in Example 1 on the viability of 293T cells.

[0027] Figure 2 Schematic diagram of the effects of lavender essential oil, marjoram essential oil, lemon balm essential oil, valerian essential oil, and vetiver essential oil in Example 1 on the viability of 293T cells.

[0028] Figure 3 Schematic diagram of the agonist effect of formula PSLO in Example 1 on 5-HTR1A. Among them, 5-HT is 5-hydroxytryptamine. Compared with the normal group, *P<0.05, **P<0.01, ***P<0.001.

[0029] Figure 4 Schematic diagram of the agonist effect of formula PSLMM in Example 1 on 5-HTR1A.

[0030] Among them, 5-HT is 5-hydroxytryptamine.

[0031] Figure 5 Schematic diagram of the agonist effect of formula PSLVV in Example 1 on 5-HTR1A. Among them, 5-HT is 5-hydroxytryptamine.

[0032] Figure 6 Schematic diagram of the agonist effect of different compatibility ratios of formula PSLO in Example 1 on 5-HTR1A. Among them, 5-HT is 5-hydroxytryptamine. Compared with the normal group, *P<0.05, ***P<0.001.

[0033] Figure 7Schematic diagram of the comparison results of the body weights of mice in each group before and after modeling in Example 2. Among them, compared with the normal group, ***P < 0.001.

[0034] Figure 8 Schematic diagram of the comparison results of the sleep latency and sleep duration among mice in each group after modeling in Example 2. Among them, compared with the normal group, **P < 0.01.

[0035] Figure 9 Schematic diagram of the comparison results of the body weights of mice in each group after drug administration in Example 2. Among them, PSLO①-L group, low-dose PSLO① group; PSLO①-M group, medium-dose PSLO① group; PSLO①-H group, high-dose PSLO① group. Compared with the model group, *P < 0.05, ***P < 0.001. Compared with the blank control group, ## P < 0.01, ### P < 0.001.

[0036] Figure 10 Schematic diagram of the influence results of the prescription PSLO① on the open field test of mice in each group in Example 2. Among them, PSLO①-L group, low-dose PSLO① group; PSLO①-M group, medium-dose PSLO① group; PSLO①-H group, high-dose PSLO① group. Compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001. Compared with the blank control group, # P < 0.05, ### P < 0.001.

[0037] Figure 11 Open field test activity trajectory diagram of mice in each group in Example 2. Among them, A, blank control group; B, model group; C, diazepam group; D, PSLO①-L group, low-dose PSLO① group; E, PSLO①-M group, medium-dose PSLO① group; F, PSLO①-H group, high-dose PSLO① group.

[0038] Figure 12 Schematic diagram of the influence results of the prescription PSLO① on the hypnotic effect induced by suprathreshold dose of pentobarbital sodium in mice. Among them, PSLO①-L group, low-dose PSLO① group; PSLO①-M group, medium-dose PSLO① group; PSLO①-H group, high-dose PSLO① group. Compared with the model group, **P < 0.01, ***P < 0.001. Compared with the blank control group, ## P < 0.01, ### P < 0.001.

[0039] Figure 13Schematic diagram of the effect of prescription PSLO① in Example 2 on the contents of neurotransmitters in the hypothalamus and hippocampus of insomnia mice. Among them, PSLO①-L group, low-dose group of PSLO①; PSLO①-M group, medium-dose group of PSLO①; PSLO①-H group, high-dose group of PSLO①. 5-HT, 5-hydroxytryptamine; GABA, γ-aminobutyric acid; Glu, glutamate. Compared with the model group, *P<0.05, **P<0.01. Compared with the blank control group, # P<0.05, ## P<0.01. Detailed implementation manners

[0040] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts fall within the protection scope of the present invention.

[0041] Unless otherwise stated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those of ordinary skill in the field of the present invention or the field to which the term is applied. Although any methods, conditions, substances or materials similar to or equivalent to those disclosed herein can be used in the implementation process of the present invention, the preferred methods, conditions, substances or materials are described herein.

[0042] The present invention is expected to cover all alternatives, variations and equivalents, which may be included in the existing field of the invention as defined in the claims. Those skilled in the art will recognize many methods and substances similar or equivalent to those described herein, which can be applied to the practice of the present invention. The present invention is by no means limited to the description of methods and substances.

[0043] The singular forms "a", "an" and "the" used in the specification and the appended claims include plural referents unless the context clearly dictates otherwise.

[0044] In the present invention, the term "comprising" is synonymous with "including". The terms "including", "comprising", "having", "containing" or any other variation thereof used herein are intended to cover non-exclusive inclusion. For example, a composition, step, method, article or device containing the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article or device.

[0045] As described in the background art section, current sedative drugs for improving insomnia have side effects such as low safety, drowsiness, addiction, and tolerance. Cognitive behavioral therapy has relatively high limitations for its treatment targets, has relatively high requirements for the psychological and cognitive levels of patients, and has a relatively long treatment cycle. Physical therapy has disadvantages such as high equipment costs and expensive treatment fees. To solve the above problems, the present invention provides a composition for improving sleep, and the composition (abbreviated as PSLO) includes total saponins in stems and leaves of Panax notoginseng (PNSLS), Semen Ziziphi Spinosae Extracts (SZS), Lavender Extracts (LAV), and Lavender Essential Oil (LEO);

[0046] Among them, the mass ratio among the total saponins in stems and leaves of Panax notoginseng, the Semen Ziziphi Spinosae Extracts, the Lavender Extracts, and the Lavender Essential Oil is (2.50 - 3.50):(45.00 - 55.00):(55.00 - 70.00):(4.00 - 20.00).

[0047] In the present invention, when a mass ratio, concentration, number of times, temperature, ratio, mass, time, volume percentage concentration, yield, volume, or other value or parameter is expressed in a range, a preferred range, or a range defined by a series of upper limit preferred values and lower limit preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when the range "4.00 - 20.00" is disclosed, the described range should be interpreted as including ranges such as "4.00 - 20.00", "4.00 - 18.00", "4.00 - 16.00", "4.00 - 14.00", "4.00 - 12.00", "4.00 - 10.00", "4.00 - 8.00", "4.00 - 6.00", etc. When a numerical range is described herein, unless otherwise specified, the range is intended to include its end values and all integers and fractions within the range.

[0048] The present invention is guided by the theory of traditional Chinese medicine, and is composed of total saponins in stems and leaves of Panax notoginseng (PNSLS), Semen Ziziphi Spinosae, and lavender and essential oil thereof (PSLO). The formula is explained as follows: Traditional Chinese medicine believes that the heart stores the spirit and governs blood vessels, and the liver stores blood and governs the release of qi. If the liver fails to release qi, the liver qi is stagnant and the qi is not smooth, resulting in the loss of nourishment of the mind, or the mind is disturbed and the mind is restless, causing insomnia; long-term liver qi stagnation affects the spleen and stomach, resulting in spleen and stomach dysfunction, further aggravating insomnia. Panax notoginseng tastes sweet and slightly bitter, is warm in nature, can dissolve blood stasis without hurting new blood, nourish the heart and calm the mind. Semen Ziziphi Spinosae is sweet, sour and neutral in nature, nourishes the heart and liver, can nourish the heart yin, benefit the liver blood, and calm the mind and the spirit. Lavender is warm in nature and spicy in taste, can relieve the liver and regulate qi, and can calm the mind and help sleep. The combination of these medicines can nourish the heart and liver, dredge the meridians, and calm the mind and soothe the nerves.

[0049] The total saponin of Panax notoginseng stems and leaves, the Chinese date seed extract, the lavender extract and the lavender essential oil of the invention are all edible or drinkable for humans.

[0050] In a preferred embodiment, the composition consists of total saponins from Panax notoginseng stems and leaves, Ziziphus jujuba seed extract, lavender extract and lavender essential oil.

[0051] In a preferred embodiment, the mass ratio of the total saponins of Panax notoginseng stems and leaves, the Ziziphus jujuba seed extract, the lavender extract and the lavender essential oil is (2.80-3.30): (47.00-53.00): (59.00-66.00): (4.00-20.00).

[0052] In a preferred embodiment, the mass ratio of the total saponins from the notoginseng stems and leaves, the Ziziphus jujuba seed extract, the lavender extract and the lavender essential oil is about 3.12: about 50.00: about 62.50: (4.00-20.00).

[0053] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 3.12" includes ±5% of 3.12, or from 2.964 to 3.276; "about 50.00" includes ±5% of 50.00, or from 47.50 to 52.50; "about 62.50" includes ±5% of 62.50, or from 59.375 to 65.625.

[0054] In a preferred embodiment, the mass ratio of the total saponins from the notoginseng stems and leaves, the Ziziphus jujuba seed extract, the lavender extract and the lavender essential oil is about 3.12: about 50.00: about 62.50: about 4.69.

[0055] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 3.12" includes ±5% of 3.12, or from 2.964 to 3.276; "about 50.00" includes ±5% of 50.00, or from 47.50 to 52.50; "about 62.50" includes ±5% of 62.50, or from 59.375 to 65.625; "about 4.69" includes ±5% of 4.69, or from 4.4555 to 4.9245.

[0056] In a preferred embodiment, the mass ratio among the total saponins from Panax notoginseng stems and leaves, the extract of Ziziphus jujuba seeds, the extract of lavender, and the lavender essential oil is about 3.12: about 50.00: about 62.50: about 18.75.

[0057] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 3.12" includes ±5% of 3.12, or from 2.964 to 3.276; "about 50.00" includes ±5% of 50.00, or from 47.50 to 52.50; "about 62.50" includes ±5% of 62.50, or from 59.375 to 65.625; "about 18.75" includes ±5% of 18.75, or from 17.8125 to 19.6875.

[0058] In a preferred embodiment, the mass ratio among the total saponins from Panax notoginseng stems and leaves, the extract of Ziziphus jujuba seeds, the extract of lavender, and the lavender essential oil is not 12.5: 25: 31.25: 4.69.

[0059] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 12.5" includes ±5% of 12.5, or from 11.875 to 13.125; "about 25" includes ±5% of 25, or from 23.75 to 26.25; "about 31.25" includes ±5% of 31.25, or from 29.6875 to 32.8125; "about 4.69" includes ±5% of 4.69, or from 4.4555 to 4.9245.

[0060] In a preferred embodiment, the mass ratio among the total saponins from Panax notoginseng stems and leaves, the extract of Ziziphus jujuba seeds, the extract of lavender, and the lavender essential oil is not 12.5: 25: 15.62: 18.75.

[0061] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 12.5" includes ±5% of 12.5, or from 11.875 to 13.125; "about 25" includes ±5% of 25, or from 23.75 to 26.25; "about 15.62" includes ±5% of 15.62, or from 14.839 to 16.401; "about 18.75" includes ±5% of 18.75, or from 17.8125 to 19.6875.

[0062] In a preferred embodiment, the mass ratio among the total saponins from Panax notoginseng stems and leaves, the Ziziphus jujuba seed extract, the lavender extract, and the lavender essential oil is not 3.12:25:15.62:4.69.

[0063] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 3.12" includes ±5% of 3.12, or from 2.964 to 3.276; "about 25" includes ±5% of 25, or from 23.75 to 26.25; "about 15.62" includes ±5% of 15.62, or from 14.839 to 16.401; "about 4.69" includes ±5% of 4.69, or from 4.4555 to 4.9245.

[0064] In a preferred embodiment, the in vitro administration concentration of the total saponins from Panax notoginseng stems and leaves in the composition is 2.5 - 3.5 μg / ml, for example, about 3.12 μg / ml.

[0065] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 3.12" includes ±5% of 3.12, or from 2.964 to 3.276.

[0066] In a preferred embodiment, the in vitro administration concentration of the Ziziphus jujuba seed extract in the composition is 45 - 55 μg / ml, for example, about 3.12 μg / ml or 50 μg / ml.

[0067] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 3.12" includes ±5% of 3.12, or from 2.964 to 3.276; "about 50.00" includes ±5% of 50.00, or from 47.50 to 52.50.

[0068] In a preferred embodiment, the in vitro administration concentration of the lavender extract in the composition is 55 - 70 μg / ml, for example, about 62.5 μg / ml.

[0069] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 62.50" includes ±5% of 62.50, or from 59.375 to 65.625.

[0070] In a preferred embodiment, the in vitro administration concentration of the lavender essential oil in the composition is 4 - 20 μg / ml, for example, about 4.69 μg / ml or 18.75 μg / ml.

[0071] In the present invention, "about" means a value within the range of ±5% of a specific value. For example, "about 4.69" includes ±5% of 4.69, or from 4.4555 to 4.9245; "about 18.75" includes ±5% of 18.75, or from 17.8125 to 19.6875.

[0072] In a preferred embodiment, the preparation method of the total saponins from Panax notoginseng stems and leaves comprises the following steps: (1) Weigh an appropriate amount of Panax notoginseng stems and leaves, extract with a solvent, filter and combine the filtrates; (2) Load the combined filtrate onto a macroporous resin and perform alcohol elution; and (3) Concentrate, dry, and sieve the eluate to obtain the total saponins from Panax notoginseng stems and leaves.

[0073] In a preferred embodiment, in step (1), the solvent is water. In a preferred embodiment, in step (1), the number of extractions is 1 - 5 times, for example, 3 times. In a preferred embodiment, in step (2), the macroporous resin is D101 macroporous resin. In a preferred embodiment, in step (2), the concentration of the alcohol is 0.1% - 100%, for example, 1% - 99%, for example, 5% - 95%, for example, 10% - 90%, for example, 20% - 80%, for example, 30% - 80%, for example, about 20%, for example, about 30%, for example, about 40%, for example, about 50%, for example, about 60%, for example, about 70%, for example, about 80%, for example, about 90%, for example, about 95%, for example, about 99%. In a preferred embodiment, in step (3), the drying is freeze-drying.

[0074] In a preferred embodiment, the preparation method of the Semen Ziziphi Spinosae extract comprises the following steps: (1) Weigh an appropriate amount of Semen Ziziphi Spinosae, crush and sieve, add a first solvent to soak, heat under reflux for extraction, and filter to obtain filter residues; (2) Evaporate the solvent from the filter residues to obtain defatted medicinal residues, add a second solvent, reflux for extraction, and filter to obtain a filtrate; and (3) Rotavapor-concentrate the filtrate to obtain a concentrated solution, dry it to obtain the Semen Ziziphi Spinosae extract.

[0075] In a preferred embodiment, in step (1), the sieve is a No. 4 sieve.

[0076] In a preferred embodiment, in step (1), the first solvent is petroleum ether.

[0077] In a preferred embodiment, in step (1), the ratio of the volume / mass (ml / g) of the first solvent to the wild jujube seeds is 1 to 5, for example, about 2.5.

[0078] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 2.5" includes ±5% of 2.5, or from 2.375 to 2.625.

[0079] In a preferred embodiment, in step (1), the mass of the wild jujube seeds is 50 to 100 g, for example, about 80 g.

[0080] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 80" includes ±5% of 80, or from 76 to 84.

[0081] In a preferred embodiment, in step (1), the number of times of heating under reflux is 1 to 3 times, for example, 2 times.

[0082] In a preferred embodiment, in step (1), the temperature of heating under reflux is 50 to 70 °C, for example, about 60 °C.

[0083] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 60" includes ±5% of 60, or from 57 to 63.

[0084] In a preferred embodiment, in step (1), the time of heating under reflux is 1 to 3 h each time, for example, about 2 h.

[0085] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 2" includes ±5% of 2, or from 1.9 to 2.1.

[0086] In a preferred embodiment, in step (2), the evaporation of the first solvent is carried out in a fume hood.

[0087] In a preferred embodiment, in step (2), the second solvent is ethanol.

[0088] In a preferred embodiment, the volume percentage concentration of the ethanol is 60% to 80%, for example, about 70%.

[0089] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 70%" includes ±5% of 70%, or from 66.5% to 73.5%.

[0090] In a preferred embodiment, in step (2), the ratio of the volume / mass (ml / g) of the second solvent to the defatted medicinal residues is 5 to 10, for example, about 8.

[0091] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 8" includes ±5% of 8, or from 7.6 to 8.4.

[0092] In a preferred embodiment, in step (2), the number of times of reflux extraction is 1 to 3 times, for example, 2 times.

[0093] In a preferred embodiment, in step (2), the time of reflux extraction is 1 to 3 h each time, for example, about 2 h.

[0094] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 2" includes ±5% of 2, or from 1.9 to 2.1.

[0095] In a preferred embodiment, in step (3), the temperature of rotary evaporation and concentration is 50 to 70 °C, for example, about 60 °C.

[0096] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 60" includes ±5% of 60, or from 57 to 63.

[0097] In a preferred embodiment, in step (3), the end point of rotary evaporation and concentration is that the concentrated solution has no alcohol smell.

[0098] In a preferred embodiment, in step (3), the drying is freeze-drying.

[0099] In a preferred embodiment, the yield of the wild jujube seed extract is 10% to 20%, for example, about 13.1%.

[0100] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 13.1%" includes ±5% of 13.1%, or from 12.445% to 13.755%.

[0101] In a preferred embodiment, the preparation method of the lavender extract comprises the following steps: (a) Weigh an appropriate amount of lavender, crush it, add a third solvent for the first soaking, filter to obtain a first filtrate and a filter residue; (b) Add the third solvent to the filter residue for the second soaking, filter to obtain a second filtrate; and (c) Combine the first filtrate and the second filtrate, carry out rotary evaporation and concentration to obtain a concentrated solution, and dry the concentrated solution to obtain the lavender extract.

[0102] In a preferred embodiment, the third solvent is ethanol.

[0103] In a preferred embodiment, the volume percentage concentration of the ethanol is 70% to 90%, for example, about 80%.

[0104] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 80%" includes ±5% of 80%, or from 76% to 84%.

[0105] In a preferred embodiment, in step (a), the volume / mass (ml / g) ratio of the third solvent to the lavender is 5 to 15, for example, about 10.

[0106] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0107] In a preferred embodiment, in step (a), the mass of the lavender is 80 to 120 g, for example, about 100 g.

[0108] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 100" includes ±5% of 100, or from 95 to 105.

[0109] In a preferred embodiment, in step (a), the volume of the third solvent is 800 to 1200 ml, for example, about 1000 ml.

[0110] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 1000" includes ±5% of 1000, or from 950 to 1050.

[0111] In a preferred embodiment, in step (a), the time of the first soaking is 48 to 96 h, for example, about 72 h.

[0112] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 72" includes ±5% of 72, or from 68.4 to 75.6.

[0113] In a preferred embodiment, in step (b), the time of the second soaking is 48 to 96 h, for example, about 72 h.

[0114] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 72" includes ±5% of 72, or from 68.4 to 75.6.

[0115] In a preferred embodiment, in step (b), the volume of the third solvent is 800 to 1200 ml, for example, about 1000 ml.

[0116] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 1000" includes ±5% of 1000, or from 950 to 1050.

[0117] In a preferred embodiment, in step (c), the drying is freeze-drying.

[0118] In a preferred embodiment, the yield of the lavender extract is 10% to 20%, such as about 16.6%.

[0119] In the present invention, "about" means a value within the range of ±5% of a specific value. For example, "about 16.6%" includes ±5% of 16.6%, or from 15.77% to 17.43%.

[0120] In a preferred embodiment, the composition further comprises one or more pharmaceutically acceptable excipients.

[0121] In a preferred embodiment, the excipients are selected from one or more of the following: diluents, wetting agents, binders, disintegrants, inclusion agents, flavoring agents, sustained-release agents, glidants, lubricants, dispersants, plasticizers, light-blocking agents, and antioxidants.

[0122] In a preferred embodiment, the dosage form of the composition is powder, tablet, dripping pill, capsule, film, lozenge, granule, injection, or oral liquid.

[0123] In a preferred embodiment, the composition further comprises one or more drugs and / or extracts for improving sleep.

[0124] In a preferred embodiment, the drug is selected from one or more of the following: benzodiazepine receptor agonists, antidepressant drugs, and melatonin receptor agonists.

[0125] In a preferred embodiment, the benzodiazepine receptor agonists include benzodiazepine drugs and non-benzodiazepine drugs.

[0126] In a preferred embodiment, the benzodiazepine drugs are estazolam, triazolam, and diazepam.

[0127] In a preferred embodiment, the non-benzodiazepine drugs are zolpidem, zopiclone, and eszopiclone.

[0128] In a preferred embodiment, the antidepressant drugs include noradrenergic and specific serotonergic antidepressant drugs, as well as 5-HT receptor blockers and reuptake inhibitors.

[0129] In a preferred embodiment, the noradrenergic and specific serotonergic antidepressant drug is mirtazapine.

[0130] In a preferred embodiment, the 5-HT receptor blocker and reuptake inhibitor is trazodone.

[0131] In a preferred embodiment, the melatonin receptor agonists are agomelatine and ramelteon.

[0132] According to another aspect of the present invention, there is provided a method for preparing the above composition, the method comprising mixing total saponins from Panax notoginseng stems and leaves, Ziziphus jujuba seed extract, lavender extract and lavender essential oil in the following mass ratio: (2.50 - 3.50):(45.00 - 55.00):(55.00 - 70.00):(4.00 - 20.00).

[0133] In a preferred embodiment, the mass ratio between the total saponins from Panax notoginseng stems and leaves, the Ziziphus jujuba seed extract, the lavender extract and the lavender essential oil is (2.80 - 3.30):(47.00 - 53.00):(59.00 - 66.00):(4.00 - 20.00).

[0134] In a preferred embodiment, the mass ratio between the total saponins from Panax notoginseng stems and leaves, the Ziziphus jujuba seed extract, the lavender extract and the lavender essential oil is about 3.12:about 50.00:about 62.50:(4.00 - 20.00).

[0135] In a preferred embodiment, the mass ratio between the total saponins from Panax notoginseng stems and leaves, the Ziziphus jujuba seed extract, the lavender extract and the lavender essential oil is about 3.12:about 50.00:about 62.50:about 4.69.

[0136] In the present invention, "about" means a value within the range of ±5% of a specific value. For example, "about 3.12" includes ±5% of 3.12, or from 2.964 to 3.276; "about 50.00" includes ±5% of 50.00, or from 47.50 to 52.50; "about 62.50" includes ±5% of 62.50, or from 59.375 to 65.625; "about 4.69" includes ±5% of 4.69, or from 4.4555 to 4.9245.

[0137] In a preferred embodiment, the mass ratio between the total saponins from Panax notoginseng stems and leaves, the Ziziphus jujuba seed extract, the lavender extract and the lavender essential oil is about 3.12:about 50.00:about 62.50:about 18.75.

[0138] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 3.12" includes ±5% of 3.12, or from 2.964 to 3.276; "about 50.00" includes ±5% of 50.00, or from 47.50 to 52.50; "about 62.50" includes ±5% of 62.50, or from 59.375 to 65.625; "about 18.75" includes ±5% of 18.75, or from 17.8125 to 19.6875.

[0139] In a preferred embodiment, the mass ratio among the total saponins from Panax notoginseng stems and leaves, the extract of Ziziphus jujuba seeds, the extract of lavender, and the lavender essential oil is not 12.5:25:31.25:4.69.

[0140] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 12.5" includes ±5% of 12.5, or from 11.875 to 13.125; "about 25" includes ±5% of 25, or from 23.75 to 26.25; "about 31.25" includes ±5% of 31.25, or from 29.6875 to 32.8125; "about 4.69" includes ±5% of 4.69, or from 4.4555 to 4.9245.

[0141] In a preferred embodiment, the mass ratio among the total saponins from Panax notoginseng stems and leaves, the extract of Ziziphus jujuba seeds, the extract of lavender, and the lavender essential oil is not 12.5:25:15.62:18.75.

[0142] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 12.5" includes ±5% of 12.5, or from 11.875 to 13.125; "about 25" includes ±5% of 25, or from 23.75 to 26.25; "about 15.62" includes ±5% of 15.62, or from 14.839 to 16.401; "about 18.75" includes ±5% of 18.75, or from 17.8125 to 19.6875.

[0143] In a preferred embodiment, the mass ratio among the total saponins from Panax notoginseng stems and leaves, the extract of Ziziphus jujuba seeds, the extract of lavender, and the lavender essential oil is not 3.12:25:15.62:4.69.

[0144] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 3.12" includes ±5% of 3.12, or from 2.964 to 3.276; "about 25" includes ±5% of 25, or from 23.75 to 26.25; "about 15.62" includes ±5% of 15.62, or from 14.839 to 16.401; "about 4.69" includes ±5% of 4.69, or from 4.4555 to 4.9245.

[0145] According to another aspect of the present invention, there is provided the use of the above composition in the preparation of a medicament, food and / or health product for improving sleep.

[0146] In a preferred embodiment, the composition improves sleep in one or more of the following ways: increasing intracellular calcium ion concentration, improving general behavioral abnormalities, alleviating anxiety behavior, improving sleep quality, and improving neurotransmitter metabolic disorders.

[0147] In a preferred embodiment, the general behavioral abnormalities include one or more of the following: weight loss, dull hair, and irritable state.

[0148] In a preferred embodiment, the improvement of sleep quality includes shortening sleep latency and prolonging sleep duration.

[0149] In a preferred embodiment, the improvement of neurotransmitter metabolic disorders includes one or more of the following: increasing the content of 5-hydroxytryptamine in the hypothalamus, increasing the content of 5-hydroxytryptamine in the hippocampus, increasing the content of γ-aminobutyric acid in the hypothalamus, increasing the content of γ-aminobutyric acid in the hippocampus, and decreasing the content of glutamate in the hypothalamus.

[0150] According to another aspect of the present invention, there is further provided the above composition for improving the sleep of a subject.

[0151] According to another aspect of the present invention, there is further provided a method for improving the sleep of a subject, comprising administering an effective amount of the above composition to the subject.

[0152] In the present invention, the term "subject" is a mammal. Mammals can be humans, non-human primates, mice, rats, dogs, cats, horses, or cows, but are not limited to these examples. Mammals other than humans can be advantageously used as subjects representing sleep disorders. Preferably, the subject is a human.

[0153] An "effective amount" of the pharmaceutical composition or formulation used in the present invention can achieve the desired improvement effect. The amount effective for this use will depend on, for example, the pharmaceutical composition, the mode of administration, the stage and severity of the disease being treated, the individual body weight and general health condition, as well as the judgment of the prescribing physician. The dose can be administered once a week, or once every two days or every day, or even several times a day. The dose units can be administered over a short period (e.g., several weeks to several months) or a longer period (several months to several years).

[0154] The present invention will be further illustrated below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out according to conventional conditions or the conditions recommended by the manufacturer.

[0155] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to persons skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the method of the present invention. The preferred methods and materials described herein are for illustrative purposes only.

[0156] The above-mentioned features mentioned in the present invention, or the features mentioned in the embodiments, can be combined arbitrarily. All the features disclosed in this patent specification can be used in combination with any composition form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equivalent or similar features.

[0157] Example 1 Research on the Compatibility Ratio of the Composition

[0158] 1. Drugs

[0159] 1.1 Tested Formulations

[0160] Total saponins from Panax notoginseng leaves and stems (appropriate amount of Panax notoginseng leaves and stems were weighed, sliced, extracted with water three times, the filtrates were filtered and combined, loaded onto column D101, eluted with alcohol, concentrated and dried, and sieved to obtain) were purchased from Nanjing Tianrun Biotechnology Co., Ltd. Semen Ziziphi Spinosae was purchased from Shanghai Kangqiao Chinese Herbal Pieces Co., Ltd., and lavender was purchased from Xinjiang Xinlübao Pharmaceutical Co., Ltd. Both were identified as genuine medicinal materials. Lavender essential oil (Lavender Essential Oil, product number 2449) was purchased from French PRANAROM essential oil, and marjoram essential oil (Marjoram Essential Oil, product number PEM0301), lemon balm essential oil (Melissa Essential Oil, product number PEM0702), valerian essential oil (Valerian Organic Essential Oil, product number OEV0201), vetiver essential oil (Vetiver Essential Oil, product number PEV0303) were purchased from POLI essential oil.

[0161] 1.2 Preparation of Chinese herbal extracts

[0162] 1.2.1 Preparation of Semen Ziziphi Spinosae extract

[0163] 80 g of Semen Ziziphi Spinosae was weighed, added with 2.5 times the amount of petroleum ether and soaked for 1 h, then heated under reflux at 60 °C for 2 times, 2 h each time, and filtered. The filter residue was placed in a fume hood to volatilize the solvent, and the off-white degreased medicinal residue was obtained. 8 times the amount of 70% ethanol was added, and it was refluxed and extracted 2 times, 2 h each time, filtered, the filtrates were combined, and the ethanol was evaporated by rotary evaporation at 60 °C until there was no alcohol smell. The concentrated solution was freeze-dried to obtain the powder of ethanol extract of Semen Ziziphi Spinosae (SZS), and the yield was 13.1%.

[0164] 1.2.2 Preparation of lavender extract

[0165] 100 g of lavender was weighed, added with 1 L of 80% ethanol solution, soaked for 72 h, and then the supernatant was filtered. The residue was repeated the above operation once. The two obtained extraction solutions were combined, and the ethanol was removed by rotary evaporation. The concentrated solution was freeze-dried to obtain the powder of ethanol extract of lavender (LAV), and the yield was 16.6%.

[0166] 1.3 Preparation of sample mother liquor

[0167] (1) Essential oil mother liquor: Using absolute ethanol as the solvent, prepare the mother liquor of lavender essential oil (LEO), marjoram essential oil (MAO), lemon balm essential oil (MEO), valerian essential oil (VAO), and vetiver essential oil (VEO) at 500 mg / mL, and store at -20 °C for later use. Dilute to the use concentration with the culture medium when using.

[0168] (2) Mother liquor of traditional Chinese medicine extract: Using DMSO as the solvent, prepare mother liquors of total saponins from Panax notoginseng stems and leaves (PNSLS), Semen Ziziphi Spinosae extract (SZS), and Lavandula angustifolia extract (LAV) at a concentration of 200 mg / mL respectively. Dilute to the working concentration with the culture medium before use.

[0169] 2. Experimental methods

[0170] 2.1 Effects of traditional Chinese medicine extracts and lavender essential oil on the viability of 293T / 5-HTR1A cells

[0171] The CCK-8 method was used to evaluate the effects of different concentrations of total saponins from Panax notoginseng stems and leaves, Semen Ziziphi Spinosae extract, Lavandula angustifolia extract, lavender essential oil, origanum essential oil, melissa essential oil, valerian essential oil, and vetiver essential oil on the survival rate of 293T / 5-HTR1A cells. Seed 293T / 5-HTR1A cells at a density of 5×10 4 cells / mL onto the coated 96-well plates (100 μL / well), and culture them in a cell incubator at 37 °C and 5% CO2. After 24 h, add 100 μL of culture medium containing different concentrations of total saponins from Panax notoginseng stems and leaves (250, 125, 50, 25, 12.5, 2.5, 0.25 μg / mL), Semen Ziziphi Spinosae extract (250, 125, 50, 25, 12.5, 2.5, 0.25 μg / mL), Lavandula angustifolia extract (250, 225, 200, 175, 150, 125, 62.5, 25 μg / mL), lavender essential oil (300, 150, 75, 37.5, 18.75, 9.375 μg / mL), origanum essential oil (250, 125, 100, 75, 50, 25 μg / mL), melissa essential oil (250, 125, 62.5, 31.25, 15.625, 7.8125 μg / mL), valerian essential oil (50, 37.5, 25, 12.5, 6.25 μg / mL), and vetiver essential oil (500, 400, 200, 100, 50, 25, 12.5 μg / mL) to each well. Add an equal volume of culture medium to the normal control group and the blank group. After culturing for 24 h, add 10 μL of CCK-8 reagent to each well, incubate in the incubator for 1 h, and then measure the OD value at a wavelength of 450 nm using an enzyme-linked immunosorbent assay reader. Cell survival rate (%) = (OD 实验组 - OD 空白组 ) / (OD 对照组 - OD 空白组 ) × 100%.

[0172] 2.2 Experimental grouping method

[0173] 2.2.1 Experimental grouping of PSLO

[0174] The experiment selected L 27 (313 ) Orthogonal array. The four herbs in formula PSLO: total saponins from Panax notoginseng stems and leaves (A), extract of Ziziphus jujuba seeds (B), extract of Lavandula angustifolia (C), and essential oil of Lavandula angustifolia (D) are taken as the investigated factors, and each factor is divided into 3 levels. According to the principle of degrees of freedom and the selection of orthogonal arrays, interaction columns can be set. The interaction columns are set as: A and B, A and C, A and D, B and C, B and D, C and D. Therefore, a total of 27 orthogonal design schemes are finally obtained. The orthogonal factor level table of formula PSLO is shown in Table 1.

[0175] Table 1 Formula PSLO L 27 (3 13 ) Orthogonal test factor levels

[0176]

[0177]

[0178] 2.2.2. Experimental grouping of formula PSLMM

[0179] The experiment uses L 27 (3 13 ) Orthogonal array. The five herbs in formula PSLMM: total saponins from Panax notoginseng stems and leaves (A), extract of Ziziphus jujuba seeds (B), extract of Lavandula angustifolia (C), essential oil of Origanum majorana (D), and essential oil of Melissa officinalis (E) are taken as the investigated factors, and each factor is divided into 3 levels. According to the principle of degrees of freedom and the selection of orthogonal arrays, four groups of interaction columns can be set. The interaction columns are set as: A and B, A and C, A and D, A and E. Therefore, a total of 27 orthogonal design schemes are finally obtained. The orthogonal factor level table of formula PSLMM is shown in Table 2.

[0180] Table 2 Formula PSLMM L 27 (3 13 ) Orthogonal test factor levels

[0181]

[0182] 2.2.3. Experimental grouping of formula PSLVV

[0183] The experiment uses L 27 (3 13 ) Orthogonal array. The five herbs in formula PSLVV: total saponins from Panax notoginseng stems and leaves (A), extract of Ziziphus jujuba seeds (B), extract of Lavandula angustifolia (C), essential oil of Valeriana officinalis (D), and essential oil of Vetiveria zizanioides (E) are taken as the investigated factors, and each factor is divided into 3 levels. According to the principle of degrees of freedom and the selection of orthogonal arrays, four groups of interaction columns can be set. The interaction columns are set as: A and B, A and C, A and D, A and E. Therefore, a total of 27 orthogonal design schemes are finally obtained. The orthogonal factor level table of formula PSLVV is shown in Table 3.

[0184] Table 3 Square PSLVV L 27 (3 13 ) Orthogonal test factor levels

[0185]

[0186]

[0187] 2.3 Calcium flux determination of agonists in 293T / 5 - HTR1A cell line

[0188] Inoculation and culture: Inoculate 293T / 5 - HTR1A cells at a density of 5×10 4 cells / mL onto the coated 96 - well plates (100 μL / well), and place them in a cell culture incubator at 37°C and 5% CO2. Set up experimental groups, positive control groups, and normal control groups. Add PSLO solutions of 27 different combinations to the experimental groups respectively, add 5 - HT receptor agonist Serotoninhydrochloride to the positive control group, and add the same volume of medium to the normal control group, and incubate for 24 h.

[0189] Staining: Aspirate the medium, wash the cells once with PBS, and add 100 μL of Fluo - 4 staining solution to each well. Incubate at 37°C in the dark for 30 min.

[0190] Detection: After incubation, detect the fluorescence intensity with a fluorescence microplate reader (Ex / Em = 490 / 525 nm).

[0191] By comparing the RFU (Relative fluorescence values) of the control group and the treatment group, the effect of drug stimulation can be obtained.

[0192] 2.4 Statistical methods

[0193] Use GraphPad Prism 8 software for statistical analysis. The data are all expressed as mean ± standard deviation and one - way ANOVA with Dunnett's test is used for comparison among multiple groups. P < 0.05 indicates that the difference is statistically significant.

[0194] 3. Experimental results

[0195] 3.1 Effect of traditional Chinese medicine extracts on cell viability

[0196] In this invention, the CCK - 8 method was used to determine the effect of total saponins from Panax notoginseng stems and leaves, Ziziphus jujuba seed extract, and lavender extract on cell viability after acting on 293T / 5 - HTR1A cells for 24 h at different concentrations. The results are as Figure 1As shown in the figure. The purpose of this experiment was to screen the concentration range of traditional Chinese medicine extracts that would not affect the normal growth and proliferation of cells. The drug concentration when the cell survival rate was ≥90% was regarded as the non-toxic concentration, and thus the safe concentration ranges of three traditional Chinese medicine extracts for treating cells were determined. According to the test results, the safe concentration range of total saponins from Panax notoginseng stems and leaves was 0 - 25 μg / mL, the safe concentration range of Ziziphus jujuba seed extract was 0 - 50 μg / mL, and the safe concentration range of lavender extract was 0 - 62.5 μg / mL.

[0197] 3.2 Effects of essential oils on cell viability

[0198] The CCK-8 method was used to determine the effects of lavender essential oil, marjoram essential oil, lemon balm essential oil, valerian essential oil, and vetiver essential oil on cell viability after acting on 293T / 5-HTR1A cells at different concentrations for 24 h. The results are as Figure 2 shown in the figure. The purpose of this experiment was to screen the concentration range of five candidate essential oils that would not affect the normal growth and proliferation of cells. The drug concentration when the cell survival rate was ≥90% was regarded as the non-toxic concentration, and thus the safe concentration ranges of lavender essential oil, marjoram essential oil, lemon balm essential oil, valerian essential oil, and vetiver essential oil for treating cells were 0 - 18.75 μg / mL, 0 - 50 μg / mL, 0 - 7.5 μg / mL, 0 - 12.5 μg / mL, and 0 - 12.5 μg / mL in sequence.

[0199] 3.3 Agonistic effects of formula PSLO, formula PSLMM, and formula PSLVV on 5-HTR1A

[0200] 3.3.1 Agonistic effect of formula PSLO on 5-HTR1A

[0201] The results are as Figure 3 shown in the figure. Compared with the normal control group, after stimulating 293T / 5-HTR1A cells in groups 1, 2, 3, 4, 7, 8, 9, 10, 13, 14, 15, 19, 20, 21, 22, 25, 26, 27 of formula PSLO experiment, the relative fluorescence intensity increased, and there were extremely significant differences (P < 0.001); the relative fluorescence intensity in groups 11, 12, 16, 17, 18, 23 of formula PSLO experiment increased significantly compared with the normal control group (P < 0.01 or P < 0.05). It is suggested that the intracellular calcium concentration increased after treatment with formula PSLO with different ratios, and it may be an agonist of the 5-HT 1A receptor.

[0202] As can be seen from Table 4, by visually analyzing the calcium influx detection results after stimulating 293T / 5-HTR1A cells with the other party's PSLO, and by comparing the range sizes of various factors, it is concluded that the order of the effects of various factors on activating 5-HTR1A from large to small is AB > C (lavender extract) > B (semen ziziphi spinosae extract) > BC > CD > A (total saponins from Panax notoginseng stems and leaves) > D (lavender essential oil) > BD > AD > AC. The predicted optimal level by visual analysis is A3B1C1D1, that is, total saponins from Panax notoginseng stems and leaves : semen ziziphi spinosae extract : lavender extract : lavender essential oil = 3.12 : 50 : 62.5 : 18.75, which does not appear in the orthogonal table and a verification experiment needs to be conducted.

[0203] Analysis of variance was performed on the test results to investigate whether each test factor has a significant effect on the test results. As shown in Table 5, A, B, AB, C, BC, and CD have a significant effect on calcium concentration (P < 0.05). Since the range size is AB > C > B > BC > CD > A > D > BD > AD > AC, that is, when A and B are used in combination, the effect on intracellular calcium concentration is greater than when A and B are used alone, indicating that there is a synergistic effect between A and B.

[0204] Table 4 Orthogonal test and results of the other party's PSLO

[0205]

[0206]

[0207]

[0208] Table 5 Analysis of variance table of the other party's PSLO

[0209] Factor Sum of squared deviations Degree of freedom F ratio Critical value of F Significance A (Total saponins from Panax notoginseng stems and leaves) 0.037 2 7.400 4.460 * B (Extract of Ziziphus jujuba seeds) 0.130 2 26.000 4.460 * A×B 0.531 2 106.200 4.460 * A×B 0.172 2 34.400 4.460 * C (Extract of Lavandula angustifolia) 0.233 2 46.600 4.460 * A×C 0.001 2 0.200 4.460 A×C 0.001 2 0.200 4.460 B×C 0.060 2 12.000 4.460 * D (Lavender essential oil) 0.015 2 3.000 4.460 A×D 0.003 2 0.600 4.460 B×C 0.103 2 20.600 4.460 * B×D 0.015 2 3.000 4.460 C×D 0.048 2 9.600 4.460 * Error 0.02 8

[0210] 3.3.2 Agonistic effect of the other party's PSLMM on 5-HTR1A

[0211] The results are as Figure 4 shown. Compared with the normal control group, there was no significant difference in the relative fluorescence intensity after stimulating 293T / 5-HTR1A cells in each experimental group of the other party's PSLMM, and no change in intracellular calcium concentration was caused, indicating that the other party's PSLMM with different ratios has no agonistic effect on the 5-HT1A receptor.

[0212] 3.3.3 Agonistic effect of the other party's PSLVV on 5-HTR1A

[0213] The results are as Figure 5As shown, compared with the normal control group, there were no significant differences in the relative fluorescence intensity after stimulating 293T / 5-HTR1A cells in each experimental group of Fang PSLVV, and no changes in intracellular calcium concentration were induced, suggesting that Fang PSLVV with different ratios has no agonist effect on the 5-HT1A receptor.

[0214] 3.4 Group formula ratio optimization experiment

[0215] According to the previous experimental results, the optimal combination A3B1C1D3 of Fang PSLO (total saponins from Panax notoginseng stems and leaves: extract of Ziziphus jujuba seeds: extract of Lavandula angustifolia: essential oil of Lavandula angustifolia = 3.12:50:62.5:4.69) and the predicted combination A3B1C1D1 (3.12:50:62.5:18.75) were screened out, and the optimal combination and predicted combination of the formula were compared through calcium flux detection experiments.

[0216] The results were as Figure 6 shown. Compared with the normal control group, all three ratios of Fang PSLO could cause significant changes in intracellular calcium concentration. Among them, the ratio of 3.12:50:62.5:4.69 had the best agonist effect on the 5-HT 1A receptor.

[0217] 4. Discussion and conclusion

[0218] (1) Among the 27 orthogonal design schemes of Fang PSLO, Experiment 19 (A3B1C1D3) had the strongest agonist effect on the 5-HT 1A receptor, that is, the optimal mass ratio was total saponins from Panax notoginseng stems and leaves: extract of Ziziphus jujuba seeds: extract of Lavandula angustifolia: essential oil of Lavandula angustifolia = 3.125:50:62.5:4.69. Based on the orthogonal design experiment, combined with intuitive analysis and variance analysis, the predicted optimal level by intuitive analysis was A3B1C1D1, that is, the optimal mass ratio was total saponins from Panax notoginseng stems and leaves: extract of Ziziphus jujuba seeds: extract of Lavandula angustifolia: essential oil of Lavandula angustifolia = 3.125:50:62.5:18.75, which did not appear in the orthogonal table and a verification experiment was needed.

[0219] (2) Among the 27 orthogonal design schemes of Fang PSLMM, each experimental group had no agonist effect on the 5-HT 1A receptor.

[0220] (3) Among the 27 orthogonal design schemes of Fang PSLVV, each experimental group had no agonist effect on the 5-HT 1A receptor.

[0221] (4) By comparing the optimal combination and predicted combination of Fang PSLO, the results showed that both the A3B1C1D3 and A3B1C1D1 combinations of Fang PSLO had good agonist effects on the 5-HT 1A receptor, that is:

[0222] ①Total saponins from Panax notoginseng stems and leaves: Extract of Semen Ziziphi Spinosae: Extract of Lavandula angustifolia Mill: Lavender essential oil = 3.12:50:62.5:4.69;

[0223] ②Total saponins from Panax notoginseng stems and leaves: Extract of Semen Ziziphi Spinosae: Extract of Lavandula angustifolia Mill: Lavender essential oil = 3.12:50:62.5:18.75. Among them, the ratio of 3.12:50:62.5:4.69 in Formulation ① has the best effect on activating 5-HT 1A receptor, and will be further verified through the animal experiment in Example 2.

[0224] Study on the sedative and hypnotic effects of Formulation ① in Example 2 on insomnia mice

[0225] 1. Drugs

[0226] 1.1 Test substances

[0227] The traditional Chinese medicine prescription PSLO① is composed of drugs such as total saponins from Panax notoginseng stems and leaves, extract of Semen Ziziphi Spinosae, extract of Lavandula angustifolia Mill, and lavender essential oil, and the mass ratio is total saponins from Panax notoginseng stems and leaves: extract of Semen Ziziphi Spinosae: extract of Lavandula angustifolia Mill: lavender essential oil = 3.12:50:62.5:4.69. The total saponins from Panax notoginseng stems and leaves are provided by Yunnan Kunming Pharmaceutical Group Co., Ltd. (Kunming, China), Semen Ziziphi Spinosae is purchased from Shanghai Kangqiao Chinese Herbal Pieces Co., Ltd., Lavandula angustifolia Mill is purchased from Xinjiang Xinlübao Pharmaceutical Co., Ltd., and lavender essential oil is purchased from doTERRA (Shanghai) Trading Co., Ltd.

[0228] 1.2 Positive drugs

[0229] Diazepam tablets (Shanghai Shangyao Xinyi Pharmaceutical Factory Co., Ltd., specification: 2.5 mg / tablet).

[0230] 2. Drug preparation

[0231] 2.1 Preparation of extract of Semen Ziziphi Spinosae

[0232] Same extraction method as "1.2.1 in Example 1".

[0233] 2.2 Preparation of extract of Lavandula angustifolia Mill

[0234] Same extraction method as "1.2.2 in Example 1".

[0235] 2.3 Preparation of the storage solution of PSLO① atomized liquid

[0236] Precisely weigh 46.9 mg of lavender essential oil, add 23.4 mg of Tween-80, and vortex to mix it thoroughly with the essential oil. Weigh 46.9 mg of absolute ethanol and add it to the essential oil-Tween mixture, then vortex to mix evenly. Weigh 31.2 mg of total saponins from Panax notoginseng stems and leaves, 500 mg of Ziziphus jujuba seed extract, and 625 mg of lavender extract, add 2 mL of sterilized pure water, and vortex until the extracts are completely dissolved. Mix the essential oil emulsion and the extract mixture evenly, make up the volume to 3.64 mL, with a final concentration of 330 mg / mL, and dilute it with water to the administration concentration before use.

[0237] 2.4 Grouping of administration doses

[0238] PSLO① Low dose (0.5 times the clinical dose): Dilute the stock solution of PSLO① aerosol with water at a ratio of 1:9 to obtain a diluted solution with a concentration of 33 mg / mL. Prepare it freshly before use and aerosolize for 30 min.

[0239] PSLO① Medium dose (1 times the clinical dose): Dilute the stock solution of PSLO① aerosol with pure water at a ratio of 1:4 to obtain a diluted solution with a concentration of 66 mg / mL. Prepare it freshly before use and aerosolize for 30 min.

[0240] PSLO① High dose (5 times the clinical dose): The stock solution of PSLO① aerosol needs no dilution, with a concentration of 330 mg / mL. Prepare it freshly before use and aerosolize for 30 min.

[0241] Diazepam: 2 mg / kg

[0242] 3. Animal feeding

[0243] 3.1 Animals

[0244] 72 male KM mice, SPF grade (Shanghai Vital River Laboratory Animal Co., Ltd.). The body weight of the animals at the start of the experiment was about 20 - 22 g, and the adaptation time to the environment was 5 - 7 days. The animal numbering method: Each cage was hung with an identity card with information such as the project number, experimental group, name of the experimental personnel, animal breed, and gender. The mice were numbered with ear tags.

[0245] 3.2 Environment

[0246] The environment in the animal house was maintained at a temperature of 23 ± 2 °C, a humidity of 40 - 70%, and a 12-hour light-dark cycle. The animals were housed 6 per cage, and the bedding (corn cob bedding, Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd.) was changed twice a week.

[0247] 3.3 Food and drinking water

[0248] During the experiment, the SPF mice were fed with maintenance feed (Co 60Sterilization: The water used was high-pressure sterilized filtered water purchased from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd. During the experiment, the animals had free access to food and water.

[0249] 4. Experimental methods

[0250] 4.1 Pre-experiment with suprathreshold dose of sodium pentobarbital

[0251] In the pre-experiment, the suprathreshold dose of sodium pentobarbital was first determined. Thirty mice were randomly divided into 5 groups of 6 mice each. Different doses of sodium pentobarbital (45, 50, 55, 60, 65 mg / kg, 0.15 mL / 10 g) were intraperitoneally injected in equal volumes. The number of mice that fell asleep within 30 min in each group was observed and recorded, and the sleep onset rate of the mice was calculated.

[0252] The suprathreshold dose of sodium pentobarbital is the minimum dose that causes the disappearance of the righting reflex in 100% of the mice. The mice were placed supine on a flat plate. If the mice did not turn over within 1 min, it was considered that the righting reflex disappeared and the mice could be regarded as falling asleep. After a period of time, when the righting reflex first recovered, they were immediately turned into the supine position. If it recovered again within 30 s, it was judged as awakening, and the time of the first recovery of the righting reflex was recorded as the sleep termination time. If it did not recover within 30 s, they were turned over again after the righting reflex recovered again until the end of sleep was determined. The time from drug administration to the disappearance of the righting reflex was the sleep latency (SL); the time was continuously recorded until the mice automatically turned their bodies and the righting reflex recovered within 30 s, which was the sleep duration (ST). The differences in SL and ST between the blank group and the model group were compared, and statistical analysis was performed. P < 0.05 indicated statistical significance, suggesting successful modeling.

[0253] The calculation methods of sleep latency and sleep duration are as follows:

[0254] Sleep latency = Time to enter the sleep state - Time of injecting sodium pentobarbital;

[0255] Sleep duration = Sleep termination time - Time to enter the sleep state.

[0256] The drug administration plan for the pre-experiment is shown in Table 6.

[0257] Table 6 Drug administration plan for the pre-experiment

[0258] Cage number Group Number of animals (pcs) Dose (mg / kg) Number of animals falling asleep (pcs) Sleeping rate (%) 1 Pentobarbital sodium group 6 45 0 0 2 Pentobarbital sodium group 6 50 5 83.33 3 Pentobarbital sodium group 6 55 6 100 4 Pentobarbital sodium group 6 60 6 100 5 Pentobarbital sodium group 6 65 6 100

[0259] 4.2 Establishment of PCPA insomnia mouse model

[0260] After 72 male Kunming mice were adaptively fed in the SPF-class animal house for one week, they were randomly divided into a normal group and a model group. The mice in the model group were intraperitoneally injected with PCPA suspension (400 mg / kg) for modeling, and the normal group was intraperitoneally injected with an equal volume of sterile pure water containing 2% Tween-80. Each injection was shaken well, and the intraperitoneal injection was performed from 8:30 to 10:00 every morning, once a day, for 3 consecutive days.

[0261] 4.3 Determination of the insomnia model

[0262] (1) Visual observation: 28 - 32 h after the intraperitoneal injection of PCPA suspension on the first day, observe the behaviors of the mice in each group. Compared with the normal group, if the mice show significant behavioral changes such as restlessness, continuous activity, disappearance of the circadian rhythm, decreased appetite, significant weight loss, enhanced excitability, enhanced aggressiveness, grayish-white feces, increased production of urine and feces, and dull, disordered and lackluster hair, it indicates that the insomnia model of mice is successfully established.

[0263] (2) Body weight detection: Before and after modeling, weigh the mice in each group at the same time in the morning and record the body weight. Compared with the normal group, if the body weight of the model group decreases, it indicates that the modeling is successful.

[0264] (3) Righting experiment: 24 h after the end of modeling, verify the model with the pentobarbital sodium righting experiment. The pre-experiment measured that the suprathreshold dose of pentobarbital sodium injected intraperitoneally into mice was 55 mg / kg.

[0265] 4.4 Animal grouping and drug administration

[0266] The successfully modeled mice were randomly divided into groups according to body weight: model group, diazepam group, PSLO① low-dose (PSLO①-L) group, PSLO① medium-dose (PSLO①-M) group, and PSLO① high-dose (PSLO①-H) group, with 12 mice in each group. The administration concentrations of the low, medium, and high doses of PSLO① were 0.925 g / kg, 1.85 g / kg, and 9.25 g / kg respectively, and the positive control was given intragastric administration of diazepam suspension with an administration concentration of 2 mg / kg.

[0267] The positive group was given intragastric administration of diazepam suspension every day, and the blank group, model group, and atomization administration group were given intragastric administration of an equal volume of distilled water. The low, medium, and high doses of PSLO① groups were given atomization inhalation intervention every day. Each dose group atomized an equal volume of atomized liquid with different concentrations every day, and continuous atomization drug administration was carried out for 7 d, once a day, at a fixed time every day. Use the YLS-8B small animal atomization drug administration instrument, first fill the mist, and then put the mice into the atomization box and atomize continuously for 30 min.

[0268] 4.5 Observation indicators

[0269] 4.5.1 Observation of the behavioral state of mice

[0270] Observe the behavioral changes of mice such as mental state, fur color, sleep, diet and water intake before and after modeling and drug administration dynamically, weigh and record the body weights of mice before and after modeling and after the end of drug administration.

[0271] 4.5.2 Open field test

[0272] 30 minutes after drug administration on the 6th day, place the mice in the central area of the open field box, record the activities of the mice within 5 minutes. After the test of each mouse, wipe the open field box clean with alcohol to avoid interfering with the experimental results of the next mouse. Analyze and record the total activity distance, average speed, residence time in the central area, number of times passing through the central grid, and stationary time using behavioral analysis software.

[0273] 4.5.3 Pentobarbital sodium-induced mouse sleep experiment

[0274] 30 minutes after the last drug administration, intraperitoneally inject each group of mice with a threshold dose of pentobarbital sodium at 55 mg / kg. After injection, place the mice on warm bedding, record the injection time, sleep onset time, and awakening time. Consider the disappearance of the righting reflex for 1 minute as the sleep onset criterion. The time from injection to the disappearance of the righting reflex is the sleep latency, and the time from the disappearance of the righting reflex until recovery is the sleep duration. Observe and record the sleep latency and sleep duration. The ability to right the body independently more than 3 times within 30 seconds indicates the recovery of the righting reflex, which is used as the awakening criterion. Keep the environment quiet and ventilated during the experiment. The longer the sleep latency, the more obvious the nerve hyperactivity and the more difficult it is for the mice to fall asleep; the shorter the sleep duration, the more restless the mice are and the worse their sleep condition.

[0275] 4.5.4 Detection of 5-HT, GABA, Glu, and DA levels in the hippocampus and hypothalamus of mice by ELISA

[0276] (1) Tissue sampling

[0277] 24 hours after the last drug administration, anesthetize the mice with pentobarbital sodium for brain tissue sampling. Dissect the head on ice. After removing the whole brain, quickly dissect the hypothalamus and hippocampus tissues, immediately wash them in ice-cold physiological saline, put them into 2 mL EP tubes, and then quickly freeze them in liquid nitrogen and store them in a -80 °C refrigerator for the detection of neurotransmitter content.

[0278] (2) Sample preparation

[0279] Weigh the hypothalamus and hippocampal tissues with a one-ten-thousandth balance, place them in 2 mL EP tubes, add 2 small steel beads, add the corresponding volume of PBS buffer according to the ratio of brain tissue:PBS = 1 g:9 mL, and homogenize the tissues thoroughly with a tissue grinder. After homogenization, take out the small steel beads, centrifuge the homogenate at 4 °C and 5000 g for 10 min, take the supernatant, aliquot it, and store it frozen at -80 °C. Detect the contents of neurotransmitters such as 5-HT, GABA, and Glu in the hippocampus and hypothalamus of mice according to the ELISA kit instructions.

[0280] 4.6 Statistical analysis

[0281] Use GraphPad Prism 8.0.2 software for graphing and statistical analysis. The data are all expressed as mean ± standard deviation and one-way ANOVA is used for significance testing in comparisons among multiple groups, while the t-test is used for comparisons between two groups. P < 0.05 indicates that the difference is statistically significant.

[0282] 5. Experimental results

[0283] 5.1 Evaluation of the insomnia model

[0284] 5.1.1 Observation of general behavior

[0285] The results are as Figure 7 shown. Before modeling, there were no significant differences in the body weights of mice in each group. Three days after intraperitoneal injection of the PCPA suspension, compared with the normal group, the body weights of the modeled mice were significantly reduced (P < 0.001). Compared with the normal group, the hair of the modeled mice in each group was dull and messy, the water intake increased, the body weight decreased, and the activity during the day increased; the mental state was listless and aggressive, indicating that the insomnia model was successfully established.

[0286] 5.1.2 Results of the righting experiment

[0287] The results are as Figure 8 shown. The pentobarbital sodium righting experiment was used to verify the insomnia model. Compared with the normal group, the sleep latency of the modeled mice was significantly prolonged (P < 0.01), and the sleep duration was significantly shortened (P < 0.01), suggesting that the insomnia model was successfully established.

[0288] 5.2 Effects of prescription PSLO① on insomnia mice

[0289] 5.2.1 Effects of prescription PSLO① on the general behavior of mice in each group

[0290] The results are as Figure 9As shown, after 7 days of drug administration, the body weight of the mice in the model group was significantly lower than that in the control group (P<0.001). The mice in the model group were in poor spirits, with dull and messy hair, and were irritable and aggressive. Compared with the model group, the body weights of the mice in the diazepam group, the low-dose PSLO① group, and the high-dose PSLO① group increased significantly (P<0.001 or P<0.05). Their hair was shiny, their mental state gradually recovered, and their water intake increased, indicating that the drug had a therapeutic effect on insomnia mice.

[0291] 5.2.2 Effects of Prescription PSLO① on Open Field Test of Mice in Each Group

[0292] The results showed that Figure 10 compared with the control group, the total activity distance of the mice in the model group increased extremely significantly (P<0.001), the average speed increased extremely significantly (P<0.001), and the stationary time decreased extremely significantly (P<0.001). There was no significant difference in the residence time in the central area and the number of times passing through the central grid of the mice in the model group. Compared with the model group, the total activity distance of the mice in each dose group of PSLO① was significantly shortened, the average speed of the low-dose and high-dose PSLO① groups decreased significantly, the stationary time of the mice in the low-dose PSLO group increased significantly, and there was no significant difference in the residence time in the central area and the number of times passing through the central grid of the mice in each dose group of PSLO①. Compared with the model group, there was no significant difference in each spontaneous activity index of the mice in the diazepam group. It was speculated that the open field test was carried out half an hour after the mice were intragastrically administered diazepam, and the drug had not taken effect in the mice at this time.

[0293] Figure 11 This is the activity trajectory map of the mice in the open field test. The mice in the model group had activity trajectories in each area and were active frequently. After drug intervention, the activity trajectories of the mice in each drug administration group were relatively sparse compared with the model group. It shows that each drug administration group can relieve the tension and anxiety of the mice and has a sedative and hypnotic effect.

[0294] 5.2.3 Effects of Prescription PSLO① on Righting Reflex of Mice in Each Group

[0295] The results showed that Figure 12 compared with the control group, the sleep latency of the mice in the model group was significantly prolonged (P<0.01), and the sleep duration decreased, but there was no significant difference. Compared with the model group, the sleep latency of the mice in the diazepam group and each dose group of PSLO① was significantly shortened (P<0.001 or P<0.01), and the sleep duration of the mice in the diazepam group and the high-dose PSLO① group increased significantly (P<0.001 or P<0.01).

[0296] 5.2.4 Effects of Prescription PSLO① on Contents of 5-HT, GABA and Glu in Hypothalamus and Hippocampus of Mice

[0297] The results showed that Figure 13As shown, compared with the control group, the 5-HT content in the hypothalamus and hippocampus of the model group mice was significantly decreased (P<0.05 or P<0.01), the GABA content in the hippocampus was significantly decreased (P<0.01), and the Glu content in the hypothalamus was significantly increased (P<0.05). Compared with the model group, the 5-HT content in the hypothalamus of the diazepam group and the low-dose PSLO① group mice was significantly increased (P<0.05), and the 5-HT content in the hippocampus of the diazepam group and the medium- and high-dose PSLO① group mice was significantly increased (P<0.01 or P<0.05). The GABA content in the hypothalamus of the diazepam group and the low- and high-dose PSLO① group mice was significantly increased (P<0.01 or P<0.05), and the GABA content in the hippocampus of the diazepam group and the medium- and high-dose PSLO① group mice was significantly increased (P<0.05). The Glu content in the hypothalamus of the medium-dose PSLO① group mice was significantly decreased (P<0.05).

[0298] 6. Discussion and Conclusion

[0299] The PCPA intraperitoneal injection method is the most widely used method for modeling in the study of insomnia mechanisms, and the success of modeling is determined by the righting experiment, body weight, and changes in the general behavior of mice. Compared with the blank control group mice, the changes in indicators such as prolonged sleep latency, shortened sleep time, decreased body weight, dull hair color, and poor mental state in the model group can prove the success of modeling. In terms of inducing hypnosis in mice with a suprathreshold dose of pentobarbital sodium, the PSLO① formula can shorten the sleep latency and prolong the sleep duration of mice, indicating that this formula can shorten the sleep latency and prolong the sleep duration, and its overall effect is basically the same as that of the diazepam group. Observations on the general behavior of mice found that the PSLO① formula can make the daily behavior of mice basically flexible, the hair shiny, the water intake increase, the circadian rhythm basically restored, and the body weight continuously increased, indicating that this formula can effectively regulate the mental state and behavioral performance of insomnia mice, increase their body weight, and in terms of improving the body weight of mice, the effect of diazepam is better than that of the PSLO① formula. The open field experiment is used to detect changes in the anxiety and tension of mice. The number of crossings and the residence time in the central grid of the open field experiment can reflect the degree of anxiety and depression of mice, and the total activity distance can reflect the degree of tension and anxiety of mice. The longer the total activity distance of mice, the more nervous they are. The total activity distance of the PSLO① group mice was significantly reduced, and the residence time in the central area was significantly increased compared with the model group, indicating that the PSLO① formula can relieve the anxiety and tension of mice to a certain extent, thereby improving sleep.

[0300] Existing studies have shown that insomnia is related to the metabolic disorders of neurotransmitters in the brain. Serotonin (5-HT) is involved in regulating the sleep-wake pathway in the human body and is an important central sleep-promoting substance in the brain. Studies have found that the content of 5-HT in the brain is significantly reduced during insomnia, and its increased expression contributes to sleep. Buspirone, a drug used for insomnia, is a new non-benzodiazepine. Its mechanism of improving sleep is to act on the 5-HT receptor and inhibit the spread and conduction of the arousal-promoting nuclei in the hypothalamus and brainstem, thereby playing a sleep-promoting role. The present invention has found that in the hypothalamus and hippocampal tissues, the content of 5-HT in the model group is lower than that in the blank control group. Compared with the model group, PSLO① can increase the content of 5-HT in the hypothalamus and hippocampus of insomnia mice and effectively improve sleep. Gamma-aminobutyric acid (GABA) is a neurotransmitter with inhibitory effects in the central nervous system. It can promote sleep by inhibiting the activity of the nervous system. An increase in its content can improve sleep. On the contrary, a decrease will enhance the function of the arousal-promoting nuclei and lead to sleep disorders. Diazepam is a long-acting benzodiazepine drug that plays a role in promoting sleep by enhancing the efficacy of GABA in the brain. In the brain, GABA is mainly converted from the excitatory neurotransmitter glutamate (Glu). By binding to GABA-A and GABA-B receptors, it inhibits the signal transmission between neurons, thereby regulating the excitability of neurons. Therefore, as interacting neurotransmitters, the balance between Glu and GABA is very important for the normal function of the nervous system. The results of this experiment show that the content of GABA in the hippocampal tissue of the model group is significantly lower than that in the blank control group, and the content of Glu in the hypothalamus is significantly higher than that in the blank control group. PSLO① can significantly increase the content of GABA in the hypothalamus and hippocampus of insomnia mice and reduce the content of Glu in the hypothalamus, thereby improving sleep.

[0301] In summary, by observing the effects of PSLO① on the general behavior of mice, the pentobarbital sodium suprathreshold righting reflex experiment, the open field experiment, and the content of neurotransmitters in the hypothalamus and hippocampus, the present invention has found that it can improve the sleep of PCPA-induced insomnia mice. Among them, the low-dose group has a better improvement effect on insomnia and has a certain sedative and hypnotic effect. The mechanism of action may be related to regulating the expression of neurotransmitters such as 5-HT, GABA, and Glu in the hypothalamus and hippocampus.

[0302] The above has introduced the embodiments of the present invention in detail. Specific examples are used in this article to elaborate on the principle and implementation manner of the present invention. The description of the above embodiments is only used to help understand the method and its core idea of the present invention. At the same time, changes or deformations made by those skilled in the art based on the idea of the present invention, within the specific implementation manner and application scope of the present invention, all belong to the scope protected by the present invention. In summary, the content of this specification should not be construed as a limitation to the present invention.

Claims

1. A composition for improving sleep, characterized in that, The composition includes total saponins from Panax notoginseng stems and leaves, Ziziphus jujuba seed extract, lavender extract, and lavender essential oil; Among them, the mass ratio between the total saponins from Panax notoginseng stems and leaves, the Ziziphus jujuba seed extract, the lavender extract, and the lavender essential oil is (2.50 - 3.50):(45.00 - 55.00):(55.00 - 70.00):(4.00 - 20.00).

2. The composition according to claim 1, wherein The composition consists of total saponins from Panax notoginseng stems and leaves, Ziziphus jujuba seed extract, lavender extract, and lavender essential oil; Preferably, the mass ratio between the total saponins from Panax notoginseng stems and leaves, the Ziziphus jujuba seed extract, the lavender extract, and the lavender essential oil is (2.80 - 3.30):(47.00 - 53.00):(59.00 - 66.00):(4.00 - 20.00); Preferably, the mass ratio between the total saponins from Panax notoginseng stems and leaves, the Ziziphus jujuba seed extract, the lavender extract, and the lavender essential oil is approximately 3.12:approximately 50.00:approximately 62.50:(4.00 - 20.00); Preferably, the mass ratio between the total saponins from Panax notoginseng stems and leaves, the Ziziphus jujuba seed extract, the lavender extract, and the lavender essential oil is approximately 3.12:approximately 50.00:approximately 62.50:approximately 4.69; Preferably, the mass ratio between the total saponins from Panax notoginseng stems and leaves, the Ziziphus jujuba seed extract, the lavender extract, and the lavender essential oil is approximately 3.12:approximately 50.00:approximately 62.50:approximately 18.75; Preferably, the mass ratio between the total saponins from Panax notoginseng stems and leaves, the Ziziphus jujuba seed extract, the lavender extract, and the lavender essential oil is not 12.5:25:31.25:4.69; Preferably, the mass ratio between the total saponins from Panax notoginseng stems and leaves, the Ziziphus jujuba seed extract, the lavender extract, and the lavender essential oil is not 12.5:25:15.62:18.75; Preferably, the mass ratio between the total saponins from Panax notoginseng stems and leaves, the Ziziphus jujuba seed extract, the lavender extract, and the lavender essential oil is not 3.12:25:15.62:4.69; More preferably, the in vitro administration concentration of the total saponins from Panax notoginseng stems and leaves in the composition is 2.5 - 3.5 μg / ml, such as approximately 3.12 μg / ml; More preferably, the in vitro administration concentration of the Ziziphus jujuba seed extract in the composition is 45 - 55 μg / ml, such as approximately 50 μg / ml; More preferably, the in vitro administration concentration of the lavender extract in the composition is 55 - 70 μg / ml, such as approximately 62.5 μg / ml; More preferably, the in vitro administration concentration of the lavender essential oil in the composition is 4 - 20 μg / ml, such as approximately 4.69 μg / ml or 18.75 μg / ml.

3. The composition according to claim 1, wherein The preparation method of the total saponins from Panax notoginseng stems and leaves comprises the following steps: (1) Weigh an appropriate amount of Panax notoginseng stems and leaves, extract with a solvent, filter and combine the filtrates; (2) Load the combined filtrate onto macroporous resin and perform alcohol elution; and (3) Concentrate, dry, and sieve the eluate to obtain the total saponins from Panax notoginseng stems and leaves; Preferably, in step (1), the solvent is water; Preferably, in step (1), the number of extractions is 1 to 5 times, such as 3 times; Preferably, in step (2), the macroporous resin is D101 macroporous resin; Preferably, in step (2), the concentration of the alcohol is 0.1% to 100%, such as 1% to 99%, such as 5% to 95%, such as 10% to 90%, such as 20% to 80%, such as 30% to 80%, such as about 20%, such as about 30%, such as about 40%, such as about 50%, such as about 60%, such as about 70%, such as about 80%, such as about 90%, such as about 95%, such as about 99%; Preferably, in step (3), the drying is freeze-drying.

4. The composition according to claim 1, wherein The preparation method of the semen ziziphi spinosae extract comprises the following steps: (1) Weigh an appropriate amount of semen ziziphi spinosae, pulverize and sieve, add a first solvent to soak, heat under reflux for extraction, filter to obtain filter residues; (2) Evaporate the solvent from the filter residues to obtain defatted medicinal residues, add a second solvent, reflux for extraction, filter to obtain a filtrate; And (3) Rotavapor-concentrate the filtrate to obtain a concentrated solution, and dry to obtain the semen ziziphi spinosae extract; Preferably, in step (1), the sieve is a No. 4 sieve; Preferably, in step (1), the first solvent is petroleum ether; Preferably, in step (1), the volume / mass (ml / g) ratio of the first solvent to the semen ziziphi spinosae is 1 to 5, such as about 2.5; Preferably, in step (1), the mass of the semen ziziphi spinosae is 50 to 100 g, such as about 80 g; Preferably, in step (1), the number of times of heating under reflux is 1 to 3 times, such as 2 times; Preferably, in step (1), the temperature of heating under reflux is 50 to 70 °C, such as about 60 °C; Preferably, in step (1), the time of heating under reflux is 1 to 3 h each time, such as about 2 h; More preferably, in step (2), the evaporation of the first solvent is carried out in a fume hood; More preferably, in step (2), the second solvent is ethanol; Also preferably, the volume percentage concentration of the ethanol is 60% to 80%, such as about 70%; Also preferably, in step (2), the volume / mass (ml / g) ratio of the second solvent to the defatted medicinal residues is 5 to 10, such as about 8; Also preferably, in step (2), the number of times of reflux extraction is 1 to 3 times, such as 2 times; Also preferably, in step (2), the time of reflux extraction is 1 to 3 h each time, such as about 2 h; Also more preferably, in step (3), the temperature of rotavapor concentration is 50 to 70 °C, such as about 60 °C; Also more preferably, in step (3), the end point of rotavapor concentration is that the concentrated solution has no alcohol smell; More preferably, in step (3), the drying is freeze-drying; More preferably, the yield of the wild jujube seed extract is 10% to 20%, such as about 13.1%.

5. The composition according to claim 1, wherein The method for preparing the lavender extract comprises the following steps: (a) Weigh an appropriate amount of lavender, crush it, add a third solvent for the first soaking, filter to obtain a first filtrate and filter residue; (b) Add the third solvent to the filter residue for the second soaking, filter to obtain a second filtrate; And (c) Combine the first filtrate and the second filtrate, rotary evaporate and concentrate to obtain a concentrated solution, dry the concentrated solution to obtain the lavender extract; Preferably, the third solvent is ethanol; More preferably, the volume percentage concentration of the ethanol is 70% to 90%, such as about 80%; More preferably, in step (a), the volume / mass (ml / g) ratio of the third solvent to the lavender is 5 to 15, such as about 10; More preferably, in step (a), the mass of the lavender is 80 to 120 g, such as about 100 g; More preferably, in step (a), the volume of the third solvent is 800 to 1200 ml, such as about 1000 ml; More preferably, in step (a), the time of the first soaking is 48 to 96 h, such as about 72 h; Also preferably, in step (b), the time of the second soaking is 48 to 96 h, such as about 72 h; Also preferably, in step (b), the volume of the third solvent is 800 to 1200 ml, such as about 1000 ml; Even more preferably, in step (c), the drying is freeze-drying; Even more preferably, the yield of the lavender extract is 10% to 20%, such as about 16.6%.

6. The composition according to claim 1, wherein The composition further comprises one or more pharmaceutically acceptable excipients; Preferably, the excipients are selected from one or more of the following: diluents, wetting agents, binders, disintegrants, inclusion agents, flavoring agents, sustained-release agents, glidants, lubricants, dispersants, plasticizers, light-blocking agents and antioxidants; Preferably, the dosage form of the composition is powder, tablet, dripping pill, capsule, film, lozenge, granule, injection or oral liquid.

7. The composition according to claim 1, characterized in that, The composition further comprises one or more drugs and / or extracts for improving sleep; Preferably, the drug is selected from one or more of the following: benzodiazepine receptor agonists, antidepressant drugs, and melatonin receptor agonists; More preferably, the benzodiazepine receptor agonist includes benzodiazepine drugs and non-benzodiazepine drugs; More preferably, the benzodiazepine drugs are estazolam, triazolam and diazepam; More preferably, the non-benzodiazepine drugs are zolpidem, zopiclone and eszopiclone; Even more preferably, the antidepressant drugs include noradrenergic and specific 5-HTergic antidepressant drugs and 5-HT receptor blockers and reuptake inhibitors; Particularly preferably, the noradrenergic and specific 5-HTergic antidepressant drug is mirtazapine; Particularly preferably, the 5-HT receptor blocker and reuptake inhibitor is trazodone; Specifically preferably, the melatonin receptor agonists are agomelatine and ramelteon.

8. A method for preparing the composition according to any one of claims 1 to 7, characterized in that, The method comprises mixing total saponins from Panax notoginseng stems and leaves, wild jujube seed extract, lavender extract and lavender essential oil according to the following mass ratio: (2.50 to 3.50):(45.00 to 55.00):(55.00 to 70.00):(4.00 to 20.00); Preferably, the mass ratio among the total saponins from Panax notoginseng stems and leaves, the semen ziziphi spinosae extract, the lavender extract and the lavender essential oil is (2.80 - 3.30):(47.00 - 53.00):(59.00 - 66.00):(4.00 - 20.00); Preferably, the mass ratio among the total saponins from Panax notoginseng stems and leaves, the semen ziziphi spinosae extract, the lavender extract and the lavender essential oil is approximately 3.12: approximately 50.00: approximately 62.50:(4.00 - 20.00); Preferably, the mass ratio among the total saponins from Panax notoginseng stems and leaves, the semen ziziphi spinosae extract, the lavender extract and the lavender essential oil is approximately 3.12: approximately 50.00: approximately 62.50: approximately 4.69; Preferably, the mass ratio among the total saponins from Panax notoginseng stems and leaves, the semen ziziphi spinosae extract, the lavender extract and the lavender essential oil is approximately 3.12: approximately 50.00: approximately 62.50: approximately 18.75; Preferably, the mass ratio among the total saponins from Panax notoginseng stems and leaves, the semen ziziphi spinosae extract, the lavender extract and the lavender essential oil is not 12.5:25:31.25:4.69; Preferably, the mass ratio among the total saponins from Panax notoginseng stems and leaves, the semen ziziphi spinosae extract, the lavender extract and the lavender essential oil is not 12.5:25:15.62:18.75; Preferably, the mass ratio among the total saponins from Panax notoginseng stems and leaves, the semen ziziphi spinosae extract, the lavender extract and the lavender essential oil is not 3.12:25:15.62:4.

69.

9. Use of the composition according to any one of claims 1 to 7 in the preparation of a medicament, food and / or health product for improving sleep.

10. The use according to claim 9, characterized in that, The composition improves sleep through one or more of the following ways: increasing the intracellular calcium ion concentration, improving general behavioral abnormalities, alleviating anxiety behavior, improving sleep quality and improving neurotransmitter metabolic disorders; Preferably, the general behavioral abnormalities include one or more of the following: weight loss, dull hair and irritable state; Preferably, the improvement of sleep quality includes shortening the sleep latency and prolonging the sleep duration; Preferably, the improvement of neurotransmitter metabolic disorders includes one or more of the following: increasing the content of 5 - hydroxytryptamine in the hypothalamus, increasing the content of 5 - hydroxytryptamine in the hippocampus, increasing the content of γ - aminobutyric acid in the hypothalamus, increasing the content of γ - aminobutyric acid in the hippocampus and decreasing the content of glutamate in the hypothalamus.

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