Rose water extract, preparation method and application of rose water extract in aspect of relieving depression

Abstract

The invention provides a rose water extract, a preparation method and application of the rose water extract in the aspect of relieving depression. The preparation method comprises the following steps: firstly, screening six key anti-depression active ingredients of roses, namely gallic acid, rutin, quercetin, kaempferol, gamma-aminobutyric acid and tryptophan, then researching an extraction method of the roses, taking the roses as raw materials, and carrying out steam explosion, acid water extraction and other steps to obtain a rose water extract; the rose water extract has a good effect of relieving depression.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant extracts, and relates to a rose flower water extract, a preparation method and application thereof in relieving depression. BACKGROUND

[0002] Rose flower (Flos Rosae) Rosa rugosa Thunb . ) is a dried first flower of Rosa rugosa Thunb. of Rosaceae. Its medicinal history in China can be traced back more than 2,000 years. The earliest record of its medicinal use can be found in Shiwu Bencao (Food Herbs). Rose flower has the functions of "promoting qi and relieving depression, soothing liver and stomach". According to the Pharmacopoeia, rose flower is sweet and slightly bitter in taste, warm in nature, and belongs to the liver and spleen meridians. It has the functions of promoting qi and relieving depression, and stopping pain. It is used to treat liver and stomach qi pain, irregular menstruation and other symptoms.

[0003] The acceleration of the pace of life, the continuous increase of professional competition pressure and the dramatic changes in social life environment have led to an increase in the number of patients with depression. Depression, also known as depressive disorder, is a mental disorder with high incidence, high clinical cure rate, low treatment acceptance rate and high recurrence rate. Its main feature is significant and persistent low mood, and some patients may have self-injury, suicidal behavior, and even may be accompanied by delusions, hallucinations and other psychotic symptoms.

[0004] Rose has been widely concerned in recent years for its antidepressant effect. Traditional Chinese medicine theory believes that liver stagnation is an important cause of low mood, and the aromatic components of rose can promote blood circulation and relieve liver stagnation. Modern research has found that volatile oils and polyphenols in rose can improve mood by regulating the levels of monoamine neurotransmitters such as serotonin, dopamine, and norepinephrine, as well as antioxidant pathways. Xia used animal models to evaluate the antidepressant effect of Rosa damascena essential oil and analyzed the expression levels of 5-HT signaling pathway-related proteins using molecular biology techniques (Xia, N., et al., Deciphering the antidepressant effects of Rosa damascena essential oil mediated through the serotonergic synapse signaling pathway. Journal of Ethnopharmacology, 2024. 328: p. 118007). Bradley used animal models to verify the anxiolytic effect of rose flower aromatherapy (Bradley, B.F., et al., The effects of prolonged rose odor inhalation in two animal models of anxiety. Physiology&Behavior, 2007. 92(5): p. 931-938).

[0005] The common extraction methods of rose include steam distillation, solvent extraction, and supercritical extraction, etc. The active ingredients of rose extracts extracted by different methods are significantly different, therefore, it is necessary to provide a rose extraction method for relieving depression. SUMMARY

[0006] To solve the above problems, the present application provides a rose water extract, a preparation method and its application in relieving depression. The present application first screens 6 key active ingredients of rose for treating depression: gallic acid, rutin, quercetin, kaempferol, gamma-aminobutyric acid and tryptophan, then studies the extraction method of rose. The rose water extract obtained by steam explosion and acid water extraction from rose has good effect in relieving depression.

[0007] The technical solution of the present application is: a preparation method of rose water extract, characterized by the following steps in sequence: (1) steam explosion treatment of rose flowers in a steam explosion tank under a hot steam pressure of 0.4-0.6 MPa for 20-30 seconds, and then rapid pressure relief to normal pressure; (2) acid water extraction of the rose flowers treated in step (1) in water at a material-liquid ratio of 1:25-30 (g / ml, same below) and an initial pH of 5.5-6.5, and at an extraction temperature of 79-82 ℃ for 75-80 min, and then obtaining rose flower water extract through filtration and centrifugation.

[0008] Preferably, the rose flowers used are heavy-petaled rose flowers of Shandong Pingyin.

[0009] Preferably, the extraction conditions of step (2) are: a material-liquid ratio of 1:26, an initial pH of 5.9, an extraction temperature of 81.5 ℃, and an extraction time of 77 min.

[0010] The rose flower water extract prepared by the above method has the following main active components: gallic acid, rutin, quercetin, kaempferol, gamma-aminobutyric acid and tryptophan, with the content of gallic acid being 4.38-4.62 mg / g, the content of rutin being 3.66-3.84 mg / g, the content of quercetin being 3.65-3.85 mg / g, the content of kaempferol being 0.267-0.283 mg / g, the content of gamma-aminobutyric acid being 0.47-0.53 mg / g, and the content of tryptophan being 1.18-1.32 mg / g, based on the dry matter of the rose flowers. The rose flower water extract can be used to prepare food or beverage for relieving depression.

[0011] The technical effects of the present application are: 1. The present application first screens 6 key active components of rose flowers for relieving depression: gallic acid, rutin, quercetin, kaempferol, gamma-aminobutyric acid and tryptophan; and then studies the extraction method of rose flowers, to obtain rose flower water extract through steam explosion and acid water extraction of rose flowers as raw materials, which is rich in gallic acid, rutin, quercetin, kaempferol, gamma-aminobutyric acid and tryptophan, and has a content significantly higher than that of alcohol extract; 2. The preparation method of the present application is simple, and experiments prove that the prepared rose flower water extract has a good effect of relieving depression, and can be further used to prepare food or beverage for relieving depression. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 PPI network diagram of active components of rose flowers and depression co-targets; Figure 2 Potential target diagram of active components of rose flowers and depression co-targets; Figure 3Figure for GO enrichment and KEGG pathway analysis of target; wherein, A is the GO enrichment analysis of BP, MF and CC category figure; B is the KEGG path bubble figure; Figure 4 Figure for rose-flower-effective component-potential target-disease-pathway network; Figure 5 Figure for molecular docking result of rutin; Figure 6 Figure for molecular docking result of gallic acid; Figure 7 Figure for molecular docking result of tryptophan; Figure 8 Figure for molecular docking result of quercetin; wherein, the right figure is the partial enlarged view of the left figure; Figure 9 Figure for molecular docking result of kaempferol; wherein, the right figure is the partial enlarged view of the left figure; Figure 10 Figure for molecular docking result of GABA; Figure 11 Figure for standard sample of rutin, quercetin and kaempferol at 360 nm; wherein, the retention time of rutin, quercetin and kaempferol is 25min46s, 36min12s and 42min9s, respectively; Figure 12 Figure for standard sample of gallic acid, rutin, quercetin and kaempferol at 280 nm; wherein, the retention time of gallic acid, rutin, quercetin and kaempferol is 9min26s, 25min46s, 36min12s and 42min9s, respectively; Figure 13 Figure for liquid chromatogram of gallic acid, rutin, quercetin and kaempferol in rose-flower water extract; wherein, A is the liquid chromatogram at 360 nm, and B is the liquid chromatogram at 280 nm; Figure 14 Figure for liquid chromatogram of gallic acid, rutin, quercetin and kaempferol in rose-flower alcohol extract; wherein, A is the liquid chromatogram at 360 nm, and B is the liquid chromatogram at 280 nm; Figure 15 Figure for chromatogram of GABA and blank derivatization agent; wherein, A is the chromatogram of GABA; B is the chromatogram of blank derivatization agent; the retention time of GABA is 31min52s; Figure 16 Figure for chromatogram of GABA and tryptophan in rose-flower water extract; wherein, the retention time of GABA and tryptophan is 31min52s and 46min19s, respectively; Figure 17Chromatogram of tryptophan and blank derivatization agent; wherein A is the chromatogram of tryptophan; B is the chromatogram of blank derivatization agent; the retention time of tryptophan is: 46min19s; Figure 18 Figure is the effect of rose water extract on cell morphology; wherein, A is the cell morphology of the blank group; B is the cell morphology of the damage model group; C is the cell morphology of Example 1; D is the cell morphology of Example 2; E is the cell morphology of Example 3; Figure 19 Figure is the effect of rose water extract on cell survival rate (relative to the blank group (control)); Figure 20 Figure is the effect of rose water extract on the content of substances for relieving depression. DETAILED DESCRIPTION

[0013] The present application will be further described below in conjunction with examples and drawings, but the scope of protection of the present application is not limited to the examples.

[0014] I. Confirmation of depression target and metabolic pathway analysis: Rose contains a variety of complex chemical components, and a single component may act on multiple molecular targets, which brings significant challenges to the study of its antidepressant mechanism. Network pharmacology provides a systematic solution to this problem by integrating traditional Chinese medicine ingredients and disease genes for comprehensive analysis from multiple angles and multiple levels, and demonstrates the potential therapeutic mechanism of traditional Chinese medicine. Specifically, by using network databases and computing platforms such as TCMSP (Traditional Chinese Medicine Systems Pharmacology Database), the "ingredient-disease" common targets can be systematically collected, and the biological functions of the related targets and the regulatory networks they participate in can be elucidated through GO function annotation and KEGG pathway enrichment analysis. Combined with molecular docking technology to predict the binding mode and binding energy of key ingredients and target proteins, and through experimental verification of their biological functions, the molecular mechanism of rose in relieving depression is systematically revealed. This integrated computational and experimental research strategy provides a scientific framework and methodological support for the study of the multi-target action mechanism of complex ingredients of traditional Chinese medicine.

[0015] 1.1 Website and database TCMSP database (https: / / old.tcmsp-e.com / tcmsp.php); Uniport database (https: / / www.uniprot.org); PharmMapper database (http: / / lilab-ecust.cn / pharmmapper / index.html); Venny 2.1.0 mapping platform (https: / / bioinfogp.cnb.csic.es / tools / venny); STRING database (https: / / string-db.org); Metascape database (https: / / metascape.org / gp / index.html# / main / step1); Swiss Target Prediction online website (http: / / www.swisstargetprediction.ch); GeneCards database (https: / / www.genecards.org); PharmGKB database (https: / / www.pharmgkb.org); NCBI database (https: / / www.ncbi.nlm.nih.gov); OMIM database (https: / / omim.org); Drugbank database (https: / / go.drugbank.com); PubChem database (https: / / pubchem.ncbi.nlm.nih.gov); Micro Bioinformatics online platform (https: / / www.bioinformatics.com.cn); Discovery Studio 3.0 software; AutoDock software; Cytoscapse software; PyMol software; RCSB PDB database (https: / / www.rcsb.org).

[0016] 1.2 Method 1.2.1 Collection of active ingredients of rose and traditional Chinese medicine target To screen the active ingredients and their targets of R. rugosa, the information of the active ingredients of R. rugosa was obtained from TCMSP database. To ensure the comprehensiveness and accuracy of the data, the related literature data in the authoritative databases such as PubMed, X-MOL, Web of Science (WOS), ScienceDirect (SD), and China National Knowledge Infrastructure (CNKI) were further integrated to query the active ingredients of R. rugosa, and the inaccurate data was verified. The chemical structure and SMILES number of the active ingredients of R. rugosa were queried from PubChem database, and the SMILES number was imported into Swiss Target Prediction to predict the targets of the active ingredients of R. rugosa. During the prediction, the species was limited to Homo sapiens, and the preliminary screening was performed with the standard of Probability>0, and the active ingredients without corresponding targets were deleted. Finally, the target names were unified by UniProt, and the species was limited to Homo sapiens to ensure the standardization and consistency of the data. This systematic data screening and verification process laid a reliable data foundation for subsequent network pharmacology analysis.

[0017] 1.2.2 Depression-related target collection The databases such as PharmGKB, OMIM, GeneCards, and DrugBank were systematically searched with "Depression" as the keyword to collect the targets related to depression. The targets obtained from each database were summarized, and the duplicate targets were deleted to ensure the uniqueness and accuracy of the target data. This process provided a data foundation for the subsequent target network construction and analysis.

[0018] 1.2.3 Intersection target of active ingredients of R. rugosa and depression The targets of R. rugosa and depression obtained previously, and the targets of the anti-depression monomer substances of R. rugosa and depression obtained after literature review were imported into MicroProfile and Venny 2.1.0 software, and the R. rugosa-depression Venn diagram was drawn. The intersection target was the anti-depression target of R. rugosa.

[0019] 1.2.4 PPI network of potential therapeutic targets and construction of key targets To show the key targets of rose against depression, the targets were introduced into STRING database (species limited to Homo sapiens) to construct protein-protein interaction (PPI) network. The minimum threshold of protein interaction in the database was 0.4, and the isolated targets were deleted to ensure the accuracy and reliability of data analysis. The generated PPI network diagram was exported and imported into Cytoscape for visualization processing. In Cytoscape, the Degree value of each node in the network was calculated using the CytoNCA plug-in, which reflects the importance of the protein node. Protein nodes with higher connectivity are more prominent in the visualized network. The Degree values of protein nodes were ranked in descending order to identify key nodes in the network and provide important basis for subsequent mechanism research.

[0020] 1.2.5 GO, KEGG enrichment analysis of common targets The targets screened in the previous stage were introduced into Metascape database, and the species was limited to Homo sapiens. The analysis parameters were set as follows: minimum overlap number 3, P value less than 0.01, and minimum enrichment score 1.5. GO analysis and KEGG analysis were performed on the targets. GO enrichment analysis includes biological process (BP), cellular component (CC), and molecular function (MF), which systematically describes the functional characteristics of the targets; KEGG analysis of the pathways involved in the targets elucidates the biological significance in the mechanism. This analysis process provides important functional and pathway-level theoretical basis for in-depth understanding of the multi-target mechanism of rose against depression.

[0021] 1.2.6 Construction of “Rose-Active Ingredients-Potential Targets-Disease-Pathway” network diagram The data of rose active ingredients, targets, disease targets, and signal pathways were introduced into Cytoscape to reflect different types of information through image information optimization. The color depth and shape of the nodes were differentiated according to their Type attributes (such as traditional Chinese medicine ingredients, potential targets, diseases, or pathways) to enhance the visualization effect and information transmission capacity of the network. The importance of nodes was judged by calculating the Degree value using the CytoNCA module in Cytoscape. During the calculation, the weight factor was not considered, and the “Without weight” option was selected to obtain the pure connectivity information of nodes in the topological network. This visualization and analysis method provides an intuitive and scientific tool support for systematic analysis of the complex action network of rose multi-component-multi-target-multi-pathway.

[0022] 1.2.7 Molecular docking Molecular docking is a simulation technique used to predict the optimal binding mode and interaction strength between small molecules and biological macromolecules, and is widely used to predict the interaction mode and binding force between ligands and receptors. In this study, first, the mol2 format file of active ingredients was obtained from the TCMSP database (http: / / tcmspw.com / tcmsp.php). Subsequently, the structure file of the core target protein was downloaded from the RCSB PDB database (https: / / www.rcsb.org), and the Autodock software was used to preprocess the ligand and target protein, including ligand extraction, target protein hydrogenation and dehydration, etc. The docking process of the receptor protein and the ligand compound was simulated by the Autodock software, and the binding mode and energy characteristics were analyzed. Finally, the PyMOL software was used for visual processing and conformation analysis of the docking results to intuitively show the binding site and interaction details of the ligand and receptor. This process provides reliable computational simulation support for further study of the binding mechanism of active ingredients and target proteins in rose flowers.

[0023] 1.3 Results 1.3.1 Active ingredients of rose flowers and traditional Chinese medicine target points Through network pharmacology method, search rose flower using TCMSP database, obtain target genes of rose flower and active ingredients of rose flower through ETCM and Swiss targetPrediction database. After removing duplicate data, 330 target points of rose flower were determined.

[0024] 1.3.2 Depression-related target points Through systematic retrieval of multiple authoritative databases, a total of 1240 potential target points related to depression were collected. Among them, ETCM database contributed 1166 target points, DrugBank database provided 34 target points, OMIM database contributed 1 target point, and GeneCards database provided 39 target points. In order to ensure the standardization and uniqueness of the data, the collected target points were de-duplicated, and the target points with non-standard or repeated names were removed, finally obtaining a high-quality target point dataset, laying a reliable foundation for subsequent analysis.

[0025] 1.3.3 Intersection target points of rose flower active ingredients and depression After literature review, the active ingredients of rose flower for relieving depression are: gallic acid in polyphenols, rutin, kaempferol, quercetin in flavonoids, high content of anthocyanins, γ-aminobutyric acid and tryptophan in amino acids (see Table 1).

[0026] Table 1 Intersection target points of rose flower and depression

[0027] 82 intersection targets of rose and depression were collected from the database (by rose and depression intersection targets Wayne diagram, 330 rose targets and 1240 depression related targets, 82 intersection targets), see Table 2 for specific targets.

[0028] Table 2 Intersection targets of rose and depression

[0029] 1.3.4 Protein PPI network results The 82 intersection targets screened out (as shown in Table 2) were imported into the STRING database, and the minimum confidence threshold of protein-protein interaction (PPI) was set to 0.4 (medium confidence). The PPI network was constructed. Subsequently, the generated PPI network data was imported into Cytoscape software for further analysis, and a network diagram containing 82 protein nodes and 446 interaction relationships was obtained (as shown in FIG. 1 and FIG. 2). Figure 1 and Figure 2 Based on the Degree value of each node in the network, the targets with Degree value ranking 1-28 were screened out as key targets. These targets have high topological importance in the network and may play a core regulatory role in the mechanism of rose against depression.

[0030] 1.3.5 GO and KEGG enrichment analysis Through Metascape database, functional enrichment analysis was performed on the potential targets, and 443 significantly enriched Gene Ontology (GO) items were obtained, including 315 biological processes (BP), 76 molecular functions (MF) and 52 cell compositions (CC) categories. In the BP category, the significantly enriched items mainly include positive regulation of B cell proliferation, calcium ion-mediated signal transduction and positive regulation of neuroinflammatory response. In the CC category, it mainly involves cell structures such as cytoplasm, plasma membrane and membrane protrusion. The MF category is significantly enriched in glutamate binding activity, oxidoreductase activity and homologous protein binding activity and other functions. These results show that rose may exert its antidepressant effect by regulating serotoninergic synapse, cell proliferation, inflammatory factors, metabolic processes, transcription factor activity, protein transport and other biological processes.

[0031] In addition, KEGG pathway enrichment analysis identified 83 significantly enriched signaling pathways, the most significant of which include response to steroid hormone, cellular response to lipid, gland morphogenesis, positive regulation of apoptotic process and regulation of miRNA metabolic process. These pathways are mainly related to biological processes such as inflammatory response, apoptosis and hormone regulation. The enrichment analysis results of GO items and KEGG pathways are shown in FIG. 3 and FIG. 4.Figure 3 The results provide a systematic functional and pathway-level theoretical basis for the multi-target and multi-pathway mechanism of rose in antidepressant effect.

[0032] 1.3.6 Construction of “Rose-Active Ingredient-Potential Target-Disease-Pathway” Network Diagram To reveal the complex mechanism of rose in antidepressant effect, active ingredient-target-pathway network was constructed and analyzed. The compounds showed extensive regulatory ability on multiple targets, and some targets were synergistically regulated by multiple compounds. This analysis result revealed the complex network mechanism of rose in antidepressant effect through multi-component synergistic action on multiple targets (see Figure 4 ), which provided an important basis for in-depth understanding of its pharmacological action.

[0033] 1.3.7 Verification of Network Pharmacology Analysis Results by Molecular Docking Based on the key targets screened by network pharmacology analysis, molecular docking technology was further used to evaluate the binding mode and affinity of the main active ingredients (including gallic acid, rutin, quercetin, kaempferol, γ-aminobutyric acid and tryptophan) in rose and target proteins. The molecular docking results (see Figures 5-10 ) showed that these active ingredients could form stable complexes with target proteins and showed significant binding affinity. The interaction diagram of the docking conformation of each active ingredient and target protein was displayed in detail, which revealed the key intermolecular forces such as hydrogen bond, hydrophobic interaction and π-π stacking, and provided structural biology level theoretical support for the molecular mechanism of rose in antidepressant effect.

[0034] 1.4 Conclusion Based on network pharmacology method, the mechanism of rose flower in treating depression was systematically studied. Through the integration of database retrieval and literature research, six key active ingredients including gallic acid, rutin, quercetin, kaempferol, γ-aminobutyric acid and tryptophan were screened. Based on PPI network, the core target of rose flower in treating depression was analyzed. GO analysis showed that rose flower played an antidepressant role by regulating the serotonin synapse, cell proliferation, inflammatory factors, metabolic processes, transcription factor activity, protein transport and other biological processes. KEGG pathway enrichment analysis further revealed that rose flower targets were involved in monoamine neurotransmitter, steroid hormone response, cell response to lipids, glandular morphology, positive regulation of apoptosis process and regulation of miRNA metabolic process, thereby participating in the antidepressant effect in multiple dimensions. To verify the reliability of the interaction between active ingredients and targets of rose flower, molecular docking technology was used to evaluate the binding mode and affinity of active ingredients and core target proteins. The results showed that rose flower acted on multiple targets through multi-component synergistic effect, regulating metabolic processes, inflammatory factors, cell proliferation, protein transport, transcription factor activity and other biological processes, thereby playing an antidepressant role. This finding confirmed the complex mechanism of rose flower "multi-component-multi-target-multi-pathway", providing a systematic scientific basis for further understanding its antidepressant pharmacological effects.

[0035] II. Examples and Comparative Examples Example 1 (1) The rose flowers of Pingyin heavy petal rose in Shandong Province were added to a steam explosion tank for steam explosion, and treated at a hot steam pressure of 0.5 MPa for 30 min, and then quickly released to normal pressure; (2) The steam explosion treated rose flowers were added to water, with a solid-liquid ratio of 1:25, and the initial pH was adjusted to 5.5 by an acidity regulator; the extraction temperature was 81.5 ℃, and the extraction time was 77 min; the obtained extract was filtered through four layers of gauze, and the filtrate was centrifuged at 8000 r / min for 10 min, and the obtained solution was the rose flower water extract.

[0036] Example 2 (1) The rose flowers of Pingyin heavy petal rose in Shandong Province were added to a steam explosion tank for steam explosion, and treated at a hot steam pressure of 0.5 MPa for 30 min, and then quickly released to normal pressure; (2) The steam explosion treated rose flowers were added to water, with a solid-liquid ratio of 1:27, and the initial pH was adjusted to 6.5 by an acidity regulator; the extraction temperature was 81.5 ℃, and the extraction time was 77 min; the obtained extract was filtered through four layers of gauze, and the filtrate was centrifuged at 8000 r / min for 10 min, and the obtained solution was the rose flower water extract.

[0037] Example 3 (1) The rose flowers of Pingyin heavy petal rose in Shandong Province were added to a steam explosion tank for steam explosion treatment under a hot steam pressure of 0.5 MPa for 30 min, and then quickly released to normal pressure; (2) The steam explosion treated rose flowers were added to water with a solid-liquid ratio of 1:26, and the initial pH was adjusted to 5.9 by using an acidity regulator. The extraction temperature was 81.5°C, and the extraction time was 77 min. After the extraction liquid was filtered through four layers of gauze, the filtrate was centrifuged at 8000 r / min for 10 min, and the obtained solution was the rose flower water extract.

[0038] Among them, Example 3 is the optimal condition of the response surface optimization experiment with total flavonoids and total polyphenols as indicators. The average total flavonoid content obtained by the verification experiment is 10.2 mg / g (calculated based on the dry matter of the raw rose flowers, the same below), and the total polyphenol content is 11.19 mg / g (the total flavonoids in the raw rose flowers are 13.54 mg / g, and the total polyphenols are 20.68 mg / g).

[0039] Comparative Example: alcohol extraction method (the optimal condition of the response surface optimization experiment with total flavonoids and total polyphenols as indicators) (1) The rose flowers of Pingyin heavy petal rose in Shandong Province were added to a steam explosion tank for steam explosion treatment under a hot steam pressure of 0.5 MPa for 30 min, and then quickly released to normal pressure; (2) The steam explosion treated rose flowers were added to 70% ethanol with a solid-liquid ratio of 1:17 (g / ml). The extraction temperature was 71°C, and the extraction time was 79 min. After the obtained extraction liquid was filtered through four layers of gauze, the filtrate was centrifuged at 8000 r / min for 10 min, and the obtained solution was the rose flower alcohol extract.

[0040] Note: The total flavonoid content in the rose flowers / rose flower extract was determined according to the aluminum nitrate-potassium acetate method in SN / T 4592-2016. The total polyphenol content in the rose flowers / rose flower extract was determined according to the Folin-phenol method in T / AHFIA 005-2018.

[0041] The detection methods of the contents of gallic acid, rutin, quercetin, kaempferol, γ-aminobutyric acid and tryptophan in the rose flower extract are as follows.

[0042] III. Detection of effective components in rose flowers 1. Test instruments: UltiMate 3000 high-performance liquid chromatograph, Thermo Fisher Scientific, USA; chromatographic column: SHIMSEN Superb II C18 chromatographic column (250 mm*4.6 mm, 5 μm; Shimadzu Corporation).

[0043] 2. Determination of the content of polyphenolic monomer substances in rose flowers The content of polyphenolic monomer substances in rose flowers was determined by using the HPLC method.

[0044] (1) Standard curve preparation: Prepare gallic acid, rutin, quercetin, and kaempferol gradient standard solutions (0.1-5 mg / mL), filter with microfiltration membranes, and ultrasonically treat for use.

[0045] (2) Sample solution treatment: Ultrasonically treat the rose water extracts of Examples 1-3 and the rose alcohol extracts of the comparative examples after filtering the solutions with microfiltration membranes.

[0046] (3) Selection of chromatographic conditions: Based on the ultraviolet-visible light absorption characteristics of polyphenols and flavonoids, a dual-wavelength detection mode was used, with detection wavelengths of 360 nm (suitable for the characteristic absorption of flavonoids) and 280 nm (suitable for the characteristic absorption of polyphenols). The chromatographic separation conditions were as follows: mobile phase A was methanol, and mobile phase B was 0.1% (v / v) acetic acid aqueous solution. The flow rate of the mobile phase was set to 0.7 mL / min, and a gradient elution program was used to achieve efficient separation of the target compounds. The gradient elution program was specifically set as follows: in the initial stage (0-8 min), the proportion of mobile phase B (0.1% acetic acid aqueous solution) was linearly reduced from 93% to 70%; in the second stage (8-13 min), the proportion of mobile phase B was reduced from 70% to 60%; in the third stage (13-35 min), the proportion of mobile phase B was reduced from 60% to 40%; and in the final stage (35-50 min), the proportion of mobile phase B was reduced from 40% to 20%. This gradient elution program can effectively improve the separation degree of the target compounds, reduce co-elution, and ensure that each target component peaks within a reasonable time, thereby achieving accurate qualitative and quantitative analysis of polyphenols and flavonoids in rose flowers.

[0047] (4) Calculation of monomer content: The external standard method was used to calculate the monomer content of polyphenols and flavonoids in rose water and alcohol extracts.

[0048] 3. Determination of γ-aminobutyric acid content in rose flowers The HPLC method was used to determine the content of γ-aminobutyric acid in rose flowers.

[0049] (1) Sample solution treatment: Ultrasonically treat the rose water extracts of Examples 1-3 and the rose alcohol extracts of the comparative examples after filtering the solutions with microfiltration membranes.

[0050] (2) Derivatization treatment of samples and standards: Precise 5.00 mL sample or standard solution, transfer to 50 mL volumetric flask. Then add 5.00 mL sodium bicarbonate solution (NaHCO3, 0.1 mol / L) and 5.00 mL 2,4-dinitrofluorobenzene solution (2,4-Dinitrofluorobenzene, 0.1% w / v) in turn. The reaction system was placed in a 60°C constant temperature water bath for derivatization reaction, the reaction time was 10 min. After the reaction was completed, the solution was cooled to room temperature, and the potassium dihydrogen phosphate buffer solution (KH2PO4, 0.01 mol / L, pH 6.8) was added to 50 mL scale line, and fully mixed. The obtained solution was filtered through 0.22 μm microporous filter membrane, and the filtrate was collected as the sample to be tested. At the same time, according to the same operation steps, a blank control solution without sample was prepared to eliminate background interference. After the sample was derivatized, the solution was filtered through a microfiltration membrane and ultrasonically treated for use.

[0051] (3) Standard curve preparation: Prepare γ-aminobutyric acid gradient standard solution, sample derivatization, and ultrasonically treat after microfiltration membrane filtration for use.

[0052] (4) Selection of chromatographic conditions: The chromatographic analysis conditions are optimized as follows: the chromatographic column temperature is controlled at 35°C to maintain the reproducibility of the separation process; the detection wavelength is set to 360 nm to match the maximum absorption wavelength of the target compound; the flow rate of the mobile phase is set to 1.0 mL / min, and the injection volume is 20 μL. The mobile phase system consists of binary solvents: mobile phase A is 40 mmol / L sodium acetate buffer solution (pH 6.4), and mobile phase B is acetonitrile-water (1:1, v / v) mixed solution. A gradient elution program is used to achieve efficient separation of the target compound, and the specific gradient settings are as follows: in the initial stage (0-20 min), the proportion of mobile phase A is linearly reduced from 70% to 64%; in the second stage (20-26 min), the proportion of mobile phase A is reduced from 64% to 45%; in the third stage (26-41 min), the proportion of mobile phase A is reduced from 45% to 35%; in the fourth stage (41-46 min), the proportion of mobile phase A is reduced from 35% to 10%; in the fifth stage (46-48 min), the proportion of mobile phase A is reduced from 10% to 2%; and in the final stage (48-54 min), the proportion of mobile phase A is restored from 2% to 70% to achieve column rebalancing. This gradient elution program can effectively optimize the separation efficiency of the target compound, reduce peak broadening, and ensure that each component is eluted within a reasonable retention time, thereby meeting the accuracy and reproducibility requirements of quantitative analysis.

[0053] (5) Calculation of monomer content: The content of γ-aminobutyric acid in rose water extract and alcohol extract was calculated by external standard method.

[0054] 4. Determination of tryptophan content in rose flowers The HPLC method was used to determine the content of tryptophan in rose flowers. The determination method is the same as that for the determination of γ-aminobutyric acid content.

[0055] 5. Chromatograms and detection results The liquid chromatograms of rutin, quercetin and kaempferol standard at 360 nm and 280 nm are shown in FIGS. 1 and 2, respectively. Figure 11 The liquid chromatograms of rutin, quercetin and kaempferol standard at 360 nm and 280 nm are shown in FIGS. 1 and 2, respectively. Figure 12 The liquid chromatograms of rutin, quercetin and kaempferol standard at 360 nm and 280 nm are shown in FIGS. 1 and 2, respectively. Figure 13 The liquid chromatograms of rutin, quercetin and kaempferol standard at 360 nm and 280 nm are shown in FIGS. 1 and 2, respectively. Figure 14 The liquid chromatograms of rutin, quercetin and kaempferol standard at 360 nm and 280 nm are shown in FIGS. 1 and 2, respectively. Figure 15 The chromatograms of γ-aminobutyric acid and blank derivatization agent are shown in FIGS. 3 and 4, respectively. Figure 16 The chromatograms of γ-aminobutyric acid and tryptophan in the water extract of rose flowers are shown in FIGS. 5 and 6, respectively. Figure 17 The chromatograms of γ-aminobutyric acid and tryptophan in the water extract of rose flowers are shown in FIGS. 5 and 6, respectively.

[0056] The contents of the antidepressant active monomer substances in the water extract of rose flowers of Example 3 and the alcohol extract of Comparative Example 1 are shown in Table 3 (based on the dry matter of raw rose flowers).

[0057] Table 3 Contents of antidepressant active monomer substances in the water extract of rose flowers of Example 3 and the alcohol extract of Comparative Example 1

[0058] The stability and bioavailability of the antidepressant substances in the rose flower extract were determined by a simulated gastrointestinal digestion experiment (pepsin-trypsin digestion test). After the simulated gastrointestinal digestion experiment, the water extract contained gallic acid 3 ± 0.05 mg / g and rutin 3.2 ± 0.06 mg / g.

[0059] Simulated gastrointestinal digestion experiment (pepsin-trypsin digestion test): (1) Pepsin digestion: The rose flower extract was adjusted to pH 2.0 with 0.1 mol / L hydrochloric acid solution, and then pepsin was added (enzyme addition amount was 4% of the mass of the substrate). After thorough mixing, the mixture was incubated in a 37℃ constant temperature water bath for 2 h. After the reaction was completed, the sample was immediately heated in a boiling water bath for 15 min to inactivate the enzyme activity. After cooling to room temperature, the sample was centrifuged at 4000 rpm for 15 min, and the supernatant was collected to obtain the digestion solution. (2) Trypsin digestion: the digestion solution was adjusted to pH 7.5 with 0.1 mol / L sodium hydroxide solution, trypsin was added (enzyme addition amount was 4% of the mass of the substrate), and after mixing thoroughly, it was incubated in a 37°C constant temperature water bath for 2 h. After the reaction was completed, the sample was immediately placed in a boiling water bath for 15 min to inactivate the enzyme activity, and after cooling to room temperature, it was centrifuged at 4000 rpm for 15 min, and the supernatant was collected. The pH value of the supernatant was adjusted to 8.3 with 0.1 mol / L sodium hydroxide solution, and the content of the antidepressant active substance therein was determined. This method simulates the gastrointestinal digestion environment to evaluate the stability and bioavailability of the antidepressant active substance in the rose extract during the digestion process.

[0060] The above data show that the content of the antidepressant active monomer in the water extract is generally higher than that in the alcohol extract, and the content of gallic acid in the water extract is significantly higher than that of other components, indicating that the water extraction method is more conducive to extracting polyphenolic compounds with antidepressant activity from rose flowers.

[0061] Four, rose antidepressant test Test Example 1: In this experiment, rat adrenal pheochromocytoma PC12 (1×10 6 ) was used as the research object to explore the antidepressant mechanism of rose. In order to verify the antidepressant effect of the invention, a related cell experiment was designed, and the experiment used corticosterone modeling. The simulated depression cell group was the model group, and the unmodeled group was the blank group. The samples of Examples 1, 2, and 3 were applied to the cells, and enzyme-linked immunosorbent assay was performed to observe the possible improvement of Examples 1, 2, and 3 on the cell depression model.

[0062] 1. Corticosterone and rose antidepressant samples Dimethyl sulfoxide (DMSO) was used as the solvent to prepare 100 mM corticosterone (CORT). The rose extract solutions of Examples 1-3 were concentrated by rotary evaporation, and then redissolved with an equal volume of DMEM medium for subsequent experiments.

[0063] 2. MTT method for measuring cell viability Complete medium: fetal bovine serum (FBS), penicillin-streptomycin solution (double antibiotic), and Dulbecco's high glucose medium (DMEM) were mixed in a volume ratio of 90:10:1 (DMEM:FBS:double antibiotic).

[0064] MTT method is a classic experimental method widely used to evaluate cell proliferation, survival ability, and drug toxicity. Its detection principle is based on the fact that mitochondrial dehydrogenase in living cells reduces MTT to insoluble blue-purple formazan crystals, which are soluble in dimethyl sulfoxide (DMSO) and have a maximum absorption peak at 490 nm wavelength.

[0065] The experiment will use 1×10 5 PC12 cells / mL were seeded into 96-well plates, with 100 µL of cell suspension added to each well. The plates were incubated at 37°C with 5% CO2 for 24 h. After incubation, the supernatant was removed, and control and experimental groups were set up. 100 µL of complete culture medium was added to each well of the control group, while 100 µL of sample dilution buffer (final concentration 2 mg / mL) from Examples 1-3 was added to each well of the experimental group. Six replicates were set up for each group. After incubation for another 12 h, the supernatant was removed, and 100 µL of MTT solution (0.5 mg / mL) was added. The plates were incubated at 37°C with 5% CO2 for 4 h. Subsequently, the supernatant was removed, and 100 µL of LDMSO was added, followed by shaking for 10 min until the formazan crystals were completely dissolved. The absorbance (OD value) of each well was measured at 490 nm using a microplate reader. The OD value of the control group was recorded as OD0. 空白 The OD value of the experimental group is denoted as OD. 样品 Cell viability was calculated using the following formula, and suitable sample concentrations that did not significantly inhibit PC12 cell viability were screened.

[0066]

[0067] 3. Determination of 5-HT (5-hydroxytryptamine) and DA (dopamine) levels This study used 6-well plates for cell passage culture and employed ELISA to measure the levels of 5-HT and DA factors in cells. Experimental groups included a control group, a model group, and a sample group (including aqueous extract, alcoholic extract, and digested aqueous and alcoholic extracts). After cells reached the logarithmic growth phase and their morphology stabilized, CORT was added to all groups except the control group to achieve a final concentration of 400 μM / L. This treatment was continued for 12 h, after which the original culture medium was discarded, and 2 mL of DMEM culture medium (final concentration 2 mg / mL) was added to each sample group, followed by another 12 h of culture. Cell morphological changes were observed periodically throughout the experiment. After the culture period, cells from each group were collected for subsequent analysis. Rotary evaporation was used to concentrate the rose aqueous and alcoholic extracts before and after digestion. The solvent was removed by evaporation, and the concentrated extracts were then reconstituted using DMEM culture medium to ensure that subsequent experiments were conducted under the same concentration conditions.

[0068] 4. Experimental results of this case: 1) Cell morphology like Figure 18As shown in the figure, the blank group has high cell density, intercellular connection is interlaced, adhesion is good, cell neurite differentiation is obvious, and the state is better; the model group has significantly decreased cell density, increased cell gap, and part of the cell antenna disappears and becomes round, and the differentiation is not obvious, and part of the cell is suspended and not adhered; compared with the model group, the cell density of the example 1, 2 and 3 groups is obviously increased, the antenna differentiation is increased, and the cell state is obviously improved.

[0069] 2) Cell survival rate As shown in the figure, the cell viability is determined by MTT method, and the blank group is used as a control, and the rose flower water extract of examples 1, 2 and 3 has no significant effect on cell viability and cell survival rate. Figure 19

[0070] 3) Enzyme-linked immunosorbent assay of relieving depression substance contentThe water extract, alcohol extract and water extract and alcohol extract after digestion of the rose flower are adjusted to the same mass concentration for experiment.

[0071] The results show that the number of cells in the model group is significantly reduced, the contents of 5-HT and DA are significantly decreased, and the construction of the depression cell model is successful. After the PC12 cells are intervened by the rose flower sample group, the DA levels are respectively: the rose flower water extract 46.246±1.26 ng / mL, the rose flower alcohol extract 27.314±2.13 ng / mL, the rose flower water extract after digestion 38.897±1.39 ng / mL, and the rose flower alcohol extract after digestion 25.314±1.96 ng / mL. After the PC12 cells are intervened by the rose flower sample group, the 5-HT levels are respectively: the rose flower water extract 6.974±0.89 ng / mL, the rose flower alcohol extract 4.024±0.18 ng / mL, the rose flower water extract after digestion 5.818±0.34 ng / mL, and the rose flower alcohol extract after digestion 3.471±0.32 ng / mL. As shown in the figure, compared with the model group, the 5-HT content in the cells determined by ELISA of examples 1, 2 and 3 is significantly increased, and there is no significant difference among examples 1, 2 and 3. Figure 20

[0072] The results show that the anti-depression effect of the rose flower water extract of the application is obviously better than that of the alcohol extract.​​

Claims

1. A method for preparing rose water extract, characterized by, The method comprises the following steps in sequence: (1) steam explosion of the rose flowers in a steam explosion tank under a steam pressure of 0.4-0.6 MPa for 20-30 seconds, and then rapid pressure relief to normal pressure; (2) acid water extraction of the rose flowers treated in step (1) in water at a material-to-liquid ratio of 1:25-30 and an initial pH of 5.5-6.5, at an extraction temperature of 79-82 ℃ for 75-80 min, and then filtration and centrifugation to obtain a rose flower water extract.

2. The method for preparing rose petal aqueous extract as described in claim 1, characterized in that, The rose flowers used are heavy-petaled rose flowers from Pingyin, Shandong Province.

3. The method for preparing rose petal aqueous extract as described in claim 1, characterized in that, In step (2), the material-to-liquid ratio is 1:26, and the initial pH is 5.

9.

4. The method for preparing rose water extract according to claim 1, characterized by, In step (2), the extraction temperature is 81.5 ℃, and the extraction time is 77 min.

5. The rose flower aqueous extract prepared by the method of any one of claims 1 to 4, characterized by, The main active ingredients are gallic acid, rutin, quercetin, kaempferol, γ-aminobutyric acid and tryptophan.

6. The rose flower aqueous extract according to claim 5, characterized by, In the rose flower water extract, the contents of gallic acid, rutin, quercetin, kaempferol, γ-aminobutyric acid and tryptophan are 4.38-4.62 mg / g, 3.66-3.84 mg / g, 3.65-3.85 mg / g, 0.267-0.283 mg / g, 0.47-0.53 mg / g and 1.18-1.32 mg / g, respectively, based on the dry matter of the raw rose flowers.

7. Use of the rose flower water extract of claim 5 or 6 in the preparation of a food or beverage for relieving depression.

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