CN111557985A

CN111557985A - White tea extract, preparation method and administration method thereof and application of white tea extract in depression resistance

Info

Publication number: CN111557985A
Application number: CN202010221918.4A
Authority: CN
Inventors: 高良才; 周天; 胡文浩; 谢归香; 林泽杰; 渠荟超
Original assignee: East China Normal University
Current assignee: East China Normal University
Priority date: 2020-03-26
Filing date: 2020-03-26
Publication date: 2020-08-21

Abstract

The invention belongs to the technical field of Chinese herbal medicine component extraction, and discloses a method for preparing a white tea water extract by using white tea as a raw material. The invention also provides a nasal feeding method for administering the white tea extract to a tested individual, which can effectively relieve depression-like symptoms, including improvement of anhedonia behaviors and easy despair behaviors; the white tea extract can also be used for repairing olfactory injury or loss symptom accompanied with depression. The invention provides a new idea for developing a medicament/health-care product/food for treating depression and olfactory deficiency accompanied by the depression.

Description

White tea extract, preparation method and administration method thereof and application of white tea extract in depression resistance

Technical Field

The invention belongs to the technical fields of medicinal component extraction and separation technology and food and medicine application, and relates to an extract of an effective component of white tea, a preparation method thereof, and application of a white tea water extract in effectively relieving depression and repairing olfactory damage accompanied by depression.

Background

White tea is a precious product in tea, has a long history, and most of the white tea is exported in the past, and gradually enters the field of vision of the public in China in recent years. With the development of scientific technology, the research on the health care efficacy of the white tea is more deep at home and abroad, and the knowledge intersection and penetration in various fields make breakthrough progress on the research on the white tea and follow a strict experimental research mode.

The processing technology of the white tea is simple, only comprises two stages of withering and drying, and as the thermal action time is short and the temperature is low, a plurality of effective components in the fresh tea can be reserved in the white tea, so that the white tea has more prominent effects on oxidation resistance, tumor resistance, bacteria resistance and the like compared with other teas.

The white tea, as one of six Chinese teas, contains various bioactive substances, such as tea polyphenols, amino acids, flavones, caffeine, gallic acid, catechin, chlorogenic acid, etc., and has higher contents of amino acids and caffeine compared with the rest five teas. The white tea has better pharmacological and health-care effects, and researches show that the white tea has obvious effects on delaying senility (resisting oxidation), resisting tumors, inhibiting bacteria, resisting viruses, resisting fatigue, reducing blood sugar, regulating blood fat, preventing cardiovascular and cerebrovascular diseases, regulating immunologic functions and the like.

Tea polyphenols are a general term for a mixture of many phenolic derivatives, and are the highest soluble substances in white tea. It affects the formation of color, aroma and taste of white tea, and is a functional component with the most main health care function. The molecular structure of tea polyphenol determines the proton supplying property, so that the tea polyphenol has strong reducibility, and therefore, the tea polyphenol is an rare natural antioxidant and the best substance for eliminating free radicals. It has antiaging effect ten times that of vitamin E, and has effect in delaying myocardial lipofuscin generation. Phenolic compounds can be divided into four groups according to their chemical structures: catechins (flavanols), anthoxanthins (flavonols), phenolic acids, and anthocyanidins, wherein catechins are the main components of polyphenols.

The flavonoid compound has strong antioxidation and free radical scavenging capability, has various biological activities of reducing blood fat, resisting bacteria, resisting virus, resisting tumor and the like, and is an important functional component of the white tea playing a role in health care.

The organism can generate a large amount of free radicals under stress state, and the free radicals are related to pathological processes such as aging, tumor, inflammation and the like, so that the topic of searching effective antioxidant substances from natural materials for preventing and treating diseases and ensuring the healthy operation of the organism becomes a hot topic. The white tea is used as a natural resource with antioxidant activity, has good application value in preventing and improving organism injury caused by oxygen free radicals and maintaining health, has the characteristics of rich resources, multiple effects and strong activity, and has good market development and utilization prospects in the aspects of medical care and food sanitation.

In addition, according to the research of the American cancer research foundation, the white tea is a new anti-cancer substance, can continuously inhibit and reduce the tumor of the liver cancer, and improves the immunologic function of the human body; studies at the university of pei si in the united states have shown that white tea extract can prevent the growth of bacteria that cause staphylococcal infections, streptococcal infections, pneumonia, and the like.

The caffeine and flavanols in the white tea can promote the activity of epinephrine pituitary, are powerful central nerve stimulants, can strengthen muscle contraction, eliminate body fatigue, clear head and brain, promote blood circulation, dilate blood vessels, reduce blood pressure and have obvious diuretic effect. Compared with other teas, the white tea contains more caffeine and flavanols, so the white tea has more obvious effects of excitation, fatigue resistance, diuresis and the like than other teas. Caffeine is also an important flavor substance of tea, and a complex formed after the caffeine is bound with theaflavin through hydrogen bond has delicate flavor, so that the content of caffeine is an important factor influencing the quality of tea.

The content of theanine in the white tea is higher than that of other tea, the theanine is decomposed into ethylamine in human livers, the ethylamine can mobilize human blood immune cells named as gamma T cells to resist external invasion, and then the T cells promote the secretion of interferon to form a chemical defense line for resisting infection of human bodies. The Bukowski laboratory of the medical institute of Harvard university in the United states concluded that drinking tea increased the amount of blood immune cell interferon secretion by 5 times, thereby increasing the body's ability to resist external aggressions even more.

Due to the special processing technology of the white tea, active enzyme which is necessary for human bodies and has less content of other tea is well reserved, and long-term drinking of the white tea can obviously improve lipoprotein lipase in human bodies, promote fat catabolism, effectively control insulin secretion, delay absorption of glucose, decompose redundant sugar in the bodies and promote blood sugar balance.

Depression is an affective disorder mental disease characterized by persistent low mood, loss of interest and anhedonia, and the incidence rate of the depression tends to rise year by year with the acceleration of modern life rhythm and the increase of social pressure, thereby causing serious harm to the physical and mental health of human beings and bringing heavy burden to the society and families. The pathogenesis of depression involves a plurality of complex mechanisms, which are not completely elucidated so far, and clinical diagnosis is still based on symptomatology. Aiming at different mechanisms and targets, the antidepressant drugs commonly used in clinic at present can be divided into the following categories:

monoamine hypothesis drugs

The most clinically used antidepressant drugs are those based on the "monoamines" pathogenesis of depression.

Traditional antidepressants:

(ii) monoamine oxidase inhibitors: the enzyme inhibition effect is quick, the enzyme activity is quickly recovered after the medicine is stopped, and the toxic and side effects are larger.

② tricyclic antidepressants: adverse reactions include anticholinergic side effects, central nervous system side effects, cardiovascular side effects, sexual side effects, weight gain, allergic reactions and poisoning.

③ tetracyclic antidepressant drugs: the main adverse reactions are dizziness, hypodynamia and rare granulocytopenia.

Novel antidepressant drugs:

(ii) serotonin reuptake inhibitors: at present, antidepressant drugs which are most widely applied, such as fluoxetine (great apprehension), paroxetine and the like, are contraindicated when being combined with other antidepressant drugs.

(xii) serotonin and norepinephrine reuptake inhibitors: venlafaxine, for example, has the common adverse reactions of gastrointestinal discomfort, central nervous system abnormality, visual abnormality, yawning, sweating and sexual dysfunction.

③ norepinephrine and specific serotonin reuptake inhibitor: common side effects are appetite increase, weight gain, lethargy, sedation.

Glutamic acid hypothesis medicine

AMPA receptor agonists and NMDA receptor antagonists.

Third, stress hypothesis medicine

-Corticotropin Releasing Hormone (CRH) receptor antagonists.

(ii) desipramine, amitriptyline: acts on GR (glucocorticoid receptor) directly, increases the binding capacity of the GR to glucocorticoid and restores the activity of the receptor.

The current antidepressant drugs commonly used in clinic have certain limitations, such as side effects or adverse reactions, and contraindications when the antidepressant drugs are used in combination with other antidepressant drugs. In addition, most of the conventional antidepressant drugs are orally taken, and the effective components can only play a role by crossing the blood brain barrier, so that the treatment effect of the drugs is limited to a certain extent.

However, brain imaging studies have found that the affected brain areas of depression patients often overlap with the brain areas involved in olfactory information processing. Patients with depression have a phenomenon of decreased olfactory bulb volume and are negatively associated with the severity of the symptoms of depression. The olfactory bulb-extirpated animal model of depression also shows that the olfactory bulb-extirpated rats generate depression-like behavior change and can be improved by antidepressant drug treatment. This suggests that the olfactory pathway may be a potential target for treatment of direct through depression.

The existing research shows that nasal administration as one of the brain targeting administration routes can effectively lead the medicine to bypass the blood brain barrier and be delivered to the corresponding brain in a targeted way through an olfactory pathway. The depression is closely related and overlapped with the brain area of the olfactory pathway, so the depression can be treated by nasal administration on the basis of the depression.

However, no report about the improvement of behavior symptoms of depression and the repair of olfactory damage accompanied by the same by the white tea extract is found at present.

Disclosure of Invention

The invention aims to provide a white tea extract (white tea aqueous extract) and an extraction method, a drug administration mode and application thereof.

The invention takes Fujian white tea as raw material, and the white tea extract is obtained by filtering after adopting a water extraction method. The obtained white tea extract can be used for relieving depression-like symptoms by intranasal administration, and has partial repairing effect on olfactory injury accompanied by depression.

The white tea extract provided by the invention is prepared by the following method:

(1) grinding the raw material white tea, and mixing the raw material white tea with the raw material liquid according to the mass ratio of 1: 30-1: 70 are mixed with water.

(2) And (2) heating and cooking the mixture obtained in the step (1), filtering and taking supernatant.

(3) Rapidly cooling to obtain white tea extract, and diluting for use. Can be stored in refrigerator for a long time.

In the step (1), the white tea is white tea dry tea leaves and comprises leaves and stems of the white tea. Grinding the dry tea leaves by using a mortar and a pestle, and then mixing the ground dry tea leaves with water; the water may be distilled water, mineral water, or the like.

In the step (1), according to the mass ratio of the material liquid of 1:50 are mixed with distilled water.

In the step (2), the heating and cooking temperature is 80-90 ℃; preferably, it is 85 ℃.

In the step (2), the heating and cooking time is 8-12 min; preferably, it is 10 min.

In the step (2), the heating evaporation can be completed by adopting a rotary evaporator.

In the step (3), the filtration is performed by adopting a Buchner funnel and a vacuum filtration method; the filtration can be a plurality of times of filtration, and the number of times is 2-3 times.

In the step (3), the rapid cooling is realized by using a water bath, and the temperature is rapidly cooled to 30 ℃. And (3) heating and cooking in the step (2), directly transferring to a water bath kettle without cooling at room temperature after the filtering process is finished, or putting the whole device into the water bath kettle during the filtering in the step (2).

In the step (3), the preservation temperature is-20 ℃.

In the step (3), the dilution means that the white tea extract is preferably diluted to a concentration of 0.021 g/ml. The purpose of the dilution is to dilute the white tea extract to a dosage suitable for administration, which is adjusted to a suitable concentration according to the amount administered and the body weight of the subject, taking into account a single nasal feeding dose, e.g., 0.1-0.3ml each time.

The invention also provides the white tea extract obtained by the method.

The invention also provides a method for administering the white tea extract to a subject by nasal feeding.

The invention also provides application of the white tea extract in preparing foods, health-care products or medicines for resisting depression and treating or relieving depression.

The invention also provides application of the white tea extract in preparing food, health-care products or medicines for treating or relieving anhedonia behaviors caused by depression.

The invention also provides application of the white tea extract in preparing food, health-care products or medicines for treating or relieving easy despair behaviors caused by depression.

The invention also provides application of the white tea extract in preparing food, health-care products or medicines for treating or relieving depression-like behaviors (reduced sugar water consumption, reduced forced swimming time and increased tail suspension immobility time) of mice caused by CUMS.

The invention provides application of the white tea extract in preparing food, health-care products or medicines for treating or improving or repairing olfactory injury or loss or providing olfactory protection.

The invention provides application of the white tea extract in preparing food, health-care products or medicines for treating or improving or repairing olfactory damage or deficiency accompanied by depression or providing olfactory protection for depression.

The invention provides a white tea extract for preparing a medicine for relieving the damage of olfactory ability of mice accompanied by depression caused by CUMS, such as increase of food embedding latency, reduction of olfactory sensitivity, reduction of olfactory preference and the like.

The white tea extract provided by the invention adopts an intranasal administration mode, can improve the sucrose preference of depression-like mice modeled by a CUMS (chronic unpredictable stress) method, reduces the immobility time of the depression-like mice in a forced swimming test and a tail suspension test, improves the damage of mitochondria and synapses in hippocampal cells, and increases the number of synapses. And increases the content of serotonin (5-HT) and brain-derived neurotrophic factor (BDNF) in the hippocampus of the mouse. The white tea extract prepared by the method has partial repairing effect on olfactory injury of mice modeled by a CUMS method, can play a role in protecting olfactory sensation, and has more obvious effect of higher concentration.

In the invention, the white tea extract is used as the only active ingredient or one of the active ingredients of food, health products or medicines.

In the invention, the food, the health product or the medicine is a white tea extract with a single component, or is a tablet formed by the white tea extract and one or more of a filling agent, a binding agent, a wetting agent, a disintegrating agent, an absorption enhancer, a solvent, a surfactant, a flavoring agent, a preservative, a lubricant, a sweetening agent and a pigment, or is an oral liquid preparation formed by the white tea extract and one or more of a buffering agent, an antioxidant synergist, a flavoring agent, a sweetening agent, a solvent, a surfactant and a preservative.

Wherein the food, health product or medicine further comprises a component for improving the stability of the white tea extract.

Wherein the food, health product or medicine is liquid preparation, solid preparation, spray or aerosol.

Wherein the food, health product or medicament is injection, suspension, emulsion or solution.

Wherein the food, health product or medicine is syrup.

Wherein the solid preparation is a tablet, a capsule, a granule or a medicinal granule.

The method for preparing the white tea extract has the advantages that the method is simple to operate, and the obtained white tea extract can effectively relieve depression-like symptoms, including anhedonia behaviors and improvement of easy despair behaviors; the white tea extract can also be used for repairing olfactory injury or loss accompanied by depression. The white tea extract can be directly and efficiently administered in brain by nasal feeding, and provides a new idea for developing medicaments, health-care products and foods for treating depression and olfactory deficiency accompanied by the depression.

Drawings

FIG. 1 is a flow chart showing the whole of examples 1 to 14.

FIG. 2 is a graph of the effect of white tea extract of examples 4, 5, 6, 7 on CUMS modeling depression-like behavior in mice; a is the effect of nasal feeding of white tea extract on the CUMS modeled mouse sugar water consumption test (example 4); b is the effect of nasal feeding of white tea extract on the CUMS modeled forced swimming test in mice (example 5); c is the effect of nasal feeding of white tea extract on the CUMS modeled mouse tail suspension test (example 6); d is the effect of nasal feeding of white tea extract on the open field test in CUMS modeled mice (example 7).

FIG. 3 is a graph of the effect of white tea extract of examples 8, 9, 10 on the olfactory ability of CUMS modeled mice; a is the effect of nasal feeding of white tea extract on the CUMS modeled mouse food embedding test (example 8); b is the effect of nasal feeding of white tea extract on the CUMS modeled mouse olfactory susceptibility test (example 9); c is the effect of nasal feeding of white tea extract on the CUMS modeled olfactory avoidance test in mice (example 10).

FIG. 4 is the effect of nasal feeding of white tea extract on the microstructure of the hippocampus of CUMS-modeled mice of example 11; a is an electron microscope photo of the influence of the white tea extract on the microstructure of the hippocampus of each group of mice; and B is the influence of nasal feeding of the white tea extract on the number of synapses of the hippocampus of the CUMS modeling mouse.

FIG. 5 is the effect of nasal feeding of white tea extract on the CUMS modeled olfactory bulb microstructure of mice in example 11; a is an electron microscope photo of the influence of the white tea extract on the micro-structure of the olfactory bulbs of each group of mice; and B is the influence of nasal feeding of the white tea extract on the number of synapses in the olfactory bulb of the CUMS modeling mouse.

FIG. 6 is the effect of nasal feeding of white tea extract on CUMS modeling mouse hippocampal brain neurotrophic factor (BDNF) gene expression level of example 12; a is an mRNA expression strip of BDNF in the hippocampus evaluated by agarose gel electrophoresis; b is the mRNA level of BDNF in hippocampus between groups.

FIG. 7 is the effect of nasal feeding of white tea extract on the expression levels of brain neurotrophic factor (BDNF) and Olfactory Marker Protein (OMP) genes in the olfactory bulb of CUMS modeled mice in example 13; a is mRNA expression bands of OMP and BDNF in an olfactory bulb evaluated by agarose gel electrophoresis; b is the mRNA level of OMP in olfactory bulb among groups; c is the mRNA level of BDNF in the olfactory bulb between groups.

FIG. 8 is a first step in the CUMS modeling of the effect of nasal feeding of white tea extract on neurotransmitter secretion in the hippocampus of mice (the first step is to obtain standard chromatograms of various neurotransmitters in the mouse brain), and representative chromatograms of NE, DA, 5-HT and 5-IAA in the mouse brain are determined, in example 14.

FIG. 9 is a second step in the effect of neurotransmitter secretion in the hippocampus of example 14 (the second step is the measurement of various neurotransmitter levels in the brain samples of the groups of mice of the present invention) to model the effect of nasal feeding of white tea extract on various monoamine neurotransmitter levels in the hippocampus of mice for CUMS.

In FIGS. 1-9, WT was a mouse without CUMS modeling treatment; CU is control mice treated with chronic stress only and not dosed; LT is a mouse given a low concentration of white tea extract (20mg/kg) after chronic stress; HT is a mouse given a high concentration of white tea extract (40mg/kg) nasal feed after chronic stress treatment; CF is a positive control group of mice given the intraperitoneal injection of the drug fositin after chronic stress treatment.

Denotes p <0.05 compared to control; denotes p <0.01 compared to control; # denotes p <0.05 compared to the chronic stress group; # indicates p <0.01 compared to the chronic stress group.

Detailed Description

The present invention will be described in further detail with reference to the following specific examples and the accompanying drawings. The procedures, conditions, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art, except for the following specific volume contents, and the present invention is not particularly limited to the contents.

When the mice are nasal fed, the effective dose of the drug is kept the same for each mouse.

Example 1 preparation of white tea extract

10g of raw white tea (manufacturer: Fujian Qingsha tea shares company; brand name: old tree white tea; quality grade: special grade; net content: 210g) is ground and crushed by using a mortar, and is mixed with 500g of distilled water according to the leaf/extraction medium ratio of 1:50(w/w), and is cooked for 10min under vacuum at 85 ℃ by using a rotary evaporator, supernatant is quickly filtered, and then the mixture is transferred to a constant-temperature water bath kettle to be cooled to 30 ℃ to obtain a white tea extract, and the white tea extract can be stored in a refrigerator at-20 ℃.

Example 2 model construction of Chronic unpredictable stress (CUMS) mice

50 male C57BL/6J mice, 6 weeks old, were weighed 20-40g each, and animals were randomized into five groups of 10 animals each: control group (WT), chronic stress group (CU), fluoxetine group (CF), high concentration white tea group (HT), and low concentration white tea group (LT). The other groups received the CUMS treatment except the control group. Chronic unpredictable mild stress programs (CUMS) consist of multiple mild stressors: (1) grain breaking treatment for 24 hours; (2) water cut-off treatment for 24 hours; (3) cage tilting treatment at 45 degrees for 24 hours; (4) light/dark cycle inversion processing; (5) performing dirty squirrel cage treatment for 24 hours; (6) forced swimming for 5 minutes at 4 ℃; (7) treating the tail of the clamp for 1 minute. In order to make the pressure sources and types unpredictable, the stress sources are arranged in a random fashion, which may occur at any time during the three week modeling process. Control mice were housed in a separate room without contact with stressed mice.

Example 3 dosing treatment of CUMS-modeled mice

After modeling of the three-week-old CUMS mice, the high-concentration white tea group and the low-concentration white tea group were administered with 40mg/kg and 20mg/kg of white tea extract by nasal feeding for one week, while the fluoxetine group was intraperitoneally injected with 20mg/kg of fluoxetine for one week, and the chronic stress group was not administered. For nasal feeding, mice were held in their hands and dosed in a supine position. Dropping the medicine liquid to the front of the nostril of the mouse by a dropper, sucking the medicine liquid by free respiration of the dropper, and if the medicine liquid has a part which is not sucked, dropping the medicine liquid by the same amount to keep the dosage of each mouse the same.

The following tests of examples 4-14 were then performed.

Example 4 Effect of nasal feeding of white tea extract on the CUMS modeled sugar Water consumption test in mice

In order to evaluate the degree of depressive behavior of the mice of the control group and each experimental group, a sugar water consumption experiment was used for judgment. Each group of mice received sucrose solution acclimation training: two bottles of sucrose solution were given for 24 hours, followed by one bottle of sucrose solution and one bottle of water for 24 hours. The mice were then deprived of food and water for 12 hours and then subjected to a 24 hour sucrose preference test during which the mice were free to obtain two bottles, one of 100ml water and the other of 100ml sucrose solution at a concentration of 1% (w/v). The positions of the two bottles are randomly varied. The sucrose preference degree is calculated by the following formula: sucrose preference (%) ═ sucrose consumption volume)/(sucrose consumption volume + water consumption volume) × 100%. In this embodiment, the one bottle of sucrose solution refers to 100mL of 1% (w/v) sucrose solution; the bottle of water refers to 100ml of water.

The effect of white tea extract on the results of sugar water preference of control and experimental mice as shown in fig. 2A, the relative sucrose intake was significantly reduced (p <0.01) in chronic stress mice compared to the control group. The low and high concentration white tea groups reversed the relative sucrose intake compared to the control group, especially the high concentration white tea group reversed this significantly (p < 0.05; p < 0.01).

Example 5 Effect of nasal feeding of white tea extract on the forced swim test in CUMS-modeled mice

In order to evaluate the degree of depressive behavior of the mice of the control group and each experimental group, judgment was made using a forced swimming experiment. A single mouse was forced to swim in a cylindrical transparent glass container filled with 17cm of water, the temperature of which was room temperature, the diameter of the glass container was 18cm, and the height was 40 cm. After immersion, the mice were acclimated for the first 1 minute and then the immobility time for 5 minutes was recorded by the monitor. Immobility time refers to the time a mouse floats in water without struggling.

The effect of white tea extract on the forced swimming test results of the control and experimental mice is shown in fig. 2B, and compared with the control group, the immobility time of the chronic stress group mice is obviously prolonged (p < 0.05). However, nasal feeding of low-concentration white tea and high-concentration white tea significantly reduced immobility time (p < 0.05; p <0.05), indicating that the white tea extract had an antidepressant effect.

Example 6 Effect of nasal feeding of white tea extract on the tail suspension test in CUMS-modeled mice

To evaluate the degree of depressive behavior of mice of the control group and each experimental group, judgment was performed using the tail suspension experiment. The mouse is firmly fixed at the position of 1.0-1.5cm away from the tip of the tail part by using a medical adhesive tape, and is hung upside down in the box, the distance from the bottom of the box is about 30cm, three sides of the box are opaque, and the visual interference of the mouse is isolated. Mice were acclimated for the first 1 minute and the final 5 minutes of immobility time was recorded by the monitor. A mouse can be considered immobile only when it has no action at all.

The influence of the white tea extract on the tail suspension experiment results of the control group mice and the experimental group mice is shown in fig. 2C, compared with the control group mice, the immobility time of the chronic stress group mice is remarkably prolonged (p is less than 0.01), the nasal feeding high-concentration white tea has a remarkable effect (p is less than 0.01) on reversing the situation, the effect of the effect is even more than that of fluoxetine (p is less than 0.05), and the nasal feeding white tea extract has a remarkable anti-depression effect.

Example 7 Effect of nasal feeding of white tea extract on the open field test in CUMS-modeled mice

Considering that the nasal feeding white tea extract may have an influence on the spontaneous locomotor activity of the experimental mice, the immobility time in the forced swimming test and the tail suspension test can be reduced. To determine whether this condition exists, and to evaluate the degree of anxiety in the autonomic activity and mood of the mice in the control and each experimental group, a determination was made using an open field experiment. The mice were placed individually in the middle of a 40cm long, 60cm wide and 50cm high iron box, the bottom of which was divided into 25 equal squares. Mice were acclimated for the first 1 minute followed by 5 minutes with a monitor to record the number of squares crossed by all paws per mouse. The bottom of the iron box was rinsed with 75% ethanol prior to testing in each mouse.

The influence of the white tea extract on the open field experimental results of the control group and the experimental group mice is shown in fig. 2D, the influence of the nasal feeding white tea extract on the motor activity of the chronic stress mice has no significant difference (p > 0.05; p >0.05), and the fluoxetine can obviously improve the motor activity of the chronic stress mice (p <0.01), which indicates that the treatment effect of the white tea extract on depression is probably not realized by improving the hypofunction of the chronic stress mice.

Example 8 Effect of nasal feeding of white tea extract on the food embedding test in CUMS-modeled mice

To evaluate olfactory ability of mice in the control group and each experimental group, judgment was made using a food inclusion test for 2 consecutive days, all mice were starved for 24 hours before the test, and the mice were subjected to a test once a day, and in each test, one mouse was placed at the bottom of the test chamber (42 × 35 × 40cm 3540 cm)³) Was made to look for 1g of food pellets, which were buried about 0.5 cm below the surface of a 3 cm thick bedding material. The latency period for each mouse to find the food pellet is defined as the time from placing the mouse in the cage to the time the mouse finds the pellet and grabs it with the forepaws and/or teeth to bite. If a mouse can find a food pellet within 5 minutes, it can use the pellet as a reward to return to its home cage. Otherwise, there is no prize. The bedding in the test cage was cleaned between each test and the position of the food pellets was changed randomly every day. After completion of the day's tests, each mouse received 1g of food to ensure their basic survival. The experiment was repeated the first day on the next day.

The effect of white tea extract on the results of the open field experiments in the control and experimental mice is shown in FIG. 3A, where the food was found faster the next day in the differently treated mice (FIG. 3A; WT: p < 0.001; CU: p < 0.05; LT: p < 0.01; HT: p <0.01), whereas the speed of food pellets was found to be significantly slower in two days in mice that received only CUMS modeling treatment than in the control mice (day 1, p < 0.01; day 2, p < 0.01). The experimental results of the 2-day period show that the incubation period of the mice is obviously shortened after the mice are treated by the low-concentration and high-concentration white tea extract, and the incubation period is close to the result of using a positive control group. (day 1: LT: p < 0.05; HT: p < 0.01; day 2: LT: p < 0.05; HT: p < 0.01).

Example 9 Effect of nasal feeding of white tea extract on the olfactory susceptibility test of CUMS-modeled mice

This example used peanut butter as the source of flavor and corn oil as the solvent. Experiments were performed with several concentration gradients of peanut butter, 0g/mL (i.e., neat oil), 0.1g/mL, 0.05g/mL, and 0.01g/mL, respectively. In addition, the present invention prepares a 9 cm x 9 cm filter paper for carrying the odor sample. First, five groups of mice were acclimated in clean cages for 15 minutes. The mice were then transferred to a cage of the same size (ensuring the same environment), and the sample was diluted with a filter paper soaked with a concentration of peanut butter and recorded for 3 minutes using a video camera. The results of the experiment were analyzed using the Ethovision XT14 software package (Noldus, virginia, usa) and the statistical index of the analysis was the total time each mouse sniffed the filter paper sheet in 3 minutes.

The results of the experiments are shown in fig. 3B, and the analysis of the dual variance is performed using Peanut Butter (PB) concentration and treatment ("treatment" means treatment with high/low concentration white tea extract, fosiltin drug, or no drug treatment at all, etc.) as factors. There was no significant difference between groups when the neat oil odor was tested. While the interest of the mice treated with CUMS modeling only, i.e., the chronic stress group, for each gradient peanut butter was significantly lower than the control group (FIG. 3B; PB 0.01g/mL, P < 0.001; PB 0.05g/mL, P < 0.001; 0.1g/mLPB, P < 0.001). When 0.1g/mL PB was analyzed, mice in both the low and high concentration white tea groups were found to have significantly increased sniff time (p < 0.001). The investigation time for the mice in the high concentration white tea group was also significantly prolonged for 0.05 and 0.1g/mL PB (0.05g/mL PB, p < 0.05; 0.1g/mLPB, p < 0.01).

Example 10 Effect of nasal feeding of white tea extract on the olfactory avoidance test in CUMS-modeled mice

The mouse cage was drawn on the bottom of the cage by drawing a line, dividing the cage into two parts (1: 3). the mouse was free to move in the experimental cage, the filter paper with the experimental smell was fixed at the bottom of the smaller area, 2-methylbutyric acid (1.7 × 10) was used^-6mol) as a source of mouse aversion odor, diluted with distilled water. The sniff time for each concentration gradient was defined as: the time the mice sniffed the filter paper sheets soaked with distilled water minus the time the mice stayed in each area with an aversive odor filter paper sheet. The results of the experiment were analyzed using the Ethovision XT14 software package (Noldus, virginia, usa).

The experimental results are shown in fig. 3C, and the smell of 2-methylbutyric acid has no obvious influence on the mice which are only subjected to the CUMS molding treatment, namely the chronic stress group, and can not lead the mice to generate obvious evasive behaviors (p is less than 0.001). Compared with the mice in the low-concentration and high-concentration white tea groups, the avoidance time is remarkably prolonged (LT: p is less than 0.01; HT: p is less than 0.01), which indicates that the judgment capability of the olfactory happiness of the mice is reduced by CUMS molding treatment, and the nasal feeding of the low-concentration and high-concentration white tea can repair the damage in the aspect.

Example 11 influence of nasal feeding of white tea extract on CUMS modeling of mouse Hippocampus and olfactory bulb microstructures

The brains of the mice were removed from the skull and placed in 2.5% glutaraldehyde for overnight immersion. The hemisphere tissue blocks containing hippocampus and olfactory bulb were cut into 400 μm thick coronal sections, respectively. Sections were washed in 0.1Mpb and in 2.5% glutaraldehyde until processed. During processing, the sections were washed with 0.1Mpb, fixed with 1% osmium tetroxide in 0.1Mpb for 2h, and then washed with 0.1 Mpb. The hippocampus was identified with a light microscope Leica-mmaf (hitachi high tech, beijing, china) and excised from the coronal section, dehydrated in graded ethanol and acetone, and then embedded in resin. The sections were trimmed to a thickness of 70-75nm with an ultramicrotome, the sections were extracted on a 200 mesh copper grid, double-stained with uranium acetate and lead citrate, and the sections were observed with an H7700 transmission electron microscope (hitachi high tech, beijing, china).

Ten fields were randomly selected for each section for counting statistics while observing the samples of each group, and the differences between groups were compared by taking the average as the result.

As shown in fig. 4A, in hippocampal cells of chronically stressed mice, a significant change in several ultrastructures of pyramidal neurons was observed, mitochondrial damage (most common abnormality in large dendrites and synaptic terminals), ridge destruction, vacuolar degeneration and even mitochondrial membrane disruption, some synaptic clefts not being dense, compared to cells of control mice, suggesting that neural signals were affected by stress. Mitochondria in the low-concentration white tea group and the high-concentration white tea group showed less vacuolar degeneration and more intact cristae than in the chronic stress group.

In order to study the density of the synapses in the hippocampus of the control group and four experimental groups, at least 3 regions were randomly selected in each group, the number of synapses was calculated, and the average value was taken, and the area of each region was 62 μm². In calculating synapse density, all synapses in a two-dimensional transmission electron microscope slice are counted, regardless of size and shape of the synapses. FIG. 4B shows a significant reduction in synapses in hippocampus (p) in chronically stressed mice compared to control groups<0.01), the high concentration of white tea and fluoxetine both remarkably increase the synaptic density (p)<0.05；p<0.05) and there is no significant difference in the effect between the two.

Mitochondrial morphology and synaptic injury are characteristic of neural plasticity of olfactory bulb cells, much like what occurs in hippocampus. The olfactory bulbs of the control group and the four experimental groups were observed by transmission electron microscopy (FIG. 5A). Cristae disruption, vacuolar degeneration and even mitochondrial membrane disruption were detected in olfactory bulbs of CU group mice. Compared with the mice subjected to the CUMS modeling treatment only, namely the chronic stress group, the mice in the low-concentration and high-concentration white tea groups have relatively less mitochondrial vacuole degeneration, more complete ridges, denser synaptic cleft and more vesicles in the white tea treatment group.

The number of olfactory bulb spinal synapsis of five groups was subjected to one-way anova (FIG. 5B), and the results showed that the spinal synapsis of mice treated with CUMS modeling alone, i.e., chronic stress group, was significantly decreased (p <0.01), while the spinal synapsis of mice treated with either low-concentration white tea or high-concentration white tea was significantly increased (LT: p < 0.05; HT: p <0.01), relative to the control group.

Example 12 Effect of nasal feeding of white tea extract on CUMS modeling of mouse hippocampal brain neurotrophic factor (BDNF) Gene expression level

Some studies have shown that BDNF levels are reduced in brain regions of depressed patients, while antidepressant treatment can increase BDNF levels in the brain. BDNF signaling facilitates synaptic vesicle docking of the active region. BDNF plays an important role in synaptic plasticity. Chronic stress impairs hippocampal BDNF expression, and the effects of antidepressant drugs are associated with increased BDNF synthesis and activity in the hippocampus. The BDNF level is increased, so that the synaptic vesicle distribution of synaptic terminals in the hippocampus of the chronic stress mice can be adjusted, and the number of docking vesicles is increased.

The present invention uses the Trizol kit (Invitrogen, Carlsbad, California, USA) to extract total RNA to assess BDNF gene levels. cDNA was synthesized using oligonucleotide primers (Dalian Takara Biotechnology Ltd.) and amplified using specific primers. The real-time primers for BDNF were forward: 5'-tcatactcggttgcatgaagg-3' (SEQ ID No. 1); and (3) reversing: 3 '-agacctcgacctgccc-5' (SEQ ID No. 2). The PCR products were separated by electrophoresis on a 2% agarose gel, stained with ethidium bromide and photographed under UV light. The brightness of all fluorescence bands was analyzed and compared using Image-J software. The expression of the target gene is calibrated by the standard gene beta-actin.

Analysis of the results showed that fig. 6A shows agarose gel electrophoresis to assess mRNA expression bands of BDNF in the hippocampus. Figure 6B shows the mRNA levels of BDNF in the hippocampus between groups. The BDNF levels of mice in the chronic stress group were significantly reduced (P <0.01) compared to the control group, but the BDNF levels of mice given low and high concentrations of white tea (P < 0.05; P <0.01) and fluoxetine (P <0.01) were significantly increased. High concentrations of white tea increased BDNF levels even above fluoxetine.

Example 13 Effect of nasal feeding of white tea extract on CUMS modeling of brain neurotrophic factor (BDNF) and Olfactory Marker Protein (OMP) Gene expression levels in olfactory bulb of mouse

The expression of BDNF and OMP mRNA in the olfactory bulb is detected by semiquantitative RT-PCR, so as to further research the olfactory bulb damage and the change of related molecules after the CUMS mice are fed with the white tea extract through nasal feeding. The present invention uses the Trizol kit (Invitrogen, Carlsbad, California, USA) to extract total RNA to assess BDNF gene levels. cDNA was synthesized using oligonucleotide primers (Dalian Takara Biotechnology Ltd.) and amplified using specific primers. The real-time primers for BDNF were forward: 5'-tcatactcggttgcatgaagg-3' (SEQ ID No. 3); and (3) reversing: 3 '-agacctcgacctgccc-5' (SEQ ID No. 4); and real-time primer forward for OMP: 5'-CAGCAGGAAGGTTCTCCTCC-3' (SEQ ID No. 5); and (3) reversing: 5'-GAACAGCCAGGATATGCCCA-3' (SEQ ID No. 6). The PCR products were separated by electrophoresis on a 2% agarose gel, stained with ethidium bromide and photographed under UV light. The brightness of all fluorescence bands was analyzed and compared using Image-J software. The expression of the target gene is calibrated by the standard gene beta-actin.

FIG. 7A shows agarose gel electrophoresis to evaluate mRNA expression band of BDNF in olfactory bulb. The results in FIG. 7B show that OMP mRNA expression was significantly lower in mice treated with CUMS molding alone, i.e., in the chronic stress group, than in the control group, while the expression was significantly increased in mice treated with both low-concentration white tea and high-concentration white tea (LT: p < 0.05; HT: p < 0.001). BDNF was similar to OMP expression changes (FIG. 7C; p < 0.05). Although there was no significant difference between the low-concentration white tea group and the CUMS molding treatment only group, i.e., the chronic stress group, the high-concentration white tea group showed a significant difference (p < 0.001).

Example 14 Effect of nasal feeding of white tea extract on CUMS modeling neurotransmitter secretion in hippocampus of mice

The secretion of four neurotransmitters Norepinephrine (NE), Dopamine (DA), pentahydroxytryptamine (5-HT) and pentahydroxyindoleacetic acid (5-HIAA) in hippocampus was measured by high performance liquid chromatography.

The hippocampus was washed with ice-cold physiological saline and weighed. On the day of the assay, samples were prepared with 0.4mol/L perchloric acid (volume of perchloric acid: tissue weight: 10: 1). The homogenate was centrifuged at 10000rpm/min for 15min at 4 ℃. The supernatant was centrifuged for 10min under the same conditions. The supernatant was taken and made up to 1ml with 0.4mol/L perchloric acid solution. Measuring NE, DA, 5-HT and 5-HIAA by high performance liquid chromatography using a fluorescence detector. High performance liquid chromatography analysis was performed using Agilent 1100 series high performance liquid chromatography (Agilent Corp.). The analytes were separated on a Zorba XSB-C18 column at room temperature. The mobile phase was a 9:1 mixture of NaAc (containing 0.1mol/L EDTA-2Na, adjusted with HAC Ph5.1) and methanol. The flow rate was 1.0ml/min, the injection amount was 20 ul/time, the emission wavelength was 330nm, the excitation wavelength was 290nm, and the sensitivity was 2. The amount of the detection index in each sample was quantified by preparing a standard curve using standards, and NE, DA, 5-HT and 5-HIAA standards were purchased from Sigma, Inc. (St. Louis, MO, USA). Weighing NE3mg, DA2mg, 5-HT2mg and 5-HIAA2.1mg, dissolving with 0.1mol/L hydrochloric acid, diluting to 10ml, diluting 500, 1500, 2000 and 2500 times, centrifuging at 4 deg.C at 10000rpm/min for 10min, and collecting supernatant for determination.

The levels of the monoamine neurotransmitters NE, DA, 5-HT and 5-IAA in the hippocampus were evaluated by HPLC and fluorometer to study the effect of nasal feeding white tea in mild stress-induced depression, which was chronically unpredictable. FIG. 8 is a chromatogram for measuring NE, DA, 5-HT and 5-IAA in the brain of a mouse (mouse brain tissue of a standard used for preparing an isolation standard). The figure shows that NE, DA, 5-HT and 5-IAA are well separated and no interference of endogenous substances is detected. The retention times of NE, DA, 5-HT and 5-IAA were 2.195 min, 3.091 min, 7.642 min and 8.507 min, respectively.

Monoamine deficiency in depression the hypothesis that depression is caused by a deficiency of monoamines in the brain, such as NE, DA and 5-HT, therapeutic effects of antidepressants are regulated by increasing monoamine levels. In particular, 5-HT plays a crucial role in the pathophysiology of depression, the 5-HT content in the brain is closely related to happy mood, and the 5-HT concentration in the brain of a patient suffering from depression is reduced.

Figure 9 shows monoamine neurotransmitter levels in the hippocampus of each group. The data show that chronic stress significantly reduced 5-HT and NE levels (p < 0.01; p <0.01) compared to the control group (chronic stress group). Both high concentration white tea and fluoxetine can significantly improve 5-HT level (p < 0.05; p < 0.01). In addition, the ratio of 5-HIAA to 5-HT (p < 0.05; p <0.01) was significantly decreased by low-concentration white tea and high-concentration white tea, and the increase in the ratio of 5-HIAA, which is a metabolite of 5-HT, indicates that white tea increases the 5-HT content in hippocampus by decreasing the metabolism of 5-HT. None of the low concentration white tea, high concentration white tea and fluoxetine increased NE levels. Surprisingly, the DA levels in the chronic stress group were significantly higher than in the control group. The invention speculates that due to the compensatory mechanism, DA metabolism is disordered, so that DA levels are recovered to be normal after white tea and fluoxetine treatment.

Example 15 Effect of different extraction conditions on white tea extract

Selecting temperature gradients of 75 deg.C, 80 deg.C, 85 deg.C, 90 deg.C and 95 deg.C, and two extraction media of ethanol and distilled water, respectively, and steaming for 10min to obtain white tea extracts.

Thereafter, the mice subjected to CUMS treatment in accordance with example 2 were divided into groups for nasal feeding treatment, each using a high concentration, i.e., 40mg/kg, administration dose. After one week of nasal feeding, open field test was performed according to the method of example 7, sugar water consumption test was performed according to the method of example 4, olfactory evasion test was performed according to the method of example 10, and experimental effects of white tea extracts obtained under different extraction conditions were compared. As shown in tables 1-3, it is found that the white tea extract obtained by steaming at 85 ℃ for 10min and using water as the extraction medium has the best technical effect.

Table 1 shows the results of the open field test

Table 2 shows the results of the sugar water consumption test

Table 3 shows the results of olfactory evasion tests

In conclusion, the method for preparing the white tea extract is simple to operate, the obtained white tea extract is administrated intranasally, the sucrose preference of depression-like mice modeled by a CUMS (chronic unpredictable stress) method can be improved, the immobility time of the depression-like mice in forced swimming tests and tail suspension tests is reduced, the damage of mitochondria and synapses in hippocampal cells is improved, and the number of synapses is increased. And increases the content of serotonin (5-HT) and brain-derived neurotrophic factor (BDNF) in the hippocampus of the mouse. The white tea extract prepared by the method has partial repairing effect on olfactory injury of mice modeled by a CUMS method, can play a role in protecting olfactory sensation, and has more obvious effect of higher concentration.

The protection of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected.

SEQUENCE LISTING

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<120> white tea extract, preparation method, administration method and application in antidepressant

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Claims

1. A method for preparing a white tea extract, comprising the steps of:

(1) grinding and sieving the white tea leaves and the stems to obtain particles;

(2) and (2) mixing the particles in the step (1) according to the mass ratio of feed liquid of 1: 30-1: 70 mixing with water, heating, steaming, filtering, and collecting supernatant to obtain the white tea extract.

2. The method according to claim 1, wherein in the step (2), the mass ratio of the feed liquid is 1:50 are mixed with water.

3. The method according to claim 1, wherein in the step (2), the heating and cooking time is 8-12 min; and/or the heating and cooking temperature is 80-90 ℃.

4. The method of claim 1, further comprising the step of rapidly cooling and preserving the supernatant collected in step (2).

5. The method of claim 4, wherein the cooling is to cool the supernatant to a temperature of 30 ℃.

6. A white tea extract obtainable by the process of any one of claims 1 to 5.

7. A method of administering the white tea extract of claim 6 to a subject using nasal feeding.

8. Use of the white tea extract according to claim 6 for the preparation of foods, health products or medicaments for the treatment or alleviation of depression.

9. Use of the white tea extract according to claim 6 for the preparation of a food, health product or medicament for the treatment or alleviation of anhedonia caused by depression.

10. Use of the white tea extract according to claim 6 for the preparation of a food, health product or medicament for treating or alleviating a tendency to despair caused by depression.

11. Use of the white tea extract according to claim 6 for the manufacture of a food, health product or medicament for the treatment or alleviation of depressive-like behavior caused by CUMS, including reduced sugar water consumption, reduced forced swimming time, increased tail suspension immobilization time.

12. The use according to any one of claims 8 to 11 wherein the white tea extract is used as the sole active ingredient or one of the active ingredients of a food, health product or pharmaceutical.

13. The use of claim 12, wherein the food, health product or pharmaceutical is a single component white tea extract, or is a tablet comprising the white tea extract and one or more of a filler, a binder, a humectant, a disintegrant, an absorption enhancer, a solvent, a surfactant, a flavoring agent, a preservative, a lubricant, a sweetener and a pigment, or is an oral liquid formulation comprising the white tea extract and one or more of a buffer, an antioxidant synergist, a flavoring agent, a sweetener, a solvent, a surfactant and a preservative.

14. The use of claim 13, wherein the food, health product or pharmaceutical further comprises an ingredient that increases the stability of the white tea extract.

15. The use of claim 12, wherein the food, health product or pharmaceutical is a liquid formulation, a solid formulation, a spray or an aerosol.

16. The use according to claim 15, wherein the food, health product or pharmaceutical is an injection, suspension, emulsion or solution.

17. The use according to claim 15, wherein the food, health product or pharmaceutical is a syrup.

18. The use of claim 15, wherein the solid formulation is a tablet, capsule, granule or granule.