Compound hemerocallis citrina concentrated juice with mood improving effect as well as preparation method and application thereof

Abstract

The invention provides compound hemerocallis citrina concentrated juice with an emotion improving effect as well as a preparation method and application of the compound hemerocallis citrina concentrated juice. The day lily, the dried orange peel, the poria cocos, the rose and the mint are compatible, the activity of the medicinal and edible compounds is fully exerted, the synergistic effect is achieved, the number of action targets is large, and the emotion improving effect can be effectively achieved by increasing release of hippocampal neurotransmitters 5-HT and DA, reducing the level of inflammatory factors TNF-alpha and IL-1beta and other mechanisms. The compound hemerocallis citrina concentrated juice has the advantages of mood improvement, small toxic and side effects and the like, the used raw materials are cheap and easy to obtain, the production cost is low, the health-care value is high, and the compound hemerocallis citrina concentrated juice is good news for patients with depression.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, in particular to a compound daylily concentrated juice with mood-improving effect, and a preparation method and application thereof. Background Art

[0002] The pathogenesis of depression is complex, believed to be linked to genetics, neurobiology, and psychosocial and cultural factors. Multiple mechanisms interact within the disease, yet these mechanisms remain largely undefined and unifying. Consequently, the etiology and pathophysiology of depression remain largely unknown, and a definitive understanding remains elusive. Currently, the main hypotheses for the pathogenesis of depression include the monoamine neurotransmitter hypothesis, the neurotrophin hypothesis, the hypothalamic-pituitary-adrenal axis hypothesis, the immune-inflammatory hypothesis, and the gut microbiome hypothesis.

[0003] Western medicine is currently the main treatment for depression, including tricyclic antidepressants and selective serotonin reuptake inhibitors. However, the cure rate is less than 50%, and it is associated with adverse reactions such as nausea, weight gain, and gastrointestinal reactions. Furthermore, while the mechanisms and targets of Western medicine for depression are relatively clear, depression is a multifactorial disease, and a single mechanism or target cannot fully cure it. Therefore, the advantages of Traditional Chinese Medicine (TCM) in treating depression, such as its multiple pharmacological components, multi-target therapy, and minimal toxicity and side effects, are gradually becoming apparent. The term "depression" was first mentioned in the "Medical Biography: Depression", but discussions of emotionally induced illnesses were already found in the "Suwen: Jutonglun Pian". Depression falls under the TCM category of "depression" and "visceral agitation", and TCM believes that treatment should focus on soothing the liver, relieving depression, and promoting blood circulation and qi. Summary of the Invention

[0004] The present invention aims to provide a compound daylily concentrated juice with mood-improving effect, and a preparation method and application thereof.

[0005] Daylily (Hemerocallis citrina Baroni), also known as "golden daylily" and "lemon daylily," was also known as "forget-me-not" in ancient times. It has a sweet flavor and cooling properties, and enters the heart, liver, and spleen meridians. It has the effects of relieving depression, stopping bleeding, nourishing blood, calming the liver, and acting as a diuretic. It is clinically used to treat symptoms such as edema, dysuria, and hematemesis. This invention, based on the principle of selecting traditional Chinese medicines with medicinal and edible properties, uses daylily as the main ingredient and adds four other herbs: tangerine peel, mint, rose, and poria cocos to develop a functional product for alleviating depression. The antidepressant effects of the daylily compound were investigated using in vitro experiments and various modern pharmacological research techniques, including network pharmacology, molecular biology, and histomorphology, to elucidate its underlying mechanism of action.

[0006] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a compound daylily concentrated juice with the effect of improving mood, which comprises the following components by weight: 5-15 parts of daylily powder, 1-10 parts of tangerine peel powder, 1-10 parts of Poria powder, 1-10 parts of rose powder and 3 parts of mint powder.

[0007] Preferably, the following components are included in parts by weight: 13 parts of daylily powder, 5 parts of tangerine peel powder, 10 parts of Poria powder, 10 parts of rose powder and 3 parts of mint powder.

[0008] The active ingredients of the compound daylily concentrated juice of the present invention include flavonoids and phenolic acid substances.

[0009] Furthermore, the content of the phenolic acid substances is 1.2-1.49%, and the content of the flavonoid substances is 1.2-1.56%.

[0010] In a second aspect, the present invention provides a preparation having the effect of improving mood, comprising the compound daylily concentrated juice and pharmaceutically acceptable excipients.

[0011] Preferably, the preparation is a decoction.

[0012] In a third aspect, the present invention provides a method for preparing the compound daylily concentrated juice, comprising: sieving each component separately, then uniformly mixing according to a proportion, mixing with water at a material-liquid ratio of 1 g:(10-20) mL, placing in a reflux extraction device and extracting at 80-90° C. for 1.5-2 hours (preferably mixing with water at a material-liquid ratio of 1:10, placing in a reflux extraction device and extracting at 90° C. for 2 hours); then filtering and centrifuging the obtained extract, and concentrating it to 10-20 Brix using a rotary evaporator.

[0013] The method further comprises: after concentration, detecting the total flavonoid content in the compound daylily concentrated juice to be 1.2-1.56%, the total phenolic acid content to be 1.2-1.49%, and the sedimentation rate to be 0.1-0.25%.

[0014] In a fourth aspect, the present invention provides the use of the compound daylily concentrated juice in the preparation of functional products for improving mood.

[0015] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: The present invention's composition combines daylily, tangerine peel, poria cocos, rose, and mint, fully leveraging the active properties of these medicinal and edible compounds. This combination exhibits synergistic effects and multiple targets, effectively improving mood by increasing the release of neurotransmitters 5-HT and DA in the hippocampus and reducing levels of inflammatory factors such as TNF-α and IL-1β. The present invention's concentrated daylily compound juice has the advantages of improving mood and minimizing toxic and side effects. Furthermore, the raw materials used are inexpensive and readily available, resulting in low production costs and significant health benefits, making it a promising treatment for patients with depression. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the behavioral roadmap of zebrafish in the diving experiment of the present invention. Compared with the control group, Compared with the model group, A. Zebrafish activity trajectory; B. Percentage of time spent by the zebrafish in the central and peripheral areas; C. Total distance moved by the zebrafish; D. Average movement speed of the zebrafish.

[0017] Figure 2 This is the behavioral roadmap of zebrafish in the open field test in the experimental example of the present invention. Compared with the control group, Compared with the model group, A. Zebrafish activity trajectory; B. Percentage of time spent by the zebrafish in the central and peripheral areas; C. Total distance moved by the zebrafish; D. Average movement speed of the zebrafish.

[0018] Figure 3 Graph showing the relationship between concentration and viscosity in the experimental examples of the present invention.

[0019] Figure 4 Graph showing the relationship between concentration and total flavonoid content in the experimental examples of the present invention.

[0020] Figure 5 Graph showing the relationship between concentration and total polyphenol content in the experimental examples of the present invention.

[0021] Figure 6 The relationship between concentration and physical and chemical properties in the experimental examples of the present invention is shown in Figure 1. A. Changes in pH value with increasing concentration; B. Changes in sedimentation rate with increasing concentration.

[0022] Figure 7 The results of the behavioral test of mice in the experimental example of the present invention are shown in Table 2. Compared with the control group, Compared with the model group, A. Mouse weight change graph; B. Mouse tail suspension immobility time; C. Mouse activity trajectory map in open field test; D. Total distance moved by mice in the open field; E. Mouse immobility time in the open field; F. Average movement speed of mice in the open field; G. Mouse immobility time in forced swimming.

[0023] Figure 8 This is the H&E staining result of the mouse hippocampus in the experimental example of the present invention.

[0024] Figure 9 The results of the test of 5-HT, BDNF and inflammatory factors in the hippocampus of mice in the experimental example of the present invention are compared with the control group. Compared with the model group, A. TNF-α content; B. IL-1β content; C. IL-6 content; D. BDNF content; E. 5-HT content.

[0025] Figure 10 The protein expression and quantification of the mouse hippocampus in the experimental example of the present invention are compared with the control group. Compared with the model group, A. PI3K, AKT, and BDNF protein expressions in the mouse hippocampus; B. PI3K to β-Actin ratio; C. AKT to β-Actin ratio; D. BDNF to β-Actin ratio. DETAILED DESCRIPTION

[0026] The invention provides a compound daylily concentrated juice with mood-improving effect, a preparation method and application thereof.

[0027] The first purpose of the present invention is to provide a compound daylily concentrated juice with the effect of improving mood, which comprises the following components by weight: 6-15 parts of daylily powder, 1-10 parts of tangerine peel powder, 1-10 parts of Poria powder, 1-10 parts of rose powder and 3 parts of mint powder.

[0028] Furthermore, the daylily powder is yellow powder.

[0029] Furthermore, the active ingredients of the compound daylily concentrated juice include phenolic acids and flavonoids.

[0030] Furthermore, the contents of the phenolic acids and flavonoids are 1.2-1.49% and 1.2-1.56% respectively, which are greater than the contents of other active substances.

[0031] Furthermore, the eggplant peel composition also includes tangerine peel powder, Poria powder, rose powder and mint powder. By weight, the compound daylily concentrated juice includes the following components: 6-15 parts of daylily powder, 1-10 parts of tangerine peel powder, 1-10 parts of Poria powder, 1-10 parts of rose powder and 3 parts of mint powder.

[0032] The present invention discovered that daylily extracts primarily contain active ingredients such as flavonoids, phenolic acids, and amino acids; tangerine peel primarily contains flavonoids, such as hesperidin, nobiletin, and neohesperidin, as well as volatile oils; Poria cocos contains polysaccharides, triterpenes, sterols, proteins, amino acids, and fatty acids; rose contains flavonoids, vitamins, amino acids, and volatile oils; and mint also has the effect of soothing the liver and relieving depression. These ingredients have antidepressant, antitumor, antioxidant, antibacterial, and anti-inflammatory effects. Combining these five herbs can interact with each other to significantly improve mood.

[0033] The present invention can purchase high-quality materials and then process them by oneself. The specific preparation method is as follows: (1) grind daylily, tangerine peel, poria, rose, and mint, and pass through a 60-mesh sieve for later use. (2) Mix the above medicinal materials in proportion to a material-liquid ratio of 1:10, extract at 90°C for 2 hours, evaporate and concentrate until the wall is sticky, then freeze-dry in a freeze dryer, collect the extract, freeze-dry, grind, and pass through an 80-mesh sieve for later use.

[0034] The second purpose of the present invention is to study the mechanism of the mood-improving effect of the compound daylily concentrated juice.

[0035] The third object of the present invention is to provide a method for preparing the above-mentioned compound daylily concentrated juice, comprising: sieving each group of powders separately, then mixing them uniformly according to a proportion, mixing them with water at a material-liquid ratio of 1g:10mL, and extracting at 90°C for 2h.

[0036] The fourth purpose of the present invention is to provide the use of the above-mentioned compound daylily concentrated juice in the preparation of a health product for improving mood.

[0037] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0038] In the following examples, daylily, tangerine peel, poria, rose, and mint were all purchased from commercially available high-quality materials, and then subjected to water extraction to obtain extracts. Raw material preparation: (1) Crush the daylily, tangerine peel, poria, rose, and mint medicinal materials into powder and pass through a 60-mesh sieve for later use. (2) Mix the above medicinal material powders in a uniform ratio, add 10 times the amount of water, and extract at 90°C for 2 h. Evaporate and concentrate the extract until it sticks to the wall, then freeze-dry it, collect the freeze-dried extract, grind it through an 80-mesh sieve, and set aside.

[0039] Example 1 The present embodiment provides a compound daylily concentrated juice with the effect of improving mood, which is composed of the following components: 6 parts of daylily, 3 parts of dried tangerine peel, 4 parts of poria, 5 parts of rose and 3 parts of mint. 10 times the amount of water is added and extracted at 90°C for 2 hours. The extract is evaporated and concentrated until it sticks to the wall and then freeze-dried. The extract is collected, freeze-dried, crushed and passed through an 80-mesh sieve for later use.

[0040] Example 2 The present embodiment provides a compound daylily concentrated juice with the effect of improving mood, which is composed of the following components: 7 parts of daylily, 6 parts of dried tangerine peel, 8 parts of poria, 10 parts of rose and 3 parts of mint. 10 times the amount of water is added and extracted at 90° C. for 2 hours. The extract is evaporated and concentrated until it sticks to the wall and then freeze-dried. The extract is collected, freeze-dried, crushed and passed through an 80-mesh sieve for later use.

[0041] Example 3 The present embodiment provides a compound daylily concentrated juice with the effect of improving mood, which is composed of the following components: 8 parts of daylily, 9 parts of dried tangerine peel, 1 part of poria, 4 parts of rose and 3 parts of mint. 10 times the amount of water is added and extracted at 90°C for 2 hours. The extract is evaporated and concentrated until it sticks to the wall and then freeze-dried. The extract is collected, freeze-dried, crushed and passed through an 80-mesh sieve for later use.

[0042] Example 4 The present embodiment provides a compound daylily concentrated juice with the effect of improving mood, which is composed of the following components: 9 parts of daylily, 1 part of dried tangerine peel, 5 parts of poria, 9 parts of rose and 3 parts of mint. 10 times the amount of water is added and extracted at 90°C for 2 hours. The extract is evaporated and concentrated until it sticks to the wall and then freeze-dried. The extract is collected, freeze-dried, crushed and passed through an 80-mesh sieve for later use.

[0043] Example 5 The present embodiment provides a compound daylily concentrated juice with the effect of improving mood, which is composed of the following components: 10 parts of daylily, 4 parts of dried tangerine peel, 9 parts of poria, 3 parts of rose and 3 parts of mint. 10 times the amount of water is added and extracted at 90°C for 2 hours. The extract is evaporated and concentrated until it sticks to the wall and then freeze-dried. The extract is collected, freeze-dried, crushed and passed through an 80-mesh sieve for later use.

[0044] Example 6 The present embodiment provides a compound daylily concentrated juice with the effect of improving mood, which is composed of the following components: 11 parts of daylily, 7 parts of dried tangerine peel, 2 parts of poria, 8 parts of rose and 3 parts of mint. 10 times the amount of water is added and extracted at 90° C. for 2 hours. The extract is evaporated and concentrated until it sticks to the wall and then freeze-dried. The extract is collected, freeze-dried, crushed and passed through an 80-mesh sieve for later use.

[0045] Example 7 The present embodiment provides a compound daylily concentrated juice with the effect of improving mood, which is composed of the following components: 12 parts of daylily, 10 parts of dried tangerine peel, 6 parts of poria, 2 parts of rose and 3 parts of mint. 10 times the amount of water is added and extracted at 90°C for 2 hours. The extract is evaporated and concentrated until it sticks to the wall and then freeze-dried. The extract is collected, freeze-dried, crushed and passed through an 80-mesh sieve for later use.

[0046] Example 8 The present embodiment provides a compound daylily concentrated juice with the effect of improving mood, which is composed of the following components: 13 parts of daylily, 2 parts of dried tangerine peel, 10 parts of poria cocos, 7 parts of rose and 3 parts of mint. 10 times the amount of water is added and extracted at 90° C. for 2 hours. The extract is evaporated and concentrated until it sticks to the wall and then freeze-dried. The extract is collected, freeze-dried, crushed and passed through an 80-mesh sieve for later use.

[0047] Example 9 The present embodiment provides a compound daylily concentrated juice with the effect of improving mood, which is composed of the following components: 14 parts of daylily, 5 parts of dried tangerine peel, 3 parts of poria, 1 part of rose and 3 parts of mint. 10 times the amount of water is added and extracted at 90° C. for 2 hours. The extract is evaporated and concentrated until it sticks to the wall and then freeze-dried. The extract is collected, freeze-dried, crushed and passed through an 80-mesh sieve for later use.

[0048] Example 10 The present embodiment provides a compound daylily concentrated juice with the effect of improving mood, which is composed of the following components: 15 parts of daylily, 8 parts of dried tangerine peel, 7 parts of poria, 6 parts of rose and 3 parts of mint. 10 times the amount of water is added and extracted at 90° C. for 2 hours. The extract is evaporated and concentrated until it sticks to the wall and then freeze-dried. The extract is collected, freeze-dried, crushed and passed through an 80-mesh sieve for later use.

[0049] Experimental example Evaluation tests were conducted on the activity of the compound daylily concentrated juice provided in each example in improving depression: 1. Test methods 1. Model Establishment: Wild-type AB zebrafish (6 months old) were selected and divided into a blank control group, a reserpine (20 μg / mL) model group, a reserpine and fluoxetine hydrochloride (10 μg / mL) positive drug group, and a treatment group with varying ratios of reserpine and compound daylily extract. Except for the blank control group, all other groups were immersed in reserpine solution for 20 minutes daily for 7 consecutive days. Behavioral experiments were then conducted in a novel tank and open field. After the behavioral experiments, all fish brains were dissected, and ELISA was used to measure the levels of 5-HT, DA, TNF-α, and IL-1β in each group.

[0050] 2. Administration: After the treatment with reserpine, the daylily compound treatment groups with different concentrations were added with solutions of corresponding concentrations and soaked for 7 consecutive days.

[0051] 3. Testing: After establishing the model and treatment, new water tank and open field behavioral experiments were conducted. The behavioral results are shown in Figure 1 After the behavioral experiment, all fish brains were dissected, and the levels of 5-HT, DA, TNF-α, and IL-1β in zebrafish of each group were detected by ELISA. The content of neurotransmitters and inflammatory factors is shown in Table 1.

[0052] 4. Extraction process optimization (1) Determination of total flavonoid content The content was determined by the aluminum nitrate-sodium nitrite colorimetric method.

[0053] (2) Orthogonal experiment Accurately weigh the powder of each medicinal material, place the powder in a reflux extraction device, and perform extraction, concentration, dissolution, and content determination one by one according to orthogonal table 1.

[0054] Table 1 Factor levels

[0055] (3) Ointment yield Dry the evaporating dish to constant weight, weigh the evaporating dish, accurately pipette 50 mL of the extract into the evaporating dish, evaporate to dryness in a water bath, and then dry in a 105°C oven to constant weight, i.e., the difference between two consecutive weighing results at an interval of 1 h should not exceed 3 mg. Record the weight at this time and calculate the paste yield.

[0056] 5. Concentration process and concentration endpoint determination Preparation process of compound daylily concentrated juice with different concentrations: 1 Brix compound daylily extract was used as raw material and concentrated by rotary evaporation (temperature 60℃, rotation speed 100 r / min) to obtain compound daylily concentrated juice with different concentration levels (3 Brix~24Brix, interval of 3 Brix), which was refrigerated at 4℃ for later use.

[0057] The viscosity of compound daylily concentrated juice with different concentrations (3 Brix-24 Brix, with a measurement interval of 3 Brix) was measured. A curve diagram showing the relationship between the concentration of compound daylily concentrated juice and its corresponding viscosity change was constructed. The viscosity mutation point during the concentration process of compound daylily concentrated juice was analyzed, and the concentration corresponding to this point was the concentration end point.

[0058] 6. Determination of total flavonoids and total phenols content Compound daylily concentrated juices with different concentrations (3 Brix-24 Brix, with a measurement interval of 3 Brix) were uniformly adjusted to a concentration of 1 Brix, and the contents of total flavonoids and total phenols were determined. The changes in the main components of compound daylily concentrated juices with different concentrations were studied, and the relationship between the components of compound daylily concentrated juice and the concentration degree was analyzed.

[0059] 7. Determination of physical properties and nutritional components (1) pH determination First, check that the glass electrode is transparent and free of cracks and that there is sufficient liquid in the reference electrode. Then, turn on the instrument and preheat it for 3-5 minutes to ensure that the circuit is stable. Use the three-point calibration method, using pH 4.01, 7.00, and 9.21 standard buffer solutions (the temperature is the same as the test solution) in sequence. Follow the calibration sequence of neutral (pH 7.00) first, then acidic (pH 4.01) or alkaline (pH 9.21) buffer solutions according to the acidity or alkalinity of the test solution. The calibration slope should be within the range of 95%-105%. The test solution should be adjusted to 25±0.5℃ (temperature compensation error ≤0.1) in a constant temperature water bath. Rinse the electrode with pure water and dry it with filter paper, then immerse it in the test solution and let it stand for 1-2 minutes until the reading stabilizes. Finally, measure twice in parallel. If the error of the two results is ≤0.1, take the average value as the final pH value.

[0060] (2) Stability determination 20 mL of compound daylily concentrate juice was placed into the LUMiSizer and the sedimentation rate was measured. The parameters were: scanning intensity 1x, scanning temperature 25°C, rotation speed 4000 rpm, scanning for 1 hour, scanning every 30 seconds, and a total of 120 scans.

[0061] (3) Nutritional composition determination The nutritional components of the compound daylily concentrated juice were determined according to the methods and standards in Table 2.

[0062] Table 2 Nutritional component testing methods and standards

[0063] 8. Establishment of mouse depression model The three doses of compound daylily concentrate (high, medium, and low doses, respectively) were set at 2, 1, and 0.5 times the clinical dose. Using a conversion factor of 12.33, the dosing concentrations for compound daylily concentrate (H, M, and L) were 4.8, 2.4, and 1.2 g / kg / day, respectively. The prescribed dosage of fluoxetine hydrochloride is 20 mg, equivalent to a dose of 4.2 mg / kg in mice.

[0064] Preparation of fluoxetine hydrochloride solution: Accurately weigh 10 mg of fluoxetine hydrochloride powder, add appropriate amount of distilled water to dissolve it into 0.42 mg / mL solution, and place it in a 4℃ refrigerator until used.

[0065] Preparation of lipopolysaccharide (LPS): Accurately weigh 3 mg of LPS powder and dissolve it in an appropriate amount of physiological saline to make a 1 mg / mL solution. Place it in a refrigerator at 4°C until use.

[0066] After a 7-day acclimation period, mice were randomly divided into six groups using a random number table: control group, model group, positive drug group, compound daylily concentrated juice-H, compound daylily concentrated juice-M, and compound daylily concentrated juice-L groups. Drugs were administered by gavage daily at 9:00 AM. The control and model groups received an equal volume of distilled water. Except for the control group, mice in all other groups were restrained in a homemade modified centrifuge tube for 6 hours daily for 21 days, ensuring they did not asphyxiate. On day 22, model animals received an intraperitoneal injection of LPS.

[0067] 9. Mouse behavioral evaluation (1) Weight The animals were weighed once on the day of grouping as day 0, and then every 5 days.

[0068] (2) Tail suspension test (TST) Before the experiment begins, the mouse is fixed with tape and hung on the tail suspension box, keeping the mouse's body suspended in the air and unable to touch the left and right sides and the bottom of the box. The time the mouse remains motionless within 3 minutes is recorded.

[0069] (3) Open field test (OFT) Before the experiment, mice were placed in the central compartment of an open-field box with dimensions of 50 cm (length) × 50 cm (width) × 40 cm (height) to adapt to the box environment for 1 min. The activity status of mice in each group was then detected and analyzed using a behavioral analysis system. The recording time was 3 min, and the observation indicators were the movement distance (cm), stationary time (s), and center movement time (s) of the mice in the open-field box. After each test, the test area was cleaned with 75% alcohol to avoid interference of odor on the experimental results.

[0070] (4) Forced swim test (FST) Before the experiment began, mice were placed in an opaque plastic cylinder with a bottom diameter of 21 cm and a height of 46 cm to acclimate for 1 minute. After acclimatization, the mice's immobility time was recorded for 5 minutes. During the experiment, the water temperature was maintained at 23-25°C and the depth was 30 cm. After the experiment, the mice were removed, dried, and placed in cages.

[0071] 10. Sample Collection After the behavioral experiment, blood was collected from the eyeballs of all mice, precooled to 4°C, centrifuged at 3000 rpm for 15 min, and the upper serum was aspirated with a pipette and stored at -80°C for later use.

[0072] After blood was collected from the eyeballs, 3 mice in each group were selected to separate the whole brain tissue, which was fixed in 4% paraformaldehyde and set aside for use.

[0073] The remaining seven mice in each group were decapitated. The skin was cut open from both sides of the ears toward the front. The skin was peeled back, and the skull was carefully cut and peeled off with small rongeurs, taking care not to damage the brain tissue. After peeling, the nerve fibers connecting to the skull base were severed with ophthalmic forceps, and the brain tissue was removed. The brain tissue was placed on an ice tray, and the hippocampus was removed according to the mouse brain stereotaxic map. It was then immersed in liquid nitrogen and frozen, and then stored in a -80°C ultra-low temperature freezer.

[0074] 11. Hippocampal pathology examination The fixed brain tissue was removed, embedded, and sectioned. The sections were then dewaxed using xylene and graded alcohol. Next, the sections were stained with hematoxylin, differentiated with hydrochloric acid and ethanol, and blued with 1% ammonia. Next, the sections were rinsed with tap water and stained with eosin. Finally, the sections were dehydrated using graded ethanol and xylene, and then mounted with neutral glue. Once the slides were dry, they were examined under a microscope.

[0075] 12. Detection of neurotransmitters in the hippocampus and serum inflammatory factors The isolated hippocampal tissue was weighed and thoroughly homogenized with 9x PBS at a weight (g): volume (ml) ratio of 1:9 at 4°C using a homogenizer. The mixture was centrifuged for approximately 10 minutes (4°C, 12,000 rpm). The supernatant was carefully collected and aliquoted. BDNF and 5-HT levels in the mouse hippocampus were quantitatively measured using a mouse enzyme-linked immunosorbent assay (ELISA) kit.

[0076] Mouse serum samples were treated with the LEGENDplex™ Mouse Inflammation Panel (13-plex) with V-bottomPlate kit, and cytokine levels in the samples were quantified by flow cytometry.

[0077] 13. Western blot analysis of hippocampus Mouse hippocampal tissue was homogenized in RIPA lysis buffer, and protein concentration was determined using a BCA protein quantification kit. Equal amounts of protein were mixed with 5X protein loading buffer, separated by electrophoresis, and then transferred to a 0.45 μM PPVDF membrane. The membrane was blocked with 5% milk in TBST for 30 minutes at room temperature. The primary antibody was diluted in 5% skim milk in TBST and incubated overnight at 4°C. The membrane was washed three times with TBST on a shaker at room temperature for 5 minutes each. The secondary antibody was diluted 5000-fold in TBST and incubated at room temperature for 30 minutes. The membrane was then washed three times with TBST on a shaker at room temperature for 5 minutes each. Protein signal was detected using ECL reagent.

[0078] 2. Experimental Results 1. New diving experiment Zebrafish activity trajectory Figure 1 A. Compared with the blank control group, the time spent exploring the upper area of the water tank in the reserpine model group was significantly shorter ( P <0.001); compared with the reserpine model group, the treatment group spent significantly more time exploring the upper area of the sink ( P <0.001)( Figure 1 B). Compared with the blank control group, the total swimming distance of the reserpine model group was significantly decreased ( P <0.001). Compared with the reserpine model group, the total swimming distance of the treatment group increased significantly ( P <0.001)( Figure 1 C). Compared with the blank group, the movement speed of zebrafish in the model group was significantly reduced; compared with the model group, the movement speed of zebrafish in the treatment group was significantly increased ( P <0.001)( Figure 1 D). The new diving experiment showed that different proportions of compound daylily concentrated juice could improve the activity of zebrafish.

[0079] 2. Open field experiment Zebrafish activity trajectory Figure 2 A. Compared with the blank control group, the total swimming distance of the reserpine model group was significantly decreased ( P <0.001), indicating that reserpine affected the activity of zebrafish; compared with the reserpine model group, the total swimming distance of the treatment group increased significantly ( P <0.01, P <0.001)( Figure 2 B). Compared with the blank control group, the swimming speed of the reserpine model group decreased significantly ( P <0.001). Compared with the reserpine model group, the swimming speed of the treatment group increased significantly ( P <0.01, P <0.001)( Figure 2 C). The open field test showed that different proportions of compound daylily concentrated juice could improve the activity of zebrafish.

[0080] 3. Biochemical indicators Compared with the blank control group, the levels of inflammatory factors TNF-α and IL-1β in the reserpine model group were significantly increased, while those in the treatment group were significantly decreased. Compared with the blank control group, the levels of 5-HT and DA in the reserpine model group were significantly decreased, while those in the treatment group were significantly increased (Table 3).

[0081] Table 3 Zebrafish TNF-α, IL-1β, 5-HT and DA test results

[0082] Note: Compared with the control group, # P <0.05, ## P <0.01, ### P <0.001; compared with the model group, .

[0083] 4. The uniform design method was used to investigate the effect of compound daylily concentrate on the levels of TNF-α, IL-1β, 5-HT, and DA in the zebrafish brain, and to screen its optimal compatibility dose. The levels of TNF-α, IL-1β, 5-HT, and DA in the zebrafish brain were used as dependent variables Y1, Y2, Y3, and Y4, respectively, and daylily, tangerine peel, Poria cocos, and rose were used as independent variables x1, x2, x3, and x4. Linear regression analysis using the DPS data processing system revealed a P > 0.05, indicating no statistical significance. Therefore, a quadratic polynomial stepwise regression analysis was used, and the fitting equation was shown in Table 4: Table 4 Quadratic polynomial regression equation for uniform design experiment Since R1>R2>R3>R4, and the amount of mint is 3g, the optimal ratio is finally selected as daylily: tangerine peel: poria: rose: mint = 13:5:10:10:3.

[0084] 5. Extraction process optimization The results of the orthogonal experiment are shown in Table 5.

[0085] Table 5 Orthogonal experiment results

[0086] Comparing the range of each factor, it can be seen that factor A has the greatest impact on the test results, followed by B→C. Therefore, the order of influence of each factor on flavonoid extraction efficiency is A>B>C. The optimal extraction process obtained through orthogonal experiment is A2B3C2, that is, the material-liquid ratio is 1:10, and extraction is carried out at 90℃ for 2 h.

[0087] 6. Determination of concentration endpoint During the production of concentrated juice, from the production level, the viscosity of the concentrated material changes greatly, which requires high precision of the equipment; from the product level, when the concentration is low, it is easy to spoil, which increases the storage cost; when the concentration is too high, the fluidity is poor, which makes transportation difficult; therefore, determining the concentration end point of the concentrated juice is of great significance to actual industrial production. Figure 3As shown in the figure, the viscosity of the compound daylily concentrated juice increases with increasing concentration. At concentrations between 3 Brix and 15 Brix, the viscosity changes gently from 5.31 mPa·s to 11.50 mPa·s. As the concentration increases from 15 Brix to 18 Brix, the viscosity increases dramatically, from 11.50 to 31.05 mPa·s. In conclusion, 15 Brix is the point at which the viscosity of the compound daylily concentrated juice suddenly increases, thus defining it as the concentration endpoint.

[0088] 7. Determination of total flavonoids and total phenols The total flavonoids content in the compound daylily concentrated juice at different concentration levels Figure 4 As shown in the results, the total flavonoid content decreased with increasing concentration during the evaporation process. Throughout the entire concentration process, the total flavonoid yield from vacuum heat concentration decreased from 1.56% to 1.22%, a decrease of 0.34%. This decrease in yield may be due to changes in the flavonoid structure in the compound daylily concentrated juice caused by prolonged heating.

[0089] Changes in total phenol content in compound daylily concentrated juice Figure 5 As shown in the data, the total phenol content in the concentrated compound daylily juice decreases with the increase of concentration degree, but the total phenol content changes relatively slowly in the initial stage of concentration. As the concentration time increases, the total phenol content decreases rapidly, from the initial yield of 1.4873% to 1.21%. This phenomenon may be caused by the thermal instability of phenol, which decomposes under long-term heat, resulting in a decrease in content.

[0090] 8. Determination of physical properties and nutritional components Changes in pH value of compound daylily concentrated juice Figure 6 As shown in Figure A, the pH value of the compound daylily concentrated juice decreases with the increase of concentration degree, indicating that the pH value decreases as the heating time increases. The influence of the sedimentation rate of the compound daylily concentrated juice at different concentration levels was measured using a fully automatic stability analyzer. Figure 6 As shown in B, the higher the concentration degree, the lower the sedimentation rate.

[0091] The nutritional components of the compound daylily concentrated juice are shown in Table 6.

[0092] Table 6 Nutritional component testing methods and standards

[0093] 9. Evaluation results of depressive-like behavior in mice The weight changes of mice Figure 7 As shown in A, CRS combined with LPS significantly changed the weight change trend of mice, and the weight gain slowed down ( P<0.01), and the compound daylily concentrated juice reversed this trend after treatment. Although it was not significant, there was a clear trend of change, among which the high-dose group and the low-dose group had more significant effects. The TST results of mice showed that the compound daylily concentrated juice significantly reversed the increase in immobility time caused by CRS combined with LPS ( P <0.001) Figure 7 B. The trajectory of mouse OFT activity Figure 7 As shown in C, CRS combined with LPS significantly reduced the movement distance and movement speed of mice in the open field ( P <0.01, P <0.001), significantly increased the immobility time of mice in the open field ( P <0.01, P <0.001), and the movement distance and movement speed of mice were significantly increased after treatment with compound daylily ( P <0.01, P <0.001), reduced the immobility time of mice ( P <0.01, P <0.001) ( Figure 7 , DF). The FST results of mice showed that the immobility time of mice in the CRS combined with LPS group was significantly longer than that in the blank group ( P <0.01, P <0.001), compound daylily treatment reduced the immobility time of mice in a dose-dependent manner, among which the high-dose group, the medium-dose group and the model group showed extremely significant differences ( P <0.001) ( Figure 7 G). Overall, the compound daylily treatment can effectively reverse the depressive symptoms in mice induced by CRS combined with LPS.

[0094] 10. H&E staining results The results are as follows Figure 8 The brain tissue structure of the blank group was normal, the neurons in the CA1 region were arranged neatly and orderly, the cell body outlines were clear, the shape was regular, and no obvious degeneration was observed; the brain tissue structure of the model group was abnormal, some neurons in the CA1 region were degenerated, the cell body shape was irregular, wrinkled and darkly stained; there was no significant difference in the CA1 region of the hippocampus of mice in the positive drug, low, medium and high dose groups compared with the blank group, the cell bodies were large, plump, round and regular in shape, the cells were arranged neatly and the outline boundaries were clear.

[0095] The neurons in the CA3 region of the blank group were arranged neatly and orderly, with clear cell body outlines and regular shapes, and no obvious degeneration was observed; the brain tissue structure of the model group was abnormal, and the CA3 neurons were loosely arranged and the gaps were enlarged while degeneration was observed; the number of neurons in the CA3 region of the hippocampus of mice in the positive drug, low, medium, and high dose groups increased, and the arrangement was relatively neat and regular, with clear edges and multiple cell layers.

[0096] The neurons in the DG region of the blank group were arranged neatly and orderly, with clear cell body outlines and regular shapes, and no obvious degeneration was observed; the brain tissue structure of the model group was abnormal, with disordered arrangement of cells in the DG region and widened spacing between cells, changes in cell morphology, condensation of cell nuclei, and darker staining, and a large number of neuronal degeneration were observed; the cells in the DG region of the hippocampus of mice in the positive drug, low-, medium-, and high-dose groups were densely arranged with normal morphology, occasionally with dark cells, and no obvious abnormalities were observed compared with the blank group.

[0097] 11. Results of hippocampal neurotransmitter and serum inflammatory factor levels The levels of 5-HT, BDNF and inflammatory factors in the hippocampus of mice Figure 9 As shown. After CRS combined with LPS modeling, the levels of related pro-inflammatory factors (TNF-α, IL-6 and IL-1β) in serum were significantly increased ( P <0.001), while the levels of 5-HT and BDNF in the hippocampus were significantly increased ( P <0.01). After treatment with compound daylily concentrated juice, the levels of TNF-α, IL-6, and IL-1β in serum decreased, and the inhibitory effect reached a very significant level ( P <0.001). After treatment with different concentrations of compound daylily concentrated juice, the levels of 5-HT and BDNF in the hippocampus were increased, and there were significant differences between the high-dose group, the medium-dose group and the model group ( P <0.01, P <0.001). Although the difference in the low-dose group was not significant, there was a trend toward increased BDNF levels. These results suggest that compound daylily concentrate has a significant inhibitory effect on the release of proinflammatory cytokines in the hippocampus induced by CRS combined with LPS, and can also promote increases in 5-HT and BDNF levels.

[0098] 12. Western blot analysis results of mouse hippocampus Protein test results such as Figure 10 As shown. The results showed that compared with the control group, the phosphorylation expression levels of PI3K and AKT in the hippocampus of mice induced by CRS combined with LPS were downregulated, while treatment with compound daylily concentrated juice reversed the abnormalities of these two indicators caused by CRS combined with LPS. BDNF is a downstream molecule of the PI3K / AKT signaling pathway. Its downregulation is closely related to apoptosis, and the development of depression is always accompanied by cell apoptosis. Western blot was used to detect the expression levels of BDNF and apoptosis-related proteins in the hippocampus. The results showed that the protein expression (or phosphorylation) level of BDNF was reduced in the hippocampus of CRS mice, and this phenomenon could be reversed by treatment with compound daylily concentrated juice.

[0099] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A compound daylily concentrated juice having the effect of improving mood, characterized in that: The invention comprises the following components by weight: 5-15 parts of daylily powder, 1-10 parts of tangerine peel powder, 1-10 parts of tuckahoe powder, 1-10 parts of rose powder and 3 parts of mint powder.

2. The compound daylily concentrated juice according to claim 1, characterized in that: The invention comprises the following components in parts by weight: 13 parts of daylily powder, 5 parts of tangerine peel powder, 10 parts of tuckahoe powder, 10 parts of rose powder and 3 parts of mint powder.

3. The compound daylily concentrated juice according to claim 1, characterized in that: The active ingredients of the compound daylily concentrated juice include flavonoids and phenolic acid substances.

4. The compound daylily concentrated juice according to claim 3, characterized in that: The content of the phenolic acid substances is 1.2-1.49%, and the content of the flavonoid substances is 1.2-1.56%.

5. A preparation having the effect of improving mood, characterized in that: The invention comprises the compound daylily concentrated juice according to any one of claims 1 to 4 and pharmaceutically acceptable excipients.

6. The preparation according to claim 5, characterized in that The preparation is a decoction.

7. The method for preparing the compound daylily concentrated juice according to any one of claims 1 to 4, characterized in that: include: Each component is sieved separately, then mixed evenly according to the proportion, mixed with water at a solid-liquid ratio of 1: (10-20), placed in a reflux extraction device at 80-90℃ for 1.5-2h; then the obtained extract is filtered and centrifuged, and concentrated to 10-20 Brix using a rotary evaporator.

8. The method according to claim 7, characterized in that The product was mixed with water at a solid-liquid ratio of 1:10 and placed in a reflux extraction device for extraction at 90°C for 2 hours.

9. The method according to claim 7 or 8, characterized in that The method further comprises: after concentration, detecting the total flavonoid content in the compound daylily concentrated juice to be 1.2-1.56%, the total phenolic acid content to be 1.2-1.49%, and the sedimentation rate to be 0.1-0.25%.

10. Use of the compound daylily concentrated juice according to any one of claims 1 to 4 in the preparation of a functional product for improving mood.

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