Use of rosemary hydrosol in preparing medicine for treating insomnia and / or food for improving sleep function

Abstract

The present invention provides the use of rosemary hydrosol in preparing a medicine for treating insomnia and / or a food for improving sleep function. The present invention detects the hippocampus of rats, and uses Elisa kits, Western-Blot experiments, Rt-PCR technology and immunohistochemistry to measure 5-HT1AR, PKA, cAMP, ADCY5 and GABAA enriched in the serotonergic synaptic pathway, and determines that rosemary hydrosol achieves the effect of treating insomnia by acting on the serotonergic synapse. It can be developed into a functional food or medicine that can relieve the symptoms of insomnia patients, regulate the emotions of insomnia patients, and reduce the risk of depression.

Description

Technical Field

[0001] The invention relates to application of rosemary hydrosol in preparing medicine for treating insomnia and / or food for improving sleep function. Background Art

[0002] Sleep is an essential part of human health and a bodily function necessary for human survival. However, most people's sleep duration and sleep quality are not ideal. Insufficient sleep is an important public health issue in today's era. Insufficient sleep will increase the mortality rate of patients with cardiovascular disease and cancer. In addition, insomnia patients will suffer from depression, dizziness, fatigue, physical discomfort, anxiety and other physical diseases.

[0003] At present, the treatment of insomnia includes psychological behavioral intervention, drug therapy and acupuncture therapy. Psychological intervention behavior has individual differences and great uncertainty; drug therapy includes benzodiazepines, sedative antidepressants, antihistamines, antipsychotics, etc. Although the effect is good, there are many side effects, such as drowsiness, nausea, dizziness, indigestion, mild mania, and addiction. Long-term use will cause physical dependence, which will aggravate the side effects; acupuncture treatment can improve central inhibition function and help people fall asleep better, but acupuncture will cause people to have pain reactions, and some patients will have local hematoma and headache during acupuncture. Therefore, safe, effective and low-side effect insomnia drugs need to be developed. Remarkably, phytochemicals have been shown to have excellent sleep-promoting effects, especially Chinese herbal medicine resources that have been used for more than 2,000 years. Studies have shown that medicinal plants used to treat depression and insomnia include valerian, chamomile, rosemary and lavender.

[0004] Rosemary (Rosmarinus officinalis L.) is an herb originating from the Mediterranean basin and is a member of the Lamiaceae family. Due to its strong adaptability, it can be grown all over the world. Rosemary extract can be used as an additive in the food and pharmaceutical industries and is authorized as a food additive in multiple food categories in the European Union at a high content. Rosemary is classified as generally safe by the FDA in the United States and approved as a naturally occurring food preservative by the European Food Safety Authority (EFSA). Its main chemical components are α-pinene, eucalyptol, rosmarinic acid, camphor, etc. Studies have shown that rosemary has good antibacterial, anti-inflammatory, antioxidant, anti-tumor, antidepressant, and antianxiety biological activities, and its antioxidant activity is at least equal to or better than other synthetic antioxidants in foods at the molecular level. Rosemary capsules have the effect of treating insomnia, and rosemary volatile oil has the effect of improving anxiety, depression and sleep disorders. Rosemary can treat emotional anxiety and depression caused by insomnia. Many folk applications make rosemary into tea to play the role of treating insomnia. However, some studies have shown that the ingredients in rosemary have a refreshing effect. Zhang Yu et al., On the Application of Essential Oil Aromatherapy in the Diagnosis and Treatment of Insomnia [J], The 13th Academic Seminar and the General Meeting of the Health and Rehabilitation Branch of the Chinese Association of Traditional Chinese Medicine, 2015, 207-210 disclosed that rosemary essential oil has a refreshing effect. The dual-directional regulation mechanism of refreshing and promoting sleep in rosemary makes the effective part of rosemary for treating insomnia unclear, which affects its practical application in treating insomnia. Summary of the invention

[0005] In order to solve the above problems, the present invention provides the use of rosemary hydrosol in preparing medicine for treating insomnia and / or food for improving sleep function.

[0006] Furthermore, the rosemary hydrosol is a liquid obtained by distilling rosemary medicinal materials with water and removing essential oil from the distillate.

[0007] Furthermore, the medicine is a preparation prepared with rosemary hydrosol as the active ingredient and pharmaceutically acceptable excipients;

[0008] The food is a preparation prepared by taking rosemary hydrosol as an active ingredient and adding auxiliary materials acceptable to food.

[0009] Furthermore, the preparation is an oral preparation.

[0010] Furthermore, the oral preparation is a solution, pill, paste or capsule.

[0011] Furthermore, the medicine is a medicine for improving sleep quality; and the food is a food for improving sleep quality.

[0012] Furthermore, the drug is a drug that increases the level of 5-HT, GABA and / or DA in serum; the food is a food that increases the level of 5-HT, GABA and / or DA in serum.

[0013] Furthermore, the drug is a drug that increases the expression level of 5-HT1AR, ADCY5, GABAA, cAMP and / or PKA in hippocampal tissue; the food is a food that increases the expression level of 5-HT1AR, ADCY5, GABAA, cAMP and / or PKA in hippocampal tissue.

[0014] The present invention has been proved by animal experiments that, compared with the main active ingredients of rosemary disclosed in the prior art and the western medicine diazepam, rosemary hydrosol has a more obvious effect of improving insomnia. The present invention detects the hippocampus of rats, and uses Elisa kits, Western-Blot experiments, Rt-PCR technology and immunohistochemistry to measure 5-HT1AR, PKA, cAMP, ADCY5 and GABAA enriched in the serotonergic synapse pathway, and determines that rosemary hydrosol achieves the effect of treating insomnia by acting on Serotonergic synapse, which can be developed into a functional food or medicine with the ability to relieve the symptoms of insomnia patients, regulate the emotions of insomnia patients, and reduce the risk of depression.

[0015] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0016] The following is a further detailed description of the above contents of the present invention through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Extraction device diagram

[0018] Figure 2 Structures of 19 compounds

[0019] Figure 3 RNA-seq analysis results (A: PCA map; B: volcano map; C: heat map; D: KEGG map; E: GO map; F: GSEA-KEGG map; G: GSEA-GO map)

[0020] Figure 4GC-MS and network pharmacology results (A. GC-MS ion mass spectrum; B. intersection gene target map; C. Cytoscape network map; D. PPI network map; E. KEGG enrichment analysis; F. GO enrichment analysis)

[0021] Figure 5 KEGG and GO result diagram (A, C, E, G are the results of KEGG, BP, CC, MF before sorting; B, D, F, H are the results of KEGG, BP, CC, MF after sorting)

[0022] Figure 6 Molecular docking result diagram (A and B are the 1,7,7-trimethylbicyclo[2.2.1]heptan-2-one component and Propofol positive drug images of the docking of protein 6X40, the target of GABRB2; C and D are the 3-Methyl-4-isopropylphenol component and Piperazine positive drug images of the docking of protein 7A5V, the target of GABRB3; E and F are the Caryophyllene component and Clozapine positive drug images of the docking of protein 7E2Y, the target of HTR1A. G and H are the Citronellol component and Mesalamine positive drug images of protein 1DIY, the target of PTGS1; I is the heat map of molecular docking).

[0023] Figure 7 Open field test and kit results (A and B are the results of the open field test; C, D, and E are the results of the Elisa kit for 5-HT, GABA, and DA)

[0024] Figure 8 Immunohistochemistry results

[0025] Fig. 9 RT-PCR and Western-blot results (AE is the result of RT-PCR; F is the analysis result of Western-Blot)

[0026] Fig.10 Mechanism diagram DETAILED DESCRIPTION

[0027] The raw materials and equipment used in the specific implementation manner of the present invention are all known products, which are obtained by purchasing commercially available products. Among them, the preparation process of the rosemary hydrosol involved in the present invention is mature and can be prepared by referring to the conventional methods of the prior art.

[0028] Embodiment 1, the preparation of rosemary hydrosol

[0029] Weigh 100 g of rosemary medicinal material, add 1200 mL of water, and extract by steam distillation. After 2 hours, collect rosemary hydrosol, separate and remove the rosemary essential oil, and obtain 500 mL of hydrosol in total.

[0030] Embodiment 2, the preparation of rosemary hydrosol

[0031] Weigh 100 g of rosemary medicinal material, add 1200 mL of water, and extract by ultrasonic-assisted steam distillation. After 2 hours, collect rosemary hydrosol, separate and remove the rosemary essential oil, and obtain a total of 500 mL of hydrosol.

[0032] Embodiment 3, the preparation of rosemary hydrosol

[0033] Weigh 100 g of rosemary medicinal material, add 1200 mL of water, and extract by microwave-assisted steam distillation. After 2 hours, collect rosemary hydrosol, separate and remove the rosemary essential oil, and obtain 500 mL of hydrosol in total.

[0034] Embodiment 4, the preparation of rosemary hydrosol

[0035] Weigh 100 g of rosemary medicinal material, add 1200 mL of water, and extract by enzymatic assisted steam distillation. After 2 hours, collect rosemary hydrosol, separate and remove the rosemary essential oil, and obtain 500 mL of hydrosol in total.

[0036] The beneficial effects of the present invention are illustrated by experiments below.

[0037] Experimental Example 1 Study on the Effect of Rosemary Hydrosol on Insomnia Rats

[0038] (I) Preparation of drugs

[0039] Take rosemary medicinal materials, add 12 times the amount of water, distill for 2 hours, take rosemary hydrosol, separate and remove the rosemary essential oil, and obtain a total of 500mL of hydrosol.

[0040] (II) Animal dosing and grouping

[0041] 80 SPF adult male SD rats were selected and randomly divided into 8 groups after three days of adaptive feeding, with 10 rats in each group. They were blank group, model group, positive drug group (diazepam 0.92mg / kg), rosemary hydrosol ultra-low dose group (5mg / mL), rosemary hydrosol low dose group (10mg / mL), rosemary hydrosol medium dose group (12.5mg / mL), rosemary hydrosol high dose group (15mg / mL) and rosemary hydrosol ultra-high dose group (30mg / mL). Except for the blank group, the other 7 groups were intraperitoneally injected with 300mg / kg of 4-Chloro-DL-phenylalanine (PCPA) once a day for three consecutive days to establish a rat insomnia model. From the fourth day, the blank group and the model group were gavaged with 1% Tween 80 aqueous solution every day, the positive drug group was gavaged with 1% Tween 80 aqueous solution containing diazepam (0.92 mg / Kg), and the drug group was gavaged with 1% Tween 80 aqueous solution containing rosemary hydrosol (5 mg / mL, 10 mg / mL, 12.5 mg / mL, 15 mg / mL, 30 mg / mL). The gavage treatment lasted for 6 days in total.

[0042] (III) Detection of physiological and biochemical indicators of rats

[0043] After the experiment, the rats were anesthetized, blood was collected from the abdominal aorta, and the blood was centrifuged at 4°C, 3000r / min for 10min to separate the serum, collect and store. After the rats were euthanized, the whole brains of the rats were stripped and placed on ice, and the hippocampus was separated. The serum of the rats was taken and the expression levels of 5-HT, GABA, and DA in the serum of the rats were detected by ELISA kit.

[0044] (IV) Experimental results

[0045] The results of the kit are shown in Table 1. Compared with the normal group, the serum 5-HT and GABA in the insomnia model group decreased (p < 0.05), and DA decreased significantly (p < 0.001). Compared with the model group, the serum 5-HT and GABA in the diazepam group and the low, medium and high groups of rosemary hydrosol increased to varying degrees (p < 0.05 and p < 0.01); compared with the model group, the serum DA in the diazepam group and the low, medium and high groups of rosemary hydrosol increased to varying degrees except for the low-dose group (p < 0.05 and p < 0.01); compared with the model group, the serum 5-HT, GABA and DA in the ultra-low-dose group of rosemary hydrosol decreased to varying degrees (p > 0.05); the serum 5-HT, GABA and DA in the ultra-high-dose group of rosemary hydrosol increased to varying degrees (p > 0.05). The results showed that the low, medium and high groups of rosemary hydrosol could significantly increase the contents of 5-HT, GABA and DA in insomnia rats. It was preliminarily judged that rosemary hydrosol had a good therapeutic effect on insomnia rats.

[0046] Table 1 Test of rosemary hydrosol on 5-HT, GABA and DA in rat serum

[0047]

[0048]

[0049] Note: *p<0.05 **p<0.01 ***p<0.001

[0050] Experimental Example 2: Exploring the components of rosemary hydrosol in treating insomnia in rats

[0051] (I) Identification of Rosemary Hydrosol Compounds

[0052] GC-MS analysis of rosemary hydrosol yielded 23 active ingredients, including 20 qualified ingredients identified using the internal standard (hexadecane). The results are shown in Table 2.

[0053] Table 2 GC-MS analysis of rosemary hydrosol

[0054]

[0055]

[0056] 2. Component Mining

[0057] Quality control research on the monomer components revealed that there may be four key ingredients in rosemary hydrosol for treating insomnia, namely 1,7,7-trimethylbicyclo[2.2.1]heptan-2-one, Citronellol, 3-Methyl-4-isopropylphenol and Caryophyllene.

[0058] (III) Determination of the content of key ingredients

[0059] The absolute contents of the four components were calculated using internal standards to determine the contents of the four components in the low, medium and high dose groups of rosemary hydrosol in the treatment of insomnia rats. The results are shown in Table 3.

[0060] Table 3 Content of rosemary hydrosol ingredients

[0061]

[0062] In summary, the results show that rosemary hydrosol has four key ingredients for treating insomnia. The content of 1,7,7-trimethylbicyclo[2.2.1]heptan-2-one is 0.0699-0.104851μg; the content of Citronellol is 0.008971-0.013456μg; the content of 3-Methyl-4-isopropylphenol is 0.011497-0.017245μg; and the content of Caryophyllene is 0.004183-0.006274μg.

[0063] Experimental Example 2: Exploring the therapeutic effects of the four ingredients and their combinations on insomnia rats

[0064] (I) Preparation of drugs

[0065] Take rosemary medicinal materials, add 12 times the amount of water, distill for 2 hours, take rosemary hydrosol, separate and remove the rosemary essential oil, and obtain a total of 500mL of hydrosol.

[0066] (II) Animal dosing and grouping

[0067] 80 SPF adult male SD rats were selected and, after three days of adaptive feeding, randomly divided into 8 groups, with 10 rats in each group. They were blank group, model group, positive drug group (diazepam 0.92 mg / Kg), 1,7,7-trimethylbicyclo[2.2.1]heptan-2-one group (0.104851 mg / Kg), Citronellol group (0.013456 mg / Kg), 3-Methyl-4-isopropylphenol group (0.017245 mg / Kg), Caryophyllene group (0.006274 mg / Kg) and compatibility group (four ingredients: 1,7,7-trimethylbicyclo[2.2.1]heptan-2-one (0.104851 mg / Kg), Citronellol (0.013456 mg / Kg), 3-Methyl-4-isopropylphenol (0.017245 mg / Kg) and Caryophyllene (0.006274 mg / Kg)). Except for the blank group, the other 7 groups were intraperitoneally injected with 300 mg / kg of 4-chlorophenylalanine (PCPA) once a day for three consecutive days to establish a rat insomnia model. From the fourth day, the blank group and the model group were gavaged with 1% Tween 80 aqueous solution every day, the positive drug group was gavaged with 1% Tween 80 aqueous solution containing diazepam (0.92 mg / Kg), and the drug-treated groups were gavaged with 1,7,7-trimethylbicyclo[2.2.1]heptan-2-one (0.104851 mg / Kg), Citronellol (0.013456 mg / Kg), 3-Methyl-4-isopropylphenol (0.017245 mg / Kg), Caryophy The mice in the combination group were intragastrically administered with 1% Tween 80 aqueous solution containing 1,7,7-trimethylbicyclo[2.2.1]heptan-2-one(0.104851mg / Kg), Citronellol(0.013456mg / Kg), 3-Methyl-4-isopropylphenol(0.017245mg / Kg) and Caryophyllene(0.006274mg / Kg). The treatment lasted for 6 days.

[0068] (III) Detection of physiological and biochemical indicators of rats

[0069] After the experiment, the rats were anesthetized, blood was collected from the abdominal aorta, and the blood was centrifuged at 4°C, 3000r / min for 10min to separate the serum, collect and store. After the rats were euthanized, the whole brains of the rats were stripped and placed on ice, and the hippocampus was separated. The serum of the rats was taken and the expression levels of 5-HT, GABA, and DA in the serum of the rats were detected by ELISA kit.

[0070] (IV) Experimental results

[0071] The results of the kit are shown in Table 4. Compared with the normal group, the serum 5-HT and GABA in the insomnia model group decreased (p < 0.01), and DA decreased significantly (p < 0.001). Compared with the model group, the serum 5-HT, GABA, and DA in the diazepam group increased to varying degrees (p < 0.01); compared with the model group, except for the 1,7,7-trimethylbicyclo[2.2.1]heptan-2-one group, the serum GABA content increased slightly (p < 0.05); the serum 5-HT, GABA, and DA in the other 1,7,7-trimethylbicyclo[2.2.1]heptan-2-one group, Citronellol group, 3-Methyl-4-isopropylphenol group, Caryophyllene group, and the combination group increased, but there was no difference (p > 0.05). This shows that the four monomer components have no obvious improvement effect on insomnia rats, and the combination of the four components has no effect on insomnia rats.

[0072] Table 4 Test of the four components on 5-HT, GABA and DA in rat serum

[0073]

[0074] Note: *p<0.05 **p<0.01 ***p<0.001

[0075] In summary, the four monomer components and their combination had no improvement effect on insomnia rats. Rosemary hydrosol containing 1,7,7-trimethylbicyclo[2.2.1]heptan-2-one (0.0699-0.104851μg), Citronellol (0.008971-0.013456μg), 3-Methyl-4-isopropylphenol (0.011497-0.017245μg) and Caryophyllene (0.004183-0.006274μg) had a significant improvement effect on insomnia rats.

[0076] Example 3 Study on the Treatment of Insomnia with Rosemary (Rosmarinus officinalis L.) Hydrosol Based on Serotonergic Synapse

[0077] 1. Materials and Methods

[0078] 1.1 Extraction of Rosemary Hydrosol

[0079] 1200ml of water was added to the bottom of the reactor, and 100g of rosemary medicinal materials were weighed on the partition of the three-necked reactor. The extract was extracted by steam distillation. After two hours of distillation, the rosemary hydrosol was collected and the rosemary essential oil was separated and removed, and a total of 500ml of hydrosol was obtained. The extraction device is shown in the figure below ( Figure 1 ).

[0080] 1.2 Identification of Rosemary Hydrosol Compounds

[0081] Extraction was performed according to the method in 1.1, and the determination method of volatile oils in the Chinese Pharmacopoeia (2020 edition) (General Rule 2204) was referred to. The xylene layer was taken and hexadecane internal standard was added for gas chromatography-mass spectrometry (GC-MS) determination. The gas chromatography conditions were as follows: Agilent HP-5ms (30m×250μm×0.25μm) capillary column, carrier gas He, no split, injection volume 1.5μL, flow rate 1mL / min. The starting temperature was 40℃ (retention time 1min), 5℃ / min to 90℃, 10℃ / min to 140℃, and 20℃ / min to 250℃. The mass spectrometry conditions were as follows: ionization source EI, ionization energy 70eV, transfer line temperature 280℃, ion source temperature 230℃, quadrupole temperature 150℃, solvent delay time 11min, scanning mass range 30–550amu. The data used was processed by data analysis software, and the threshold was adjusted to 24 to screen the content of rosemary hydrosol. The NIST standard library was used to search and the components were screened according to the matching degree, retention index and relevant literature. The retention index is defined as n-alkanes (C8-C 40 ) under the same conditions. According to the retention index of normal alkanes, the retention index of rosemary hydrosol is as follows:

[0082]

[0083] Among them, t R is the retention time, X is the compound to be analyzed, K and K+1 are the carbon number of the two n-alkanes before and after the analyte, that is, t R (K)<t R (X)<t R (K+1).

[0084] 1.3 Animal experiments

[0085] Nine SPF male rats weighing (200±20) g were purchased from the Experimental Animal Center of the School of Medicine of Xi'an Jiaotong University, with an animal license number of SCXK (Shaanxi) 2018-001. Under experimental conditions (temperature 25±2℃, relative humidity 50±5%), the animals were fed for 1 week. This study was approved by the Animal Ethics Committee of Shaanxi University of Chinese Medicine. SD rats were randomly divided into 3 groups, 3 in each group, including a blank control group, a model group, and a rosemary administration group. Except for the blank group, the other two groups were intraperitoneally injected with 300 mg / Kg PCPA (DL-4-chlorophenylalanine) for three consecutive days to replicate the insomnia rat model. Starting from the fourth day, the blank control group and the model group were gavaged with 1% Tween 80 aqueous solution; the rosemary hydrosol administration group was gavaged with 12.5 mg / mL rosemary hydrosol; the administration was carried out for 6 days, and the sleep recovery of rats in each group was observed. After the experiment, all rats were anesthetized with 10% chloral hydrate and euthanized. The whole brain of the rat was removed and placed on ice, and the hippocampus was isolated, frozen in liquid nitrogen and quickly transferred to a -80°C freezer.

[0086] 1.4 NA-seq analysis

[0087] RNA-seq is the sequencing of genes in organisms. Total RNA was extracted from hippocampal tissues under 1.3, and mRNA was enriched and then reverse transcribed into double-stranded cDNA. After repairing the double ends of cDNA, adapters were added and the machine library was constructed by PCR amplification. Bioinformatics analysis was performed using the real-time interactive online data analysis platform Omicsmart (http: / / www.omicsmart.com). Genes that met the conditions of FDR<0.05 and log2|FC|≥2 were considered differentially expressed genes. Based on the results of differentially expressed genes, GO and KEGG Pathway enrichment analysis was performed.

[0088] 1.5 Network pharmacology analysis based on “ingredient-target”

[0089] 1.5.1 Acquisition of Rosemary Hydrosol Ingredient Targets and Insomnia Disease Targets

[0090] The PubChem (http: / / pubchem.ncbi.nlm.nih.gov / ) database, SwissTargetPrediction database and Metatarget database were used to predict the targets of rosemary hydrosol and screen out the targets of rosemary active ingredients. The keyword "insomnia" was entered into the Genecards (https: / / www.genecards.org / ), DisGeNET (https: / / www.disgenet.org / ) and OMIM (https: / / omim.org / ) databases to obtain the target genes related to insomnia diseases, which were integrated and deleted.

[0091] 1.5.2 Insomnia-Rosemary Hydrosol Ingredients Network Construction

[0092] The rosemary active targets, insomnia disease targets and RNA-seq differential gene targets were input into the online Vennydiagram (http: / / bioinformatics.psb.ugent.be / webools / Venn / ) platform, and the intersection was taken. These common targets are the key targets for explaining the effective ingredients of rosemary hydrosol in treating insomnia. The rosemary hydrosol ingredient-target was imported into the Cytoscape3.7.2 software (http: / / www.cytoscape.org / ) to construct a rosemary ingredient-target network, so that the relationship between the effective ingredients and common targets in rosemary hydrosol is clearer. In the network diagram, the network was screened under the degree of freedom condition to determine the interaction relationship and importance between the rosemary active ingredients and the disease cross-target. The sorted intersection target genes were imported into the online String (http: / / string-db.org / ) database, the research species was selected as "Homo sapiens", the confidence was selected as 0.7, and the PPI network diagram was obtained. EnsemblID and log2fc of the intersection genes were used for KEGG and GO enrichment analysis.

[0093] 1.6 Establishment of weight coefficient

[0094] Rosemary hydrosol contains a variety of ingredients that are effective in treating insomnia, but the relative content of medicinal ingredients varies from place to place and manufacturer to manufacturer. OB is an important parameter for weighing the metabolic process of drugs in the body. Therefore, the OB value is included in the scope of investigation and a corresponding relationship is established with the ingredient content:

[0095] A x =B x *C x

[0096] Where Ax Indicates the amount of component x involved in drug metabolism in the body, B x Indicates the relative content of ingredient x in rosemary hydrosol, C x Represents the oral bioavailability value of ingredient x.

[0097]

[0098] In the above formula, M x Represents the sum of the scores of each target contained in component x.

[0099] N=(M1+M2+…+M n )

[0100] The above formula represents the sum of the scores of each component participating in pathway N.

[0101] The establishment of the weight coefficient successfully introduced the key factor of component content. The pathways were reordered according to the data results of the weight coefficient, making the experimental results more reliable.

[0102] 1.7 Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis

[0103] In order to further clarify the targets and pathways of rosemary hydrosol in the treatment of insomnia, first, the sequenced differentially expressed genes were subjected to KEGG enrichment analysis, and then GO and KEGG enrichment analysis were performed using R software. The analysis results were reordered according to the weight coefficients, and the weight coefficient of each pathway was the sum of the weight coefficients of all targets in the specific pathway.

[0104] 1.8 Molecular docking

[0105] The targets enriched in the pathway were selected as the research objects, the 3D structures of the key targets were screened out through the PCSB-PDB database, the 2D structures of the active ingredients were downloaded from PubChem, and molecular docking was performed using the Discovery Studio Client4.5 software.

[0106] 1.9 Pharmacodynamics experiments

[0107] 1.9.1 Animal model preparation and grouping

[0108] 60 SPF male SD rats, weighing (200±20) g, were purchased from the Experimental Animal Center of the School of Medicine of Xi'an Jiaotong University, animal license number SCXK (Shaanxi) 2018-001. Under experimental conditions (temperature 25±2°C, relative humidity 50±5%), the animals were adaptively fed for 1 week. This study was approved by the Animal Ethics Committee of Shaanxi University of Chinese Medicine. SD rats were randomly divided into 6 groups, 10 in each group, including a blank control group, a model group, a positive group, a low-dose group of rosemary hydrosol, a medium-dose group, and a high-dose group. During the modeling period, except for the blank group, all other groups were intraperitoneally injected with 300 mg / Kg PCPA (DL-4-chlorophenylalanine) (MACKLIN D831376.) for three days. Compared with the blank group, the remaining groups all showed disappearance of circadian rhythm, stronger aggression, and unkempt hair, indicating that the insomnia model was successfully established. From the fourth day, the blank control group and the model group were gavaged with 1% Tween 80 aqueous solution; the diazepam group was gavaged with 0.092 mg / mL diazepam solution; the low-dose rosemary hydrosol group was gavaged with 10 mg / mL rosemary hydrosol; the medium-dose rosemary hydrosol group was gavaged with 12.5 mg / mL rosemary hydrosol; and the high-dose rosemary hydrosol group was gavaged with 15 mg / mL rosemary hydrosol. The drugs were administered for 6 days respectively, and the sleep recovery of rats in each group was observed.

[0109] 1.9.2 Open field test

[0110] After the animal treatment experiment, the open field experiment was conducted. Measurement conditions: Keep the behavioral laboratory quiet and ventilated, dim the light indoors, and reflect the surgical light from the ceiling to the experimental operation area; the experimenter should stay away from the open field experiment analysis box (the operating angle should not see the animals in the box). Before the experiment, the animals need to adapt to the measurement room for more than 10 minutes. In the animal behavior analysis system, the open field analysis box is divided into 9 identical small grids from the upper left and numbered in different colors from left to right. No. 5 is the central area, and the rest are peripheral grids. In each experiment, pinch the rat's tail 2 / 3 of the distance from the root and gently put it into grid No. 5. Observe its activity within 5 minutes on the monitor. After each rat test, remove the feces, wipe the bottom and inner wall of the test box with 75% alcohol, and clean it. After the fan is dried, put the next one in. Each rat is measured only once. After the test, the video is analyzed and the software is counted to record the number of times each rat stands and passes the central grid.

[0111] 1.9.3 Sample Collection

[0112] After the open field experiment, all rats under 1.9.1 were euthanized and anesthetized with 10% chloral hydrate. 5 mL of blood was collected from the abdominal aorta. The blood was centrifuged at 4°C and 3000 r / min for 10 min. The upper serum was then collected and stored. After the rats were euthanized, the whole brains of the rats were stripped and placed on ice. The hippocampus was separated, half of which was fixed with paraformaldehyde, and the other half was frozen in liquid nitrogen and quickly transferred to a -80°C refrigerator.

[0113] 1.9.4 Enzyme-linked immunosorbent assay

[0114] The ELISA method was used to detect the contents of serotonin (5-HT), γ-aminobutyric acid (GABA), and dopamine (DA) in each group of rats. The double-antibody one-step sandwich enzyme-linked immunosorbent assay was performed according to the instructions of the kit. Finally, the absorbance (OD) value was determined at a wavelength of 450 nm using a microplate reader, and a standard curve was drawn to calculate the contents of 5-HT, GABA, and DA in the samples.

[0115] 1.9.5 Immunohistochemistry

[0116] The hippocampus tissue was sliced ​​and soaked in xylene twice, then hydrated with ethanol gradient (1000%, 95%, 85%, 75%) and double distilled water for 3 minutes each, and high-pressure antigen repair was performed in 0.01M sodium citrate buffer solution for 20 minutes to 30 minutes, and 3% H2O2 was added to block endogenous peroxidase, and the wet box was incubated for 10 minutes. Non-immune and normal sheep serum was added to block non-specific antigens, and the wet box was incubated for 30 minutes at 37°C, and then the primary antibody was added dropwise, incubated overnight at 4°C, and then taken out and washed 3 times at room temperature, each time for 3 minutes, and then HRP-labeled secondary antibody (rabbit antibody) was added and incubated at 37°C for 60 minutes. The slices were then stained with DAB, rinsed with tap water, counterstained with hematoxylin, and differentiated with 0.1% hydrochloric acid alcohol. The degree of staining was observed under a microscope, and finally dehydrated, transparent and sealed.

[0117] 1.9.6 Western-Blot Experiment

[0118] The hippocampal tissues of rats in each group were collected and RIPA lysis buffer was added until completely lysed. The samples were centrifuged at 4°C and 12000g for 15min, and the supernatant was taken as the protein extract. According to the SDS-PAGE gel, a separation gel of appropriate concentration was prepared, 15μg of protein was loaded, electrophoresis was performed, and then transferred to a PVDF membrane, sealed with 5% skim milk powder, added with primary antibody, incubated at 4°C overnight, and washed with TBST 3 times, 5min each time. HRP-labeled secondary antibody diluted 1:1000 was added, incubated with the membrane at 37°C for 1h, then washed with TBST, and developed with ECL reagent. The target bands were analyzed using a gel image processing system to analyze and calculate the relative expression of the target protein.

[0119] 1.9.7 Real-time fluorescence quantitative polymerase chain reaction (RT-PCR)

[0120] Total DNA was extracted by TRlzol method, reverse transcription reaction program: 42℃, 60 minutes; 70℃, 5 minutes; real-time PCR reaction program: 95℃, 10 minutes (95℃, 15 seconds; 55℃, 45 seconds) × 40; 95℃, 15 seconds; 60℃, 1 minute; 95℃, 15 seconds; 60℃, 15 seconds. Data were analyzed using the instrument's own software (ABI Prism 7300SDS Software) using 2 -ΔctΔct Methods The relative expression level of each mRNA was calculated. Primers are shown in Table 5.

[0121] Table 5 Rt-PCR primer list

[0122]

[0123] 1.10 Statistical analysis

[0124] All experimental data in this study were statistically analyzed using SPSS19.0 software. The results are expressed as mean ± standard deviation One-way analysis of variance was used to determine the differences between the groups, with p < 0.05 and p < 0.01 indicating a significant difference.

[0125] 2. Results

[0126] 2.1 GC-MS analysis of rosemary hydrosol

[0127] In this study, GC-MS was used to analyze the active ingredients of rosemary hydrosol and the ion mass spectrum of the hydrosol was obtained ( Figure 4 A), combined with the NIST database and retention index screening, a total of 23 active ingredients were obtained, and 19 qualified rosemary hydrosol ingredients were identified. The specific compound structures are shown in Figure 2 .

[0128] 2.2 RNA-seq analysis results

[0129] The online analysis platform of Kidio Bio was used to set FDR≤0.05 and the difference multiple to 1. PCA Figure 3 A, a total of 1579 differentially expressed gene targets were obtained. Among them, compared with the model, there were 945 up-regulated genes and 613 down-regulated genes in the blank. The volcano plot of differentially expressed genes is shown in Figure 1. Figure 3 B. The heat map of differentially expressed genes is shown in Figure 3C. KEGG and GO enrichment were performed for the differentially expressed genes between the blank and model groups, and pathways related to nervous system diseases were analyzed. The pathways enriched by KEGG included GABAergic synapse, Nicotine addiction, cAMP signaling pathway, Serotonergic synapse, etc. (e.g. Figure 3 D); GO enrichment analysis includes BP, CC, MF, and the pathways involved include cellular process, biological regulation, binding, cell part, etc. (such as Figure 3 E). Based on the sequencing results, GSEA analysis was performed on all genes in a single comparison group using the KEGG and GO databases to enrich for diseases related to the nervous system. For both the blank and model groups, the GSEA-KEGG results showed that the enriched pathways included Amyotrophic lateral sclerosis, Serotonergic synapse, Neurotrophin signaling pathway, etc. (e.g. Figure 3 F); GSEA-GO results showed that the pathways involved included protein activation cascade, complement activation, etc. (e.g. Figure 3 G).

[0130] 2.3 Selection of insomnia targets and network analysis

[0131] By using PubChem database combined with Swiss Target Prediction and Meta Tar Fisher database, a total of 533 rosemary hydrosol targets were obtained. And 2705 insomnia disease targets were obtained from GeneCards, DisGeNet and OMIM databases. Taking the intersection of rosemary hydrosol component targets, insomnia disease targets and sequenced differentially expressed genes, 29 targets for rosemary hydrosol to treat insomnia were obtained as shown in the figure ( Figure 4 B) The intersection gene target data were imported into Cytoscape3.7.2 software to construct the "ingredient-target" network diagram of rosemary hydrosol for the treatment of insomnia. The results are as follows Figure 4 As shown in C, the yellow ellipse represents the active ingredient of rosemary hydrosol, the green diamond represents the key gene, and the connecting line indicates the relationship between the ingredient and the gene. The more the number of connections, the more important the ingredient or gene is. The 29 intersection genes were imported into string to construct the PPI network diagram. The results are as follows Figure 4D. The PPI network diagram consists of 29 nodes with an average degree of freedom of 1.45. The node size represents the degree value, and the larger the value, the more important it is. PPI shows that ALB, GABAR2, GBARB3 and other genes have large degree values ​​and play a role in the treatment of insomnia. The Ensembl ID and log2fc values ​​of the 29 intersection genes were subjected to KEGG and GO enrichment analysis. KEGG results ( Figure 4 E) showed that the involved pathways include Nicotine addiction, Neuroactive ligand-receptor interaction, GABAergic synapse, Serotonergic synapse, etc. GO enrichment analysis such as Figure 4 As shown in F.

[0132] 2.4GO and KEGG enrichment analysis

[0133] GO and KEGG enrichment analysis was performed on 29 genes using R software. GO analysis included 87 biological processes, 45 cell components and 61 molecular functional pathways. KEGG enriched 18 pathways. These pathways include neuroactive ligand receptor interaction, serotonin synapse, GABAergic synapse and cAMP signaling pathway. After calculating the weight coefficient, the results were comprehensively considered to determine that the serotonin synapse pathway was the most important pathway. The results are shown in Figure 5 .

[0134] 2.5 Molecular docking results

[0135] Through molecular docking, the active ingredients and key targets of rosemary hydrosol for treating insomnia were connected. The results showed that the docking results of key target proteins were better than those of positive drugs, indicating that rosemary hydrosol has active ingredients in treating insomnia. Figure 6 shown.

[0136] 2.6 Study on the therapeutic effect of rosemary hydrosol on insomnia rats

[0137] After PCPA intraperitoneal injection, except for the blank group, the other groups showed the disappearance of circadian rhythm, stronger aggression, and unkempt hair, indicating that the model was successful; the blank group was flexible and had regular circadian activities. After treatment with the positive drug group and different doses of rosemary hydrosol groups, the aggression decreased and the circadian rhythm emerged.

[0138] 2.7 Open field test results

[0139] All rats were subjected to an open field test. The results showed that the number of standing and passing through the center of the model group was significantly reduced compared with the normal group (p < 0.001). Compared with the model group, the number of standing and passing through the center of the rats in the diazepam group and rosemary hydrosol group increased (p < 0.05 and p < 0.01). Figure 7 A, B.

[0140] 2.8 Serum levels of 5-HT, GABA, and DA

[0141] The levels of 5-HT, GABA, and DA in serum were measured by ELISA kits. The results showed that ( Figure 7 C, D, E), compared with the normal group, the serum 5-HT and GABA in the insomnia model group decreased (p < 0.05, n = 6), and DA decreased significantly (p < 0.001, n = 6). Compared with the model group, the serum 5-HT and GABA in the diazepam group and rosemary hydrosol group increased to varying degrees (p < 0.05 and p < 0.01, n = 6). Compared with the model group, the serum DA in the diazepam group and rosemary hydrosol group increased to varying degrees (p < 0.05 and p < 0.01) except for the low-dose group (p > 0.05).

[0142] 2.9 Expression of 5-HT1AR, ADCY5, GABAA and PKA in hippocampus (immunohistochemistry results)

[0143] The expression levels of 5-HT1AR, ADCY5, GABAA and PKA in the hippocampus of rats were determined by immunohistochemistry experiments. Compared with the normal group, the expression levels of 5-HT1AR, ADCY5, GABAA and PKA in the model group were significantly reduced (p < 0.001). Compared with the model group, the 5-HT1AR, ADCY5, GABAA and PKA in the diazepam group and the rosemary hydrosol group were increased to varying degrees (p < 0.05 and p < 0.01). The results showed that rosemary hydrosol can increase the expression of 5-HT1AR, ADCY5, GABAA and PKA in the hippocampus tissue of insomnia rats, thereby improving their sleep quality, such as Figure 8 .

[0144] 2.10 Western-Blot analysis

[0145] The protein expression levels of 5-HT1AR, ADCY5, GABAA and PKA in the rat hippocampus were determined by Western-Blot experiments. Compared with the normal group, the expression levels of 5-HT1AR, ADCY5, GABAA and PKA in the model group were significantly reduced (p < 0.01, n = 3). Compared with the model group, 5-HT1AR, ADCY5, GABAA and PKA in the diazepam group and the low-, medium- and high-dose groups of rosemary were increased to varying degrees. Among them, there were differences between the medium-dose group of rosemary and the model group (p < 0.05, n = 3). The results show that rosemary hydrosol can promote the 5-HT pathway and treat insomnia. The results are as follows Fig. 9 F.

[0146] 2.11 Real-time fluorescence quantitative polymerase chain reaction (RT-PCR)

[0147] The expression levels of 5-HT1AR, ADCY5, GABAA and PKA mRNA in the hippocampus of rats were determined by Rt-PCR experiments. The results showed that compared with the normal group, the expression levels of 5-HT1AR, ADCY5, GABAA and PKA in the model group were significantly decreased (p < 0.001, n = 3), and the expression level of cAMP was decreased (p < 0.01, n = 3). Compared with the model group, the expression levels of 5-HT1AR, cAMP and PKA in the diazepam group and the rosemary hydrosol group increased to varying degrees, and there were differences (p < 0.05 and p < 0.01); the expression level of ADCY5 increased differentially except for the low-dose rosemary group (p < 0.05); the expression level of GABAA increased differentially except for the high-dose rosemary hydrosol group (p < 0.05). The results are as follows Fig. 9 AE.

[0148] The above results show that the active ingredients of rosemary hydrosol, 1,7,7-trimethylbicyclo[2.2.1]heptan-2-one, 3-Methyl-4-isopropylphenol, Caryophyllene and Citronellol, synergistically improve the symptoms of insomnia. Among them, Caryophyllene may achieve the purpose of treating insomnia by upregulating 5-HT1AR expression, increasing 5-HT content, and upregulating ADCY5, cAMP, PKA and GABA involved in the serotonergic synaptic pathway. Its mechanism of action is shown in the figure below. Fig.10 .

[0149] 3. Discussion

[0150] As we all know, insomnia is highly correlated with depression, and conventional drugs for treating mental illnesses can cause dependence in patients, so people are increasingly interested in the study of herbal medicines for insomnia. Herbal extracts and their components, such as dianthus, forsythia, and rosemary, have anti-insomnia and anti-depression effects in animal models. This study explored the mechanism of action for the treatment of insomnia by oral administration of rosemary hydrosol. Based on network pharmacology, using the construction of "weight coefficient" as a carrier, using RNA-seq, introducing key indicators of oral bioavailability and ingredient content, and performing pathway enrichment sorting, a comprehensive review of literature information determined that serotonergic synapses are the key pathways for rosemary hydrosol to treat insomnia.

[0151] The serotonergic system plays an irreplaceable role in the treatment of insomnia and depression. Serotonin (5-HT) is an important brain neurotransmitter that plays a vital role in emotions, cognition, sleep, reproduction and motor functions, and is also related to the pathogenesis of various mental illnesses. 5-HT is the first known substance in sleep neuroscience that promotes sleep. Serotonergic drugs have been shown to be effective in treating anxiety disorders without the side effects of benzodiazepines. Studies have shown that 5-HT in the brain is mainly distributed in the dorsal raphe nucleus (DRN) and the raphe magnus in the brainstem raphe nuclei, and has many receptors, among which 5-HT1A and 5-HT1B receptor subtypes are related to the inhibition of adenylate cyclase (ADCY5). Flesinoxan (5-HT1A agonist) microinjection into the DRN can enhance REM sleep. Studies have shown that DA (dopamine) dysfunction can induce insomnia. In the cerebrospinal fluid of patients with severe depression, the content of DA metabolites is reduced, which is consistent with the results of the ELISA kit for DA serum content detection in this study. Oral administration of rosemary hydrosol can increase the DA content of insomnia rats and improve insomnia symptoms. The 5-HT1A binding potential is reduced in the hippocampus of depressed patients. Depression can cause insomnia, indicating that 5-HT1AR is an important factor causing depression and insomnia. As a signaling molecule, 5-HT can bind to G protein-coupled receptors on the cell membrane surface, thereby triggering adenylate cyclase (ADCY5), leading to the production of the second messenger cyclic adenosine monophosphate (cAMP) in the cell. cAMP activates protein kinase A (PKA) to phosphorylate proteins, and then acts on GABA, increasing the GABA content and promoting sleep. On the contrary, when the 5-HT content decreases, cAMP decreases, which will downregulate the transmission of extracellular signals to the cells, causing the expression of the PKA-(cyclic adenosine monophosphate response element binding protein) CREB-(brain-derived neurotrophic factor) BDNF pathway to be downregulated, resulting in adverse psychological changes and leading to insomnia and depression. Therefore, when cAMP activates PKA, CREB phosphorylation is used to regulate transcription, and BDNF gene expression increases, mediating the cell's response to external stimuli, thereby affecting people's psychological and behavioral characteristics. Garcia's research shows that in a highly anxious environment, activating 5-HT neurons in the DRN-(bed nucleus of the stria terminalis) BNST pathway can reduce anxious behavior and promote sleep.

[0152] In this study, PCPA suspension was intraperitoneally injected into SD rats to replicate the insomnia model. After the experiment, the hippocampus of the rats was taken for detection. Elisa kits, Western-Blot experiments, Rt-PCR technology and immunohistochemistry were used to measure 5-HT1AR, PKA, cAMP, ADCY5 and GABAA enriched in the serotonergic synaptic pathway. The results showed that the content of each index of insomnia rats was reduced, and the indexes of rats treated with rosemary hydrosol were improved to varying degrees compared with the model group rats, indicating that rosemary hydrosol has a corresponding effect on the treatment of insomnia. The reliability of the combination analysis of "weight coefficient" and network pharmacology was better proved by the verification of animal experiments. In the study of insomnia, 5-HT and GABA are involved in the sleep-wake cycle, and the inhibition of 5-HT1AR-mediated cAMP / PKA signaling pathway in the hippocampus can cause cognitive dysfunction. GABA is a key target for many clinical drugs to treat insomnia. For example, diazepam regulates GABA by binding to GABAA receptors to treat insomnia. PKA activator 8-Br-cAMP has antidepressant activity. In order to further determine the effective ingredients in rosemary hydrosol that affect serotonergic synapses, the active ingredients of rosemary hydrosol corresponding to the four targets of GABRB2, GABRB3, HTR1A and PTGS1 were selected and the corresponding positive drugs were used as ligands for molecular docking to verify the relationship between the active ingredients of rosemary hydrosol and the targets of insomnia disease. After analysis, it was shown that 1,7,7-trimethylbicyclo[2.2.1]heptan-2-one, 3-Methyl-4-isopropylphenol, Caryophyllene and Citronellol as components of rosemary hydrosol can play a role in treating insomnia. Rosemary hydrosol plays a role in treating insomnia through the synergistic effect between the ingredients.

[0153] In summary, the study shows that rosemary hydrosol can treat insomnia by acting on serotonergic synapse. It can be developed into a functional beverage that can relieve the symptoms of insomnia patients, regulate their emotions, and reduce the risk of depression.

Claims

1. Use of rosemary hydrosol as the sole active ingredient in the preparation of a medicine for treating insomnia, or a food for improving sleep, characterized in that: The medicine is an oral preparation prepared by taking rosemary hydrosol as the active ingredient and adding pharmaceutically acceptable excipients; The food is a preparation made of rosemary hydrosol as an active ingredient and supplemented with food-acceptable auxiliary materials; The key components of the rosemary hydrosol are camphor, citronellol, 3-methyl-4-isopropylphenol and caryophyllene.

2. The use according to claim 1, characterized in that The rosemary hydrosol is a liquid obtained by distilling rosemary medicinal materials with water and removing essential oil from the distillate.

3. The use according to claim 1, characterized in that The oral preparation is a solution, a pill, an ointment or a capsule.

4. The use according to claim 1, characterized in that The medicine is a medicine for improving sleep quality; the food is a food for improving sleep quality.

5. The use according to claim 1, characterized in that: The drug is a drug that increases the level of 5-HT, GABA and / or DA in serum; the food is a food that increases the level of 5-HT, GABA and / or DA in serum.

6. The use according to claim 1, characterized in that: The drug is a drug that increases the expression level of 5-HT1AR, ADCY5, GABAA, cAMP and / or PKA in hippocampal tissue; the food is a food that increases the expression level of 5-HT1AR, ADCY5, GABAA, cAMP and / or PKA in hippocampal tissue.

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