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 present invention relates to a preparation method and application of a lily flower mixed polypeptide oral liquid for relieving depression, belonging to the field of bioactive small molecules Background Art

[0002] Depression is a common mental illness, manifested as long-term low mood, irritability, decreased attention, etc., which is the result of the combined action of social, psychological and biological factors. It is estimated that 5% of adults globally suffer from depression, and the incidence rate among women is about 50% higher than that among men. Worldwide, more than 10% of pregnant and newly delivered women suffer from depression, which is an important inducement for suicide. The World Health Organization has discussed mental health issues many times and approved the extension of the Comprehensive Mental Health Action Plan to 2030 in 2019 to promote and improve the global mental health situation. The methods for treating depression include psychotherapy and drug therapy or a combination of both. There are various types of commonly used antidepressant drugs in clinical practice, such as monoamine oxidase inhibitors, selective serotonin reuptake inhibitors, etc., which improve an individual's mood and behavior by affecting neurotransmitters in the brain. However, the long-term use of these antidepressant drugs may produce a series of side effects, such as headache, nausea, constipation and sexual dysfunction, etc. More and more studies have shown that a variety of natural organic compounds in plants, such as polypeptides, polysaccharides, alkaloids, etc., can relieve depressive symptoms while reducing side effects, which is of great significance for improving the quality of life of depressive patients.

[0003] The lily flower is a plant of the genus Hemerocallis in the family Liliaceae. Its unopened and newly opened flower buds are used as medicine, with medicinal value and health care effects. It is mainly produced in Gansu, Hunan, Shanxi, Sichuan, Hubei and other places. It contains a variety of chemical components, mainly including flavonoids, volatile oils, polyphenols, anthraquinones, alkaloids and other types. Among them, the content of flavonoids and their glycosides is relatively high, which are its main active substances, with the effects of clearing heat and promoting diuresis, relieving chest depression, cooling blood and detoxifying. Modern research shows that the lily flower has effects such as antidepressant, anti-tumor, sedative sleep, anti-inflammatory and antibacterial.

[0004] Proteins are biological macromolecules composed of polypeptide chains, which are digested in the body and then absorbed by the human body to exert their nutritional value and application value. At present, there has been no report on the research of the polypeptide activity produced by the digestion of lily flower protein. The present invention develops a lily flower mixed polypeptide with antidepressant activity through the preparation of a lily flower mixed polypeptide oral liquid and the use of experiments on a mouse model of depression. Summary of the Invention

[0005] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a preparation method and application of a mixed polypeptide oral liquid of daylily for relieving depression, so as to solve the problems raised in the above background technology. The present invention develops an antidepressant mixed polypeptide oral liquid derived from daylily, with clear structure and good health care effect.

[0006] To achieve the above purpose, the present invention is realized through the following technical solutions: A daylily antidepressant polypeptide oral liquid, the oral liquid is an oral liquid containing mixed polypeptides and has antidepressant efficacy.

[0007] Preferably, the raw material of the daylily antidepressant polypeptide oral liquid is dried daylily.

[0008] A preparation method of a mixed polypeptide oral liquid of daylily for relieving depression, the method includes the following steps,

[0009] (1) Take dried daylily, grind it into powder with a high-speed grinder, and fully dissolve it in distilled water;

[0010] (2) Adjust the pH with NaOH, stir magnetically at 55 °C, centrifuge, collect the supernatant, adjust the pH with HCl, let it stand, centrifuge, collect the precipitate, adjust the pH of the precipitate to neutral, wash with distilled water, and freeze-dry to obtain crude daylily protein;

[0011] (3) Dissolve the crude daylily protein in deionized water, stir magnetically at 55 °C for 10 min, adjust the pH to an appropriate range, add protease, mix well, and carry out water bath enzymatic hydrolysis; after the enzymatic hydrolysis is completed, terminate the reaction in a boiling water bath, cool to room temperature, centrifuge the enzymatic hydrolysate, collect the supernatant, and freeze-dry to obtain the freeze-dried powder of mixed polypeptides of daylily;

[0012] (4) Mix the freeze-dried powder of mixed polypeptides, water with auxiliary materials such as honey, citric acid and potassium sorbate in proportion, homogenize, and then carry out pasteurization and aseptic filling.

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

[0014] Further, the specific experimental conditions of step (2) are: adjust the pH to 10 with NaOH, stir magnetically for 5 h, centrifuge at 10000 r / min for 10 min, adjust the pH to 4 with HCl, and let it stand for 1 h.

[0015] Furthermore, the specific experimental conditions of step (3) are as follows: the ratio of crude protein of daylily to deionized water is 1:10. Stir magnetically for 10 min, adjust the pH to 2, add a pepsin solution with a mass concentration of 5%, mix well, place it in a constant temperature oscillator, and enzymatically hydrolyze it in a water bath at 37°C for 2 h; then adjust the pH to 7.5, add trypsin with a mass concentration of 5%, mix well, and enzymatically hydrolyze it in a water bath at 37°C for 4 h; then adjust the pH to 8, add enterokinase with a mass concentration of 5%, mix well, and enzymatically hydrolyze it in a water bath at 37°C for 2 h. Terminate the reaction in a boiling water bath at 100°C for 10 min, and centrifuge the enzymatic hydrolysate at 4°C and 10,000 r / min for 10 min.

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

[0017] Furthermore, the specific experimental conditions of step (4) are as follows: mix the mixed polypeptide, water, and excipients honey, citric acid, and potassium sorbate in a ratio of 30%:60%:8%:1%:1%, and the pasteurization condition is to treat at 75°C for 15 seconds.

[0018] Furthermore, the application of an oral liquid of daylily antidepressant polypeptide or the daylily oral liquid obtained by the preparation method of the above - mentioned mixed polypeptide of daylily for relieving depression in antidepressant drugs, foods, or health products.

[0019] Advantages of the present invention: For the preparation method and application of the above - mentioned mixed polypeptide oral liquid of daylily for relieving depression, the antidepressant mixed polypeptide used is derived from daylily, and the mixed polypeptide oral liquid of daylily has very good antidepressant effects. Therefore, the antidepressant mixed polypeptide oral liquid prepared by the present invention has the advantages of small side effects, definite curative effect, and high cost - performance. At the same time, since daylily has a wide source and is a common crop in vegetable planting agriculture, the present invention can improve the overall value of vegetable planting industry and increase farmers' income. Description of the Drawings

[0020] Figure 1 It is a picture of the effect of the mixed polypeptide oral liquid of daylily prepared by the present invention on the SPT of CUMS mice;

[0021] Figure 2 It is a picture of the effect of the mixed polypeptide oral liquid of daylily prepared by the present invention on the total distance of OFT of CUMS mice;

[0022] Figure 3 It is a picture of the effect of the mixed polypeptide oral liquid of daylily prepared by the present invention on the central activity time of OFT of CUMS mice;

[0023] Figure 4 Picture of the effect of the mixed polypeptide oral liquid of daylily prepared according to the present invention on the TST of CUMS mice;

[0024] Figure 5 Picture of the effect of the mixed polypeptide oral liquid of daylily prepared according to the present invention on the FST of CUMS mice;

[0025] Figure 6 Picture of the effect of the mixed polypeptide oral liquid of daylily prepared according to the present invention on the body weight of CUMS mice;

[0026] Figure 7 Picture of the effect of the mixed polypeptide oral liquid of daylily prepared according to the present invention on the concentrations of inflammatory factors and brain-derived neurotrophic factor in CUMS mice. Detailed implementation manners

[0027] To make the technical means, creative features, achieved purposes and effects of the present invention easy to understand, the present invention will be further described below in conjunction with specific implementation manners.

[0028] Example 1: A mixed polypeptide oral liquid for antidepressant of daylily and its preparation method, comprising the following steps:

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

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

[0031] (3) Dissolve the crude daylily protein in deionized water, stir magnetically at 55 °C for 10 min, adjust the pH to an appropriate range, add protease, mix well, and carry out water bath enzymolysis; 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 freeze-dried powder of the mixed polypeptide of daylily;

[0032] (4) The specific experimental conditions are: the mixed 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 to treat at 75 °C for 15 seconds.

[0033] In order to make the purpose and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with animal experiments. It should be understood that the specific experiments described here are only used to explain the present invention and are not used to limit the present invention.

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

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

[0036] 1. Animal grouping, establishment of a chronic unpredictable stress depression model (CUMS) and administration treatment: 30 male C57BL / 6 mice were randomly divided into a normal blank control group (CON), a chronic stress model group (CUMS), and a Daylily mixed with polypeptides administration group (DMP, 300 mg / kg). After the mice were acclimated to the environment for 7 days, except for the CON blank control group, the other groups were induced to establish a mouse depression model using daytime and nighttime stressors continuously for 2 weeks. During the establishment of the CUMS model, 11 stressors were randomly assigned, with 2 stressors per day, and the same stressor needed to be spaced at least 1 day apart, and each stressor needed to appear at least 3 times. The main stressors were: fasting (24 h), water deprivation (24 h), tail clamping (2 min), cage shaking (5 min), wet bedding (24 h), 45° cage tilt (24 h), circadian rhythm reversal (24 h), restraint (2 h), bright light exposure to the eyes (5 min), swimming in 4°C cold water and 45°C hot water. To ensure the smooth measurement of subsequent behavioral indicators, all mice were fasted and water-deprived on the last day of modeling, i.e., the 14th day of intervention. While constructing the CUMS model, the DMP group was intragastrically administered a dose of 300 mg / kg of Daylily mixed polypeptide every day at a fixed time.

[0037] 2. Experimental methods

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

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

[0040] (3) Tail suspension test (TST): The test was conducted on the 14th day of the experiment. The mice were allowed to acclimatize to the test environment for 1 h. At the start of the experiment, the mice were hung head-down 1 cm from the tip of the tail on an H-shaped stand with a width of 20 cm and adjustable height, with the mouse's head 10 cm above the tabletop. The timer was started for 6 min, and the immobile time of the mice in the last 4 min was counted.

[0041] (4) Forced swimming test (FST): The test was conducted on the 14th day 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 allowed to acclimatize to the test environment for 1 h before the test. After the acclimatization time ended, the mice were taken out, and pure water at 23 - 25 °C was poured into the beaker to a depth of 12 cm. The mice were then placed in the water, and the camera was immediately turned on to record the swimming state of the mice for 6 min. The immobile time in the last 4 min was statistically analyzed.

[0042] (5) The body weights of the mice were measured and recorded on the 1st, 7th, and 14th days when the CUMS model was constructed, and the differences between groups and the weight changes of each group of mice 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 ended, blood was collected from the eye socket of the mice, centrifuged, and the supernatant was taken for measuring the mass concentrations of inflammatory factors TNF-α, IL-6, and IL-1β in the serum according to the instructions of the ELISA kit. The mouse brain tissue obtained by dissection was rinsed with pre-cooled PBS to remove residual blood, weighed, and then the tissue was minced. The minced tissue and the corresponding volume of PBS were added to a glass homogenizer at a weight-to-volume ratio of 1:9 and ground thoroughly on ice. Finally, the homogenate was centrifuged, and the supernatant was taken for measuring the concentration of BDNF in the brain tissue according to the instructions of the ELISA kit.

[0044] 3. Experimental results

[0045] (1) Effect of DMP on the SPT of mice: To evaluate the effect of DMP on anhedonia in depressed mice, the SPT experiment was conducted on CUMS mice. From Figure 1 It can be seen that: Compared with the CON control group, the sugar intake rate of the mice in the CUMS model group decreased significantly (P < 0.01), and the excitatory ability to the sweetness of the sugar water weakened, indicating that the model was successfully constructed. After treatment with DMP, compared with the model group, the sugar intake rate of the sugar water increased extremely significantly (P < 0.01), indicating that the mixed polypeptide of daylily can weaken the depressive behavior of mice during the modeling process.

[0046] (2) Effects of DMP on the OFT of mice: To evaluate the effects of DMP on the exploratory behavior and anxiety-depressive state of depressive mice in a new environment, the OFT experiment was conducted on CUMS mice. From Figure 2 It can be seen that: compared with the CON control group, the total distance traveled by the mice in the CUMS model group decreased in the open field (P<0.05), indicating that the exploratory ability of the mice in the new environment weakened, suggesting that the model was successfully constructed. After treatment with DMP, compared with the model group, the activity range of the mice increased (P<0.05), indicating that the mixed polypeptide oral liquid of daylily can weaken the depressive and anxious behaviors of mice during the modeling process. Similarly, from Figure 3 It can be seen that after treatment with DMP, compared with the model group, the activity time of the mice in the center of the open field increased (P<0.05), which also indicates that the mixed polypeptide oral liquid of daylily can weaken the depressive and anxious behaviors of mice during the modeling process and improve the exploratory ability of mice.

[0047] (3) Effects of DMP on the TST of mice: To evaluate the effects of DMP on the motor tendency and motor ability of depressive mice, the TST experiment was conducted on CUMS mice. From Figure 4 It can be seen that compared with the control group, the immobility time of the mice in the model group during the TST increased significantly (P<0.01), indicating that the motor tendency of the mice weakened after modeling and the depressive behavior was obvious. Compared with the model group, the immobility time of the mice in the DMP treatment group during the TST decreased significantly (P<0.05), indicating that the depressive behavior of the mice was significantly improved and the motor ability was enhanced, suggesting that the mixed polypeptide oral liquid of daylily effectively reversed the learned helplessness caused by depression in mice.

[0048] (4) Effects of DMP on the FST of mice: To evaluate the effects of DMP on the behavioral despair of depressive mice in an inescapable oppressive environment, the FST experiment was conducted on CUMS mice. From Figure 5 It can be seen that compared with the control group, the immobility time of the mice in the model group during the FST increased extremely significantly (P<0.01), indicating that the survival desire of the mice decreased significantly and the motor ability decreased significantly after modeling. However, after DMP treatment, the immobility time of the mice during the FST decreased significantly (P<0.05), indicating that DMP intervention can significantly alleviate the decrease in the survival desire of depressive mice.

[0049] (5) Effects of DMP on the body weight of mice at different stages: To analyze the changes in body weight caused by reduced appetite and depressive mood in depressive mice under DMP intervention, the body weight of mice was recorded at different times during the model construction process, such as Figure 6As shown, the average body weights of mice in each group were close at the beginning of modeling. As the modeling time increased, the differences in the average body weights of mice in each group became more and more obvious. Compared with the control group of mice, the body weights of the model group of mice were extremely significantly reduced on the 7th and 14th days (P<0.01), indicating that the model group of mice had significantly reduced appetite and continued to be in a low mood, ultimately resulting in slow weight gain. The body weights of the DMP group of mice were significantly increased on the 14th day compared with the model group (P<0.01), indicating that after the intervention of the Hemerocallis citrina Baroni mixed polypeptide oral liquid, the appetite of the mice improved significantly, and the increased food intake led to weight gain.

[0050] (6) Effects of DMP on inflammatory factors in the serum of mice and BDNF in the brain tissue: To further evaluate the effects of DMP on inflammatory factors and brain-derived neurotrophic factor BDNF in depressed mice, an ELISA kit was used to measure the concentrations of the corresponding substances. As Figure 7 shown, compared with the control group, the concentrations of the three inflammatory factors TNF-α, IL-6, and IL-1β in the model group of mice were 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 nerves or brain tissues. However, after the intervention of DMP, the above process was reversed, indicating that the Hemerocallis citrina Baroni mixed polypeptide has good anti-inflammatory and neuroprotective effects.

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

[0052] In addition, it should be understood that although this specification is described according to embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A depression-resistant polypeptide oral liquid of Hemerocallis citrina Baroni, characterized in that, The Hemerocallis citrina antidepressant oral liquid is a compound oral liquid prepared by blending.

2. The anti-depressant polypeptide oral liquid of daylily according to claim 1, characterized in that Its raw material is commercially available dried Hemerocallis citrina.

3. A preparation method of a lily mixed polypeptide oral liquid for relieving depression, characterized in that, The method comprises the following steps: (1) Take the dried Hemerocallis citrina, grind it into powder with a high-speed grinder, and fully dissolve it in distilled water; (2) Adjust the pH with NaOH, stir magnetically at 55 °C, centrifuge, collect the supernatant, adjust the pH with HCl, let it stand, centrifuge, collect the precipitate, adjust the pH of the precipitate to neutral, wash it with distilled water, and freeze-dry to obtain crude Hemerocallis citrina protein; (3) Dissolve the crude Hemerocallis citrina protein in deionized water, stir magnetically at 55 °C for 10 min, adjust the pH to an appropriate range, add protease, mix well, and perform enzymatic hydrolysis in a water bath; after the enzymatic hydrolysis is completed, terminate the reaction in a boiling water bath, cool to room temperature, centrifuge the enzymatic hydrolysate, collect the supernatant, and freeze-dry to obtain a freeze-dried powder of mixed Hemerocallis citrina polypeptides; (4) Mix the freeze-dried powder of mixed polypeptides, water, and auxiliary materials such as honey, citric acid, and potassium sorbate in proportion, homogenize, perform pasteurization and aseptic filling.

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

5. The preparation method of a lily mixed polypeptide oral liquid for relieving depression according to claim 3, characterized in that: The specific experimental conditions of step (2) are: adjust the pH to 10 with NaOH, stir magnetically for 5 h, centrifuge at 10000 r / min for 10 min, adjust the pH to 4 with HCl, and let it stand for 1 h.

6. The preparation method of a lily mixed polypeptide oral liquid for relieving depression according to claim 1, characterized in that: The specific experimental conditions of step (3) are: the ratio of crude Hemerocallis citrina protein to deionized water is 1:10, stir magnetically for 10 min, adjust the pH to 2, add a 5% pepsin solution by mass concentration, mix well, place it in a constant temperature oscillator, and perform enzymatic hydrolysis in a 37 °C water bath for 2 h; then adjust the pH to 7.5, add a 5% trypsin by mass concentration, mix well, and perform enzymatic hydrolysis in a 37 °C water bath for 4 h; then adjust the pH to 8, add a 5% enterokinase by mass concentration, mix well, perform enzymatic hydrolysis in a 37 °C water bath for 2 h, terminate the reaction in a 100 °C boiling water bath for 10 min, and centrifuge the enzymatic hydrolysate at 4 °C and 10000 r / min for 10 min.

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

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

9. Use of the Hemerocallis citrina antidepressant mixed polypeptide oral liquid obtained by the preparation method of the Hemerocallis citrina antidepressant polypeptide oral liquid according to any one of claims 1 - 2 or the Hemerocallis citrina 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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