Preparation method and application of hemerocallis citrina baroni mixed polypeptide oral liquid for relieving depression

Abstract

The invention relates to a preparation method and application of a hemerocallis citrina mixed polypeptide oral liquid for relieving depression, the protein content of hemerocallis citrina is relatively high, 100g of dried hemerocallis citrina contains about 20g of protein, and in addition, the hemerocallis citrina is rich in amino acid composition, is an excellent plant protein polypeptide source, and can be used for preparing the oral liquid. The hemerocallis citrina mixed active polypeptide is prepared by obtaining hemerocallis citrina crude protein through an alkali extraction and acid precipitation method and then carrying out an enzymolysis process by combining trypsin, pepsin and intestinal protease, and experimental results show that the hemerocallis citrina mixed active polypeptide oral liquid has a good effect of preventing and treating depression, and the hemerocallis citrina as a common vegetable food has a wide application prospect. The tea is suitable for people of all ages and has good safety. The traditional Chinese medicine composition has a very good anti-depression effect, the edible value of the day lily can be improved, the income of farmers is increased, and a contribution is made to develop a novel agricultural industry.

Description

TECHNICAL FIELD

[0001] The application relates to a preparation method and application of a day lily mixed polypeptide oral liquid for relieving depression, and belongs to the field of bioactive small molecule peptides. BACKGROUND

[0002] Depression is a common mental illness characterized by long-term low mood, irritability, and decreased attention, and is the result of the combined effects of social, psychological, and biological factors. It is estimated that 5% of adults worldwide suffer from depression, and the incidence in women is about 50% higher than that in men. Globally, more than 10% of pregnant women and women immediately after childbirth suffer from depression, which is an important inducement to suicide. The World Health Organization has repeatedly discussed mental health issues and approved the extension of the Comprehensive Mental Health Action Plan to 2030 in 2019 to promote and improve global mental health. Methods for treating depression include psychological treatment and drug treatment or a combination of the two. There are various types of antidepressants commonly used in clinical practice, such as monoamine oxidase inhibitors and selective serotonin reuptake inhibitors, which improve the mood and behavior of individuals by affecting neurotransmitters in the brain. However, long-term use of these antidepressants can produce a series of side effects, such as headache, nausea, constipation, and sexual dysfunction. More and more studies have shown that various natural organic compounds in plants, such as polypeptides, polysaccharides, and alkaloids, can relieve depressive symptoms while reducing side effects, which is of great significance to the improvement of the quality of life of patients with depression.

[0003] Day lily is a plant of the genus Hemerocallis in the Liliaceae family, and the unopened and newly opened flower buds are used as medicine, have medicinal value and health care efficacy, and are mainly produced in Gansu, Hunan, Shanxi, Sichuan, Hubei and other places. It contains various chemical components, mainly including flavonoids, volatile oils, polyphenols, anthraquinones, alkaloids and other types, among which flavonoids and their glycosides have a higher content and are the main active substances, and have the effects of clearing heat and dampness, relieving chest and relieving depression, and cooling blood and detoxifying. Modern studies have shown that day lily has the effects of relieving depression, resisting tumors, calming sleep, resisting inflammation and inhibiting bacteria.

[0004] Proteins are biological macromolecules composed of polypeptide chains, which are absorbed by the human body after digestion in the body and exert their nutritional value and application value. At present, there is no report on the activity of polypeptides produced by the digestion of day lily protein. The application develops day lily mixed polypeptide oral liquid by preparing the day lily mixed polypeptide oral liquid and using a depression mouse model experiment to develop day lily mixed polypeptides with antidepressant activity. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application aims to provide a preparation method and application of a cymbidium hybrid polypeptide oral liquid for relieving depression, so as to solve the problems in the foregoing background art.

[0006] In order to achieve the above-mentioned purpose, the present application is implemented by the following technical scheme: a cymbidium polypeptide oral liquid for relieving depression, which is an oral liquid containing a hybrid polypeptide and has an anti-depression effect.

[0007] Preferably, the raw material of the cymbidium polypeptide oral liquid for relieving depression is dried cymbidium.

[0008] A preparation method of a cymbidium hybrid polypeptide oral liquid for relieving depression, the method comprising the following steps,

[0009] (1) taking dried cymbidium, grinding into powder with a high-speed grinder, and fully dissolving with distilled water;

[0010] (2) adjusting pH with NaOH, magnetic stirring at 55 DEG C, centrifugation, collecting supernatant, adjusting pH with HCl, standing, centrifugation, collecting precipitate, adjusting pH of the precipitate to neutral, washing with distilled water, and freeze-drying to obtain cymbidium crude protein;

[0011] (3) dissolving the cymbidium crude protein in deionized water, magnetic stirring at 55 DEG C for 10 min, adjusting pH to a suitable range, adding protease, mixing, and water bath enzymolysis; after the enzymolysis is completed, boiling water bath is used to terminate the reaction, cooling to room temperature, centrifugation of the enzymolysis solution, collecting supernatant, freeze-drying, and obtaining cymbidium hybrid polypeptide freeze-dried powder;

[0012] (4) mixing the hybrid polypeptide freeze-dried powder, water, and auxiliary materials honey, citric acid, and potassium sorbate according to a proportion, homogenizing, pasteurizing, and aseptically filling.

[0013] Further, the specific experimental conditions of the step (1) are as follows: 10000-12000 rpm, grinding for 8-10 min, and the ratio of cymbidium powder to distilled water is 1:5-6.

[0014] Further, the specific experimental conditions of the step (2) are as follows: adjusting pH to 10 with NaOH, magnetic stirring for 5 h, centrifugation at 10000 r / min for 10 min, adjusting pH to 4 with HCl, and standing for 1 h.

[0015] Further, the specific experimental conditions of the step (3) are as follows: the ratio of day lily crude protein and deionized water is 1:10, magnetic stirring is performed for 10 min, the pH is adjusted to 2, a pepsin solution with a mass concentration of 5% is added, mixing is performed, the enzyme hydrolysis is performed in a constant temperature oscillator at 37 DEG C water bath for 2 h, the pH is adjusted to 7.5, a trypsin with a mass concentration of 5% is added, mixing is performed, the enzyme hydrolysis is performed at 37 DEG C water bath for 4 h, the pH is adjusted to 8, an enterokinase with a mass concentration of 5% is added, mixing is performed, the enzyme hydrolysis is performed at 37 DEG C water bath for 2 h, the reaction is terminated by boiling in 100 DEG C water bath for 10 min, and the enzyme hydrolysate is centrifuged at 10000 r / min at 4 DEG C for 10 min.

[0016] Further, the enzymes in the step (3) are pepsin, trypsin and enterokinase, the efficiency of the pepsin is 1:(25000-35000), the enzyme activity of the trypsin is greater than 250 USPu / mg, and the efficiency of the enterokinase is 1:(30000-35000).

[0017] Further, the specific experimental conditions of the step (4) are as follows: the polypeptide, water, and the auxiliary materials honey, citric acid and potassium sorbate are mixed according to the ratio of 30%:60%:8%:1%:1%, and the pasteurization condition is 75 DEG C for 15 s.

[0018] Further, the application of the day lily anti-depression polypeptide oral liquid or the day lily oral liquid prepared by the preparation method of the day lily anti-depression polypeptide oral liquid in an anti-depression drug, food or health product.

[0019] The application has the following advantages: the anti-depression polypeptide used in the preparation method and application of the day lily anti-depression polypeptide oral liquid is derived from day lily, and the day lily anti-depression polypeptide oral liquid has a very good anti-depression effect, so that the anti-depression polypeptide oral liquid prepared by the application has the advantages of small side effects, accurate curative effect and high cost performance, and the application can improve the overall value of the vegetable planting industry and increase the income of farmers because day lily is a common crop in the vegetable planting industry. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Picture of the influence of the day lily anti-depression polypeptide oral liquid prepared by the application on the SPT of CUMS mice;

[0021] Figure 2 Picture of the influence of the day lily anti-depression polypeptide oral liquid prepared by the application on the OFT total distance of CUMS mice;

[0022] Figure 3 Picture of the influence of the day lily anti-depression polypeptide oral liquid prepared by the application on the OFT central activity time of CUMS mice;

[0023] Figure 4 Picture of the effect of the daylily mixed polypeptide oral solution prepared in the application on the TST of CUMS mice;

[0024] Figure 5 Picture of the effect of the daylily mixed polypeptide oral solution prepared in the application on the FST of CUMS mice;

[0025] Figure 6 Picture of the effect of the daylily mixed polypeptide oral solution prepared in the application on the body weight of CUMS mice;

[0026] Figure 7 Picture of the effect of the daylily mixed polypeptide oral solution prepared in the application on the concentrations of inflammatory factors and brain-derived neurotrophic factors of CUMS mice. DETAILED DESCRIPTION

[0027] In order to make the technical means, creative features, purposes and effects achieved by the application easy to understand, the application will be further described below in combination with specific embodiments.

[0028] Embodiment 1: A daylily antidepressant mixed polypeptide oral solution and a preparation method thereof, comprising the following steps:

[0029] (1) Take dried daylily, grind into powder with a high-speed grinder, and dissolve in distilled water;

[0030] (2) Adjust the pH with NaOH, magnetically stir at 55°C, centrifuge, collect the supernatant, adjust the pH with HCl, stand, centrifuge, collect the precipitate, adjust the pH of the precipitate to neutral, wash with distilled water, and freeze-dry to obtain daylily crude protein;

[0031] (3) Dissolve the daylily crude protein in deionized water, magnetically stir at 55°C for 10 min, adjust the pH to a suitable range, add protease, mix well, and perform enzyme hydrolysis in a water bath; after enzyme hydrolysis, terminate the reaction in a boiling water bath, cool to room temperature, centrifuge the enzyme hydrolysate, collect the supernatant, freeze-dry, and obtain daylily mixed polypeptide freeze-dried powder;

[0032] (4) The specific experimental conditions are as follows: the daylily mixed polypeptide, water, and the auxiliary materials honey, citric acid, and potassium sorbate are mixed in a ratio of 30%:60%:8%:1%:1%, and the pasteurization condition is 75°C for 15 seconds.

[0033] In order to make the purposes and advantages of the application more clear and explicit, the application will be further described in detail below in combination with animal experiments. It should be understood that the specific experiments described herein are only used to explain the application and do not limit the application.

[0034] Unless otherwise specified, the reagents used in the following experiments are all from commercial sources, and the operation methods are all existing conventional operation methods.

[0035] Example 2: Evaluation of the antidepressant effect of a Daylily mixed polypeptide oral solution for relieving depression:

[0036] 1. Animal grouping, establishment of chronic unpredictable depression model (CUMS), and drug treatment: 30 male C57BL / 6 mice were randomly divided into normal blank control group (CON), chronic stress model group (CUMS), and Daylily mixed polypeptide (DMP) treatment group (300 mg / kg). After 7 days of environmental adaptation, all groups except the CON blank control group were induced to establish a mouse depression model using daytime stressors and nighttime stressors for 2 weeks. The 11 stressors were randomly assigned during the establishment of the CUMS model, with 2 stressors per day, at least 1 day apart for the same stressor, and each stressor appearing at least 3 times. The stressors mainly included: fasting (24 h), water deprivation (24 h), tail clamping (2 min), shaking the cage (5 min), damp bedding (24 h), 45° inclined cage (24 h), day-night reversal (24 h), restraint (2 h), bright light shining into the eyes (5 min), 4°C cold water swimming, and 45°C hot water swimming. To facilitate the subsequent behavioral index measurement, all mice were fasted and deprived of water on the last day of modeling, i.e., the 14th day of intervention. While the CUMS model was being constructed, the DMP group was given a dose of 300 mg / kg of Daylily mixed polypeptide by gavage every day.

[0037] 2. Experimental methods

[0038] (1) Sucrose preference test (SPT): On the 13th day of the experiment, all mice were fasted and deprived of water for 12 h, and were uniformly housed in single cages. Each mouse was given a bottle of 1% sucrose solution and a bottle of pure water, and the fast was continued. The positions of the sugar water bottle and the pure water bottle were exchanged midway, and the sucrose consumption and the pure water consumption were recorded within 12 h. The sugar water consumption rate was calculated (sugar water consumption rate = sugar water consumption / (sugar water consumption + water consumption) x 100%).

[0039] (2) Open field test (OFT): The test was conducted on the 14th day of the experiment in a square open box with a side length of 50 cm. Before the test, the mice were adapted to the test environment for 1 h, and a video recording function was started with a camera. The autonomous movement of the mice within 5 min was recorded. A 25 cm square centered on the center point of the open box was defined as the central region, and the movement trajectory of the mice in the last 5 min was analyzed. The evaluation indicators included the total distance traveled by the mice in the open field and the activity time in the central region of the open field.

[0040] (3) Tail suspension test (TST): The test was performed on day 14 of the experiment. The mice were adapted to the test environment for 1 h, and then the experiment began. The mice were suspended by the tail 1 cm from the tip on a H-shaped support with a width of 20 cm, which could be adjusted in height. The mice's heads were 10 cm from the table. A timer was used to time the mice's immobility for 6 min.

[0041] (4) Forced swimming test (FST): The test was performed on day 14 of the experiment. The mice were placed in a glass container with a height of 28 cm and a diameter of 15 cm. The mice were adapted to the test environment for 1 h before the test. After the adaptation period, the mice were removed and pure water at 23-25°C was added to the beaker to a depth of 12 cm. The mice were then placed in the beaker, and a camera was started to record the mice's swimming behavior for 6 min. The immobility time was analyzed for the last 4 min.

[0042] (5) On the 1st, 7th, and 14th days of the CUMS model construction, the mice's body weight was measured and recorded. The differences between groups and the changes in body weight at different stages were compared.

[0043] (6) Detection of inflammatory factors in mouse serum and brain-derived neurotrophic factor BDNF in brain tissue: After the experiment, the eyeball blood was taken and centrifuged. The supernatant was measured according to the ELISA kit instructions to measure the mass concentration of inflammatory factors TNF-α, IL-6, and IL-1β in the serum. The mouse brain tissue obtained by dissection was washed with pre-cooled PBS to remove residual blood. After weighing, the tissue was cut into small pieces. The cut tissue was added to a glass homogenizer with a corresponding volume of PBS at a weight-to-volume ratio of 1:9 and thoroughly ground on ice. Finally, the homogenate was centrifuged, and the supernatant was measured according to the ELISA kit instructions to measure the concentration of BDNF in the brain tissue.

[0044] 3. Experimental results

[0045] (1) Effect of DMP on SPT in mice: To evaluate the effect of DMP on the lack of pleasure in depressed mice, SPT was performed on CUMS mice. From Figure 1 It can be seen that: compared with the CON control group, the sugar intake rate of the CUMS model group mice decreased significantly (P<0.01), and the sweet taste excitement ability of sugar water decreased, indicating that the model was successfully constructed. After DMP treatment, the sugar intake rate of sugar water was significantly higher than that of the model group (P<0.01), indicating that the day lily mixed polypeptide can reduce the depressive behavior of mice during modeling.

[0046] (2) The effect of DMP on the OFT of mice: To evaluate the effect of DMP on the exploration behavior of depressive mice in a new environment and the anxiety and depression state, the CUMS mice were subjected to the OFT experiment. From Figure 2 It can be seen that the total distance of the CUMS model group mice in the open field decreased compared with the CON control group (P<0.05), and the exploration ability of the mice in the new environment was weakened, indicating that the model was successfully constructed. After DMP treatment, the activity range of the mice increased compared with the model group (P<0.05), indicating that the Dendranthema indicum mixed polypeptide oral liquid can reduce the depression and anxiety behavior of the mice during modeling. Similarly, from Figure 3 It can be seen that after DMP treatment, the mice in the open field center activity time increased compared with the model group (P<0.05), which also indicates that the Dendranthema indicum mixed polypeptide oral liquid can reduce the depression and anxiety behavior of the mice during modeling, and improve the exploration ability of the mice.

[0047] (3) The effect of DMP on the TST of mice: To evaluate the effect of DMP on the movement tendency and movement ability of depressive mice, the CUMS mice were subjected to the TST experiment, from Figure 4 It can be seen that compared with the control group, the TST immobility time of the model group mice increased significantly (P<0.01), indicating that the movement tendency of the mice decreased after modeling, and the depression behavior was obvious. Compared with the model group, the TST immobility time of the DMP treatment group mice decreased significantly (P<0.05), indicating that the depression behavior of the mice was significantly improved, and the movement ability was enhanced, indicating that the Dendranthema indicum mixed polypeptide oral liquid effectively reversed the learned helplessness of the mice caused by depression.

[0048] (4) The effect of DMP on the FST of mice: To evaluate the effect of DMP on the behavior despair of depressive mice in an unavoidable oppressive environment, the CUMS mice were subjected to the FST experiment, from Figure 5 It can be seen that compared with the control group, the FST immobility time of the mice in the model group increased significantly (P<0.01), indicating that the survival desire of the mice decreased significantly, and the movement ability decreased significantly after modeling. However, after DMP treatment, the FST immobility time of the mice decreased significantly (P<0.05), indicating that DMP intervention can significantly alleviate the decrease in the survival desire of the depressive mice.

[0049] (5) The effect of DMP on the body weight of mice at different stages: To analyze the changes in body weight caused by decreased appetite and depressed mood of depressive mice under DMP intervention, the body weight of the mice at different times during modeling was recorded, such as Figure 6As shown, the average body weight of each group of mice was close at the beginning of modeling, and the difference in average body weight of each group of mice became more and more obvious with the increase of modeling time. Compared with the control group of mice, the body weight of the model group of mice was extremely significantly reduced at 7 days and 14 days (P < 0.01), indicating that the model group of mice had obvious anorexia and sustained depression, which eventually led to slow body weight gain. The body weight of the DMP group of mice was significantly increased compared with the model group at 14 days (P < 0.01), indicating that after the intervention of the day lily mixed polypeptide oral liquid, the appetite of the mice was obviously improved, and the food intake increased, resulting in weight gain.

[0050] (6) The effect of DMP on inflammatory factors in the serum of mice and BDNF in the brain tissue: In order to further evaluate the effect of DMP on inflammatory factors and brain-derived neurotrophic factor BDNF in the body of depressed mice, the concentration of the corresponding substance was measured using an ELISA kit. As shown in Figure 7 compared with the control group, the concentration of the three inflammatory factors TNF-α, IL-6 and IL-1β in the model group of mice was significantly increased, and the concentration of BDNF was significantly decreased (P < 0.01), indicating that depression can increase the inflammatory response in the body and reduce the protective effect of nutritional factors on the nerve or brain tissue. However, after the intervention of DMP, the above process was reversed, which indicated that the day lily mixed polypeptide had good anti-inflammatory and neuroprotective effects.

[0051] The above shows and describes the basic principles and main features of the present application and the advantages of the present application. It is obvious for those skilled in the art that the present application is not limited to the details of the above exemplary embodiments, and the present application can be realized in other specific forms without departing from the spirit or essential characteristics of the present application. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting, and the scope of the present application is defined by the appended claims rather than the above description, and therefore all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present application. Any reference signs in the claims should not be regarded as limiting the claims involved.

[0052] In addition, it should be understood that although the present specification is described in terms of embodiments, each embodiment does not contain only one independent technical solution, and the description manner of the specification is only for the sake of clarity, and those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that those skilled in the art can understand.

Claims

1. A daylily antidepressant polypeptide oral liquid, characterized in that: The daylily antidepressant oral liquid is a formulated compound oral liquid.

2. The daylily antidepressant polypeptide oral liquid according to claim 1, characterized in that: The raw material is dried commercially available day lily.

3. A method for preparing a daylily mixed polypeptide oral liquid for relieving depression, characterized in that: The method comprises the following steps: (1) Take dried daylily, grind it into powder using a high-speed grinder, and add distilled water to fully dissolve it; (2) adjusting the pH with NaOH, stirring magnetically at 55°C, centrifuging, collecting the supernatant, adjusting the pH with HCl, allowing to stand, centrifuging, collecting the precipitate, adjusting the pH of the precipitate to neutral, washing with distilled water, and freeze-drying to obtain crude protein from daylily; (3) Dissolve the crude protein of daylily in deionized water, stir magnetically at 55°C for 10 minutes, adjust the pH to an appropriate range, add protease, mix well, and perform enzymolysis in a water bath; after the enzymolysis is completed, terminate the reaction in a boiling water bath, cool to room temperature, centrifuge the enzymolysis solution, collect the supernatant, and freeze-dry to obtain the daylily mixed polypeptide freeze-dried powder; (4) The mixed polypeptide freeze-dried powder, water and auxiliary materials honey, citric acid and potassium sorbate are mixed and homogenized in proportion, and then pasteurized and aseptically filled.

4. The method for preparing the daylily mixed polypeptide oral liquid for relieving depression according to claim 1, characterized in that: The specific experimental conditions of step (1) are: 10000-12000 rpm, grinding for 8-10 minutes, and the ratio of daylily powder to distilled water is 1:5-6.

5. The method for preparing the daylily mixed polypeptide oral liquid for relieving depression according to claim 3, characterized in that: The specific experimental conditions of step (2) are: adjusting the pH to 10 with NaOH, magnetic stirring for 5 hours, centrifugation at 10000 r / min for 10 minutes, adjusting the pH to 4 with HCl, and standing for 1 hour.

6. The method for preparing the daylily mixed polypeptide oral liquid for relieving depression according to claim 1, characterized in that: The specific experimental conditions of step (3) are as follows: the ratio of crude protein of daylily to deionized water is 1:10, magnetic stirring is performed for 10 minutes, the pH is adjusted to 2, a 5% pepsin solution is added, the mixture is mixed, and the mixture is placed in a constant temperature oscillator for enzymatic hydrolysis at 37°C in a water bath for 2 hours; the pH is then adjusted to 7.5, 5% trypsin is added, the mixture is mixed, and the mixture is enzymatic hydrolyzed at 37°C in a water bath for 4 hours; the pH is then adjusted to 8, 5% enteroproteinase is added, the mixture is mixed, and the mixture is enzymatic hydrolyzed at 37°C in a water bath for 2 hours, the reaction is terminated by boiling water bath at 100°C for 10 minutes, and the enzymatic hydrolyzate is centrifuged at 4°C at 10,000 r / min for 10 minutes.

7. The method for preparing the daylily mixed polypeptide oral liquid for relieving depression according to claim 1, characterized in that: The enzymes in step (3) are pepsin, trypsin and enteropeptide, the efficiency of pepsin is 1:(25000-35000), the enzymatic activity of trypsin is greater than 250 USPu / mg, and the efficiency of enteropeptide is 1:(30000-35000).

8. The method for preparing the daylily mixed polypeptide oral liquid for relieving depression according to claim 3, characterized in that: The specific experimental conditions of step (4) are as follows: the polypeptide, water and auxiliary materials honey, citric acid and potassium sorbate are mixed in a ratio of 30%:60%:8%:1%:1%, and the pasteurization condition is 75°C for 15 seconds.

9. Use of the daylily antidepressant mixed polypeptide oral liquid obtained by the method for preparing the daylily antidepressant polypeptide oral liquid according to any one of claims 1-2 or the daylily mixed polypeptide oral liquid for relieving depression according to any one of claims 3-8 in the preparation of antidepressant drugs, foods or health products.

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