Application of snow white water lily extract

Abstract

The invention discloses an application of a snow white water lily extract. The invention relates to the field of medicines, in particular to the preparation of medicines for treating, preventing or improving depression and anxiety disorder, medicines for protecting brain neurons or repairing brain neuron injury, medicines for protecting tissues and organs induced by LPS, medicines for reducing expression of inflammatory factors 1L-1beta and / or TNF-alpha, medicines for treating, preventing or improving depression and anxiety disorder, medicines for protecting brain neurons or repairing brain neuron injury, medicines for protecting tissues and organs induced by LPS, medicines for reducing expression of inflammatory factors The invention also relates to an application in medicines for reducing the level of a hippocampus microglial cell marker Iba-1 and / or medicines for up-regulating the expression of brain-derived neurotrophic factors in hippocampus. The snow-white water lily extract is an ethyl acetate extract of a snow-white water lily ethanol water solution extract. The preparation method is green and safe, the raw materials are sustainable, the preparation process is simple, the activity is remarkable, and a foundation is provided for using the ethyl acetate extract of the water lily ethanol aqueous solution extract as an antidepressant and an antianxiety drug or as a main component or a characteristic component of the antidepressant and the antianxiety drug for clinically treating depression and the like.

Description

Technical Field

[0001] The invention belongs to the field of natural products, and particularly relates to application of a snow-white water lily extract. Background Art

[0003] At present, the treatment strategies for depression mainly include drug therapy, psychotherapy, cognitive behavioral therapy, physical therapy and sports therapy, among which drug therapy and psychotherapy occupy a core position. Common therapeutic drugs include fluoxetine (FLX), sertraline, imipramine, paroxetine and venlafaxine, however, the use of these drugs is often accompanied by a variety of adverse reactions, such as nausea, diarrhea, fever, cramps, and in severe cases may even lead to shock or death. Because depression is a complex mental illness, its exact mechanism involves the interaction of multiple biological, psychological and sociological factors, which is still under study. In addition, depression has a high recurrence and high response rate. More than 50% of patients with depression will relapse after recovery, and 30% of patients with depression do not respond to drug treatment or respond slowly. Compared with traditional antidepressants, natural plant extracts have multiple active ingredients, multiple targets and multiple pharmacological effects, and show the advantage of less toxic side effects. Some medicinal plants, such as saffron, turmeric, St. John's wort and ginseng, show potential antidepressant effects and have shown positive effects in in vitro and in vivo experiments. Therefore, it is of great significance to screen out low-toxic, effective and safe ingredients from natural products and develop drugs or foods with antidepressant effects.

[0004] Snow-white water lily (Nymphaea candida Presl) belongs to the genus Nymphaea of ​​the family Nymphaeaceae. It is a native plant of Xinjiang. It not only has high ornamental value, but also has good edible and medicinal value. In clinical Uyghur medicine, it is mainly used to treat headaches caused by fever, fever coughs, and acute and chronic convulsions in children.

[0005] So far, people have made some progress in the study of the composition and activity of snow-white water lilies. Studies have found that snow-white water lilies contain rich active ingredients, including phenolic acids, polysaccharides, flavonoids, alkaloids and lignans, and exhibit multiple biological activities such as anti-oxidation, anti-hepatitis, hypoglycemia and hypotension. Du Lijun et al. conducted a systematic study on the chemical composition of the dried flower buds of snow-white water lilies produced in Xinjiang, isolated 10 flavonoid compounds from 95% ethanol extracts, and conducted in vitro antioxidant experiments and studies on the effects of simulated ischemia on nerve cells (Study on the Antioxidant Effects of Flavonoids in Snow-White Water Lilies and the Effects of Simulated Ischemia on Nerve Cells [J]. World Science and Technology-Modernization of Traditional Chinese Medicine, 2006.5). Zhao Jun et al. isolated seven flavonoid compounds (gallic acid, rutin, quercetin, kaempferol, quercetin-3-methyl ether, and wheat flavonoid-7-methyl ether) from snow-white water lilies by water extraction and alcohol precipitation, followed by multiple purifications using AB-8 macroporous adsorption resin and sephadex-20 columns (Study on Chemical Components of Uyghur Medicine Water Lily [J]. Chinese Journal of Traditional Chinese Medicine, 2007.32). Chinese patent application CN201610625219.X uses a method of combining macroporous resin and polyamide resin to extract flavonoid components from water lilies to prepare a total flavonoid extract of water lilies. The extract has good anti-hepatitis effects, is highly efficient, and has low toxicity, and can be used in the prevention and treatment of liver damage. However, judging from the current research progress, reports on snow-white water lily extracts are mostly limited to ingredient analysis, as well as antioxidant, anti-inflammatory, and prevention of liver damage, and there are no reports on the research and development of other product uses. There is a lot of room for exploration as to whether the extracts obtained by different extraction methods are active, what kind of activity they have, how high the activity is, and whether the extracts obtained by existing extraction methods have new biological activities. Summary of the invention

[0006] In order to develop an antidepressant drug of natural plant extract, the present invention conducts in-depth research on the snow white water lily extract, in order to explore the medicinal value of the snow white water lily extract and expand the application range of the snow white water lily.

[0007] When exploring snow-white water lily, the present invention finds that the ethyl acetate extract of the ethanol aqueous solution extract of snow-white water lily has significant antidepressant and antianxiety activities, can protect or repair brain neurons, protect tissues and organs to prevent damage, reduce the expression of inflammatory factors, reduce the level of ionized calcium binding adapter molecule 1 (Iba-1), a marker of hippocampal microglia, up-regulate the expression of brain-derived neurotrophic factor (BDNF) in the hippocampus, up-regulate the NA content in the hippocampus, and the like; indicating that the ethyl acetate extract of the ethanol aqueous solution extract of snow-white water lily has good application prospects, and provides a new choice for clinical antidepressant, antianxiety and other treatments.

[0008] The invention discloses an application of a snow-white water lily extract, wherein the snow-white water lily extract can be used to prepare a medicine for treating, preventing or improving depression and anxiety.

[0009] The snow-white water lily extract can also be used to prepare drugs for protecting brain neurons (preventing and / or reducing their damage) or repairing brain neuron damage, drugs for protecting tissues and organs induced by LPS (preventing and / or reducing their damage), drugs for reducing the expression of inflammatory factors 1L-1β and / or TNF-α, drugs for reducing the level of hippocampal microglial marker Iba-1, drugs for up-regulating the expression of brain-derived neurotrophic factor in the hippocampus and / or drugs for up-regulating the norepinephrine content in the hippocampus.

[0010] The tissues and organs include one or more of the liver, lungs, intestines, etc.

[0011] The snow-white water lily extract is an ethyl acetate extract of an ethanol-water extract of a snow-white water lily. Preferably, the ethyl acetate extract of an ethanol-water extract of a snow-white water lily is an ethyl acetate extract of an ethanol-water extract of a snow-white water lily. The snow-white water lily refers to the flower of a snow-white water lily (Nymphaea candida Presl) plant, including one or more of an unopened snow-white water lily, a newly opened snow-white water lily, and a blooming snow-white water lily. The snow-white water lily includes one or more of flower parts such as petals and stamens. Preferably, the snow-white water lily is a dried flower of a snow-white water lily (Nymphaea candida Presl). Generally, fresh snow-white water lilies are picked and dried by ordinary sun drying or hot air drying to obtain dried flowers with a water content of less than 10% by mass.

[0012] Preferably, the preparation method of the ethyl acetate extract of the snow-white water lily comprises: crushing the dried snow-white water lily, refluxing extraction with an ethanol aqueous solution for multiple times, combining the extracts, removing the solvent and suspending in water, extracting with ethyl acetate for multiple times, combining the ethyl acetate extracts, removing the solvent, and obtaining the ethyl acetate extract of the snow-white water lily ethanol aqueous solution extract.

[0013] Preferably, the mass fraction of ethanol in the ethanol aqueous solution is 85%-95%, more preferably 90%.

[0014] Preferably, in each reflux extraction step, the ratio of milliliters of ethyl acetate to grams of snow-white water lily flowers is 10-20:1.

[0015] Preferably, the time of each reflux extraction is 1 hour to 1.5 hours.

[0016] Preferably, the reflux extraction is performed 2-3 times.

[0017] Preferably, the ethyl acetate extraction is performed in small amounts and multiple times, preferably 3-5 times.

[0018] Through in-depth research, the present invention found that the ethyl acetate extract of the ethanol aqueous solution extract of snow-white water lily has significant antidepressant and antianxiety effects. Multiple intraventricular injections of LPS induce depressive behavior in mice, and the antidepressant pharmacological effects of the ethyl acetate extract of the ethanol aqueous solution extract of snow-white water lily are studied; the antidepressant and antianxiety effects of the ethyl acetate extract of the ethanol aqueous solution extract of snow-white water lily are evaluated by open mine, elevated cross, tail suspension test and forced swimming test, and the toxicity of the ethyl acetate extract of the ethanol aqueous solution extract of snow-white water lily is evaluated by HE staining. In addition, the effects of the ethyl acetate extract of the ethanol aqueous solution extract of snow-white water lily on LPS-induced tissue and organ damage, the effects on the expression of inflammatory factors 1L-1β and / or TNF-α, and the effects on the content of microglia markers Iba-1, BDNF and NA in the mouse hippocampus and the changes in the density of hippocampal dendritic spines are also studied.

[0019] The present invention evaluates the effect of the ethyl acetate extract of the ethanol aqueous solution extract of the snow-white water lily on the modeling depression mice through a variety of experiments. The results show that the ethyl acetate extract of the ethanol aqueous solution extract of the snow-white water lily extracted from the natural product of the present invention has a significant improvement effect on the depression and anxiety behavior of mice, and the effect is better than the petroleum ether extract of the ethanol aqueous solution extract of the snow-white water lily; the ethyl acetate extract of the ethanol aqueous solution extract of the snow-white water lily is basically non-toxic, highly safe, and can be taken for a long time. The ethyl acetate extract of the ethanol aqueous solution extract of the snow-white water lily also has the effects of protecting or repairing brain neurons, protecting tissues and organs from damage, reducing the expression of inflammatory factors, reducing the level of hippocampal microglial marker Iba-1, up-regulating BDNF expression in the hippocampus, and up-regulating NA content in the hippocampus.

[0020] The snow white water lily extract of the present invention has a wide range of raw material sources, a simple and controllable preparation process, significant drug efficacy and good transformation prospects.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] The present invention first obtains the ethyl acetate extract of the ethanol aqueous solution extract of snow white water lily by reflux of ethanol aqueous solution and extraction with ethyl acetate, and explores the antidepressant, antianxiety and other effects of the ethyl acetate extract of the ethanol aqueous solution extract of snow white water lily for the first time. In the LPS-induced depression animal model experiment, the ethyl acetate extract of the ethanol aqueous solution extract of snow white water lily has significant antidepressant and antianxiety effects at low and high doses, and its efficacy is equivalent to or better than the antidepressant fluoxetine. In addition, the ethyl acetate extract of the ethanol aqueous solution extract of snow white water lily has no physiological toxicity, has potential protective effects on organs such as liver and lung, and can also significantly upregulate the expression of BDNF in the hippocampus of depressed mice, thereby increasing the content of BDNF in the hippocampus of depressed mice, significantly inhibiting the secretion of hippocampal microglia marker Iba-1 in the hippocampus of depressed mice, thereby reducing the level of Iba-1 in the hippocampus of depressed mice; upregulating the content of NA in the hippocampus; increasing the density of hippocampal dendritic spines, and protecting or repairing brain neurons.

[0023] The invention is green and safe, has sustainable raw materials, a simple preparation process, and significant activity, and provides a basis for the subsequent use of snow white water lily extract as an antidepressant, antianxiety drug, or as a main component or characteristic component in antianxiety drug, for clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a graph showing the effect of each experimental group on the number of crossings of mice in the center of the open field in the open field test (OFT), where the ordinate Total crossing (Number) is the total number of crossings (number);

[0025] Figure 2 This is a graph showing the effect of each experimental group in OFT on the total distance that mice move in the open field, where the ordinate Totaldistance (cm) is the total distance (cm);

[0026] Figure 3 This is a graph showing the effect of each experimental group on the number of times mice reach the open arm in the elevated plus maze test (EPM), where the ordinate open entry (Number) represents the number of times the mice reach the open arm;

[0027] Figure 4 This is a graph showing the effect of each experimental group on the time mice spend in the open arms in the EPM, where the ordinate opentime (Sec) represents the time (seconds) spent in the open arms;

[0028] Figure 5 This is a graph showing the effect of each experimental group on the immobility time of mice in the tail suspension test (TST), where the ordinate Immobility time (S) is the immobility time (seconds);

[0029] Figure 6 It is a graph showing the effect of each experimental group on the immobility time of mice in the forced swimming test (FST), where the ordinate Immobility time (S) is the immobility time (seconds);

[0030] Figure 7 Pathological sections of the heart, liver, lung, spleen, kidney, stomach, and intestine of mice in each experimental group;

[0031] Figure 8 for Figure 7 The middle part is the pathological section and the local magnified picture;

[0032] Fig. 9 Figure 2 shows the BDNF content in the hippocampus of mice in each experimental group;

[0033] Fig.10 The figures show the effects of each experimental group on the expression of Iba-1 and BDNF in the mouse hippocampus, where A is the immunofluorescence staining of Iba-1 in the hippocampal DG region; B is the semi-quantitative analysis of the relative fluorescence intensity of Iba-1 in the hippocampal DG region; C is the immunofluorescence staining of BDNF in the hippocampal DG region; D is the semi-quantitative analysis of the relative fluorescence intensity of BDNF in the hippocampal DG region; DAPI is 4',6-diamidino-2-phenylindole, Merge means merge, and the rightmost columns of A and C are enlarged views of the areas in the Merge columns;

[0034] Fig.11 The figure shows the effect of each experimental group on the density of dendritic spines in the mouse hippocampus, where A is a skeletal figure showing the changes in the density of dendritic spines in the mouse hippocampus; B is a quantitative analysis figure showing the density of dendritic spines in the mouse hippocampus;

[0035] Fig.12 This is a graph showing the effects of each experimental group on the content of inflammatory factor IL-1β in the hippocampus of mice;

[0036] Fig.13 This is a graph showing the effects of each experimental group on the content of inflammatory factor TNF-α in the hippocampus of mice;

[0037] Fig.14 This is a graph showing the effects of each experimental group on the norepinephrine (NA) content in the mouse hippocampus;

[0038] Fig.15The figure shows the effect of each experimental group on the content of each substance in the mouse serum; among them, ALT is alanine aminotransferase, TBIL is total bilirubin, ALB is albumin, TP is serum total protein, BUN is blood urea nitrogen, CREA is creatinine, TG is triglyceride, HDL is high-density lipoprotein, and LDH is lactate dehydrogenase.

[0039] The data in the figure were analyzed using GraphPad Prism8.0 software, and the data are expressed as mean ± standard deviation (n = 10). # indicates 0.01≤P<0.05, ## indicates 0.001≤P<0.01, ### indicates 0.0001≤P<0.001, #### indicates P<0.0001, compared with the Sham group; * indicates 0.01≤P<0.05, ** indicates 0.001≤P<0.01, *** indicates 0.0001≤P<0.001, **** indicates P<0.0001, ns indicates p>0.05, compared with the LPS group or between groups. P<0.05 indicates a significant difference, and P<0.01 indicates a very significant difference. DETAILED DESCRIPTION

[0040] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can better understand the technical solution of the present invention, but it should not be construed as limiting the technical solution of the present invention.

[0041] The snow-white water lily adopts the dried flowers of the snow-white water lily (Nymphaea candida Presl), and the water content by mass is less than 10%.

[0042] All reagents and materials required for the experiment were purchased from the market unless otherwise specified. ELISA kits: IL-1β (Shenzhen Misi Biotechnology Co., Ltd. (MIKX), SZ652); TNF-α (MIKX, SZ654); NA (MIKX, SZ653); BDNF (MIKX, SZ650).

[0043] Experimental animals: 6-8 week old male C57BL / 6J mice (initial weight 16g-20g) were purchased from Guangdong Medical Experimental Animal Center. Animal production license number: SCXK (Yue) 2022-0002. Mice were adapted to the environment for one week before the experiment, with free access to food and water, maintained at room temperature of 23℃±2℃, maintained a 12h / 12h day and night pattern, and fluorescent lighting from 9:00 to 21:00.

[0044] Preparation of experimental drugs: The dried buds of snow-white water lily were crushed and refluxed with an ethanol aqueous solution with an ethanol mass fraction of 90% for 3 times. The reflux extraction time was 1 h / time, and the solid-liquid ratio was 1:10 (g / mL) each time. The extracts were combined, concentrated under reduced pressure, and then suspended in water. They were extracted with ethyl acetate for 3 times, and the extracts were combined, concentrated under reduced pressure, and dried to obtain the ethyl acetate extract of the snow-white water lily ethanol aqueous solution extract (denoted as NCEA) for later use.

[0045] The dried buds of snow-white water lily were crushed, and refluxed and extracted three times with an ethanol aqueous solution with an ethanol mass fraction of 90%, the reflux extraction time was 1 h / time, and the solid-liquid ratio was 1:10 (g / mL) each time. The extracts were combined, concentrated under reduced pressure, and then suspended in water, and then extracted three times with petroleum ether, and the extracts were combined, concentrated under reduced pressure, and dried to obtain a petroleum ether extract of the snow-white water lily ethanol aqueous solution extract (referred to as NCPE) for later use.

[0046] Peripheral bacterial endotoxin lipopolysaccharide (LPS) (Sigma-Aldrich L2880-10 mg) was dissolved in 0.9% saline to prepare LPS saline solution with a concentration of 100 ng / μL.

[0047] NCEA, NCPE and fluoxetine hydrochloride (Beyotime, ST1330-50 mg) were suspended in a 0.5% by mass CMC-Na, i.e., sodium carboxymethylcellulose (ACMEC, C91598-500 g) distilled water solution.

[0048] Experimental instruments: rotary evaporator, fully automatic brain stereotaxic instrument, spontaneous activity (open field), elevated cross maze, iron frame, 1L beaker, animal motion trajectory tracking system (EthoVision XT), small animal bone drill, etc.

[0049] Example 1 LPS-induced depression modeling, grouping and drug administration in mice

[0050] LPS-induced depression model in mice: Mice were anesthetized with isoflurane through a mask and placed in a stereotaxic apparatus (RWD, Shenzhen, China). A 26s-gauge needle (Hamilton Company, 1701RN, Romania) of a 10 μL syringe (Hamilton) was inserted into the left ventricle through a burr hole, using the coordinates relative to the anterior fontanelle: 1.0 mm outside the horizontal plane, 0.35 mm behind, and 2.4 mm below. 1 μL of saline or 1 μL of LPS saline solution was injected into the ventricle at a rate of 4 nL / sec (nanoliter / second). After the injection, the needle was stopped for 5 minutes, the needle was removed, the wound was sutured, and iodine was used for disinfection. The experiment was injected on the first, third, and fifth days (once a day, a total of three injections). 24 hours after the last injection, mice were gavaged and administered with a dose of 0.3 mL / mouse / day for 7 consecutive days.

[0051] Grouping and administration: The animals were randomly divided into seven groups (n=10 / group): blank control group (Sham group, recorded as Sham), LPS group (recorded as LPS), LPS+fluoxetine hydrochloride 20 mg / kg group (recorded as FLX20), LPS+NCEA low-dose group, i.e. 10 mg / kg group (recorded as NCEA10), LPS+NCEA high-dose group, i.e. 50 mg / kg group (recorded as NCEA50), LPS+NCPE10 mg / kg group (recorded as NCPE10), and LPS+NCPE100 mg / kg group (recorded as NCPE100).

[0052] Sham: 1 μL normal saline was injected each time; 0.5% CMC-Na normal saline solution was administered by gavage;

[0053] LPS: 1 μL of 100 ng / μL LPS saline solution was injected each time; 0.5% CMC-Na saline solution was administered by gavage;

[0054] FLX20: Each injection of 1 μL of 100 ng / μL LPS saline solution; Fluoxetine hydrochloride was suspended in 0.5% CMC-Na saline solution at a dosage of 20 mg / kg mouse body weight and administered by gavage;

[0055] NCEA10: Each injection was 1 μL of 100 ng / μL LPS saline solution; NCEA was suspended in 0.5% CMC-Na saline solution at a dosage of 10 mg / kg mouse body weight and administered intragastrically;

[0056] NCEA50: Each injection was 1 μL of 100 ng / μL LPS saline solution; NCEA was suspended in 0.5% CMC-Na saline solution at a dosage of 50 mg / kg mouse body weight and administered intragastrically;

[0057] NCPE10: Each injection of 1 μL of 100 ng / μL LPS saline solution; NCPE was suspended in 0.5% CMC-Na saline solution at a dosage of 10 mg / kg mouse body weight and administered intragastrically;

[0058] NCPE100: Each injection was 1 μL of 100 ng / μL LPS saline solution; NCPE was suspended in 0.5% CMC-Na saline solution at a dosage of 100 mg / kg mouse body weight and administered intragastrically.

[0059] Example 2: Effect of Snow Lily Extract on LPS-induced Depressive Behavior in Mice

[0060] 1. Experimental methods

[0061] 1. Open Field Test (OFT)

[0062] The open field consisted of a single white polyethylene cubic open box (50 cm × 50 cm × 40 cm), the bottom of which was evenly divided into 16 small squares. Mice were placed individually in the open box and allowed to explore freely for 5 min. Their activities were observed and the total movement distance, number of entries into the center area, and activity time in the center area of ​​the mice were recorded using an animal movement trajectory tracking system (EthoVision XT15, Nolddus, Holland). After each mouse finished the experiment, the open box was cleaned and disinfected with 75% (mass fraction) alcohol.

[0063] 2. Elevated plus maze test (EPM)

[0064] After the open field experiment, the elevated plus maze experiment was performed. The EPM test was conducted in an open-air device consisting of two open arms (30 cm × 5 cm) and two closed arms (30 cm × 5 cm × 10 cm). The two arms were crossed, and the central area (5 cm × 5 cm) was 50 cm above the ground. The mouse was placed at one end of the open arm of the maze, with its back facing the experimenter, and the mouse was allowed to freely explore the maze for 5 min. The movement trajectory in the maze was recorded using an animal motion trajectory tracking system (EthoVisionXT15, Nolddus, Holland). After each mouse finished the experiment, the maze was cleaned and disinfected with 75% (mass fraction) alcohol.

[0065] 3. Tail suspension test (TST)

[0066] The tail of each mouse was fixed to the hanging rod (1 cm from the tip) with medical tape and hung upside down. The head of the mouse was about 15-20 cm away from the experimental table. The activity of the mouse was observed for 6 min using an animal motion trajectory tracking system (EthoVision XT15, Nolddus, Holland), and the immobility time (i.e., immobility time) after 4 min was recorded.

[0067] 4. Forced Swim Test (FST)

[0068] The forced swimming test was conducted in a 1000 mL transparent plastic beaker (15 cm in height and 10 cm in diameter) filled with 10 cm deep water at 23-25°C. Mice were placed in the beaker and allowed to swim for 6 min. The animal motion trajectory tracking system (EthoVision XT15, Nolddus, Holland) was used to observe the activity of the mice for 6 min, and the resting time (i.e., immobility time) of the next 4 min was recorded.

[0069] After the above behavioral experiment was completed, the mice were blooded from the eyeballs; and the mice were divided into two groups. After one group of mice was killed by cervical dislocation, the abdomen was placed on a polystyrene foam board with the limbs fixed with a needle, the outer fur of the abdomen was clamped with tweezers, and the skin was cut from the tail to the head with scissors to expose the mouse viscera and sternum, the sternum was cut, the diaphragm was cut horizontally, the left and right ribs were turned over along both sides of the sternum, and fixed with a needle, the heart was completely exposed, the needle was inserted from the apex at an angle parallel to the midline of the heart, a small opening was cut at the upper end of the right atrium, and blood was observed to flow out, and the needle was held and uniformly pushed with a mass percentage of 0.5% NaCl aqueous solution 20mL, and then replaced with a mass percentage of 4% paraformaldehyde (PFA) solution (solvent 0.01mol / LPBS) and perfused again. After the perfusion was completed, the needle was pulled out, and the heart, liver, spleen, lung, kidney, intestines and stomach were taken out in turn. The removed tissues and organs were placed in a mass percentage of 4% paraformaldehyde solution (solvent 0.01mol / LPBS) for fixation. Dehydrate and wax-immerse in a dehydrator, then place in an embedding machine for paraffin embedding, and cut the paraffin block into 4μm slices with a pathological slicer. Bake dry in an oven, take out and set aside at room temperature, dewax the slices and put them into water, rehydrate with graded concentrations of ethanol, treat with high-definition constant staining pretreatment solution for 1min, then stain with hematoxylin stain for 3-5min, and rinse with tap water. Dehydrate with 95% alcohol by mass for 1min, and stain with eosin stain for 15s. Dehydrate and make transparent in anhydrous ethanol, n-butanol, and xylene in turn, and seal with neutral gum. Scan the slices with a slide scanner for observation.

[0070] Another batch of mice were killed by cervical dislocation, and the whole brain was immediately removed on ice. The removed brains were divided into two groups. One group was immersed in a 4% PFA solution, and the brain was removed and embedded in paraffin. The coronary artery sections were sliced ​​using a pathological slicer (4 μm). Then, the membrane was permeated with a 0.3% Triton X-100 solution (solvent 0.01 mol / L PBS), and blocked with a 3% bovine serum albumin (BSA) distilled water solution. Primary antibodies, including rabbit anti-BDNF (1:2000, GB11559) and rabbit anti-Iba-1 (1:500, Servicebio, GB113502), and secondary antibodies HRP-labeled goat anti-rabbit IgG (1:500, Servicebio, GB23303) and CY3-labeled goat anti-rabbit IgG (1:300; Servicebio, GB21303), were used for BDNF and Iba-1 staining, respectively. Finally, DAPI (Servicebio, G1012) was added to counterstain the cell nuclei, the fluorescence quencher (Servicebio, G1221) was used to quench the tissue autofluorescence, and the anti-fluorescence quenching mounting medium (Servicebio, G1401) was used to mount the slides, and the images were collected.

[0071] Another group of brains were quickly rinsed with distilled water to remove blood stains on the surface. The mouse brain tissue was directly immersed in Golgi staining fixative (Servicebio, G1069-15ML), and then incubated in the stain at room temperature in the dark for 14 days. Tissue treatment solution was added and incubated at 4°C in the dark for 3 days. The slices were sliced ​​with a vibrating slicer (60 μm), and tissue treatment solution was dripped on the surface of the slices. After drying slightly, the slices were washed with water, and Golgi developer solution was dripped for 30 minutes. The slices were sealed with glycerol gelatin, and then the slices were scanned with a slice scanner and pannoramic scanner scanning software, and photographed. Image J was used to count the density and measure the length of dendritic spines.

[0072] (II) Experimental results

[0073] The ethyl acetate extract of the ethanol-water extract of snow-white water lily improves the depressive behavior of LPS-induced mice. Figure 1-Figure 6 shown.

[0074] In the OFT experiment, compared with the Sham group, the number of crossings in the central area of ​​the open field in the LPS group was significantly reduced (p<0.01), and the total movement distance of the mice was also significantly reduced (p<0.0001), indicating that LPS successfully induced depressive behavior in mice. Compared with the LPS group, the number of crossings in the central area of ​​the mice in the LPS group was increased to varying degrees by administering FLX (20 mg / kg, p>0.05), NCEA (10 mg / kg, p<0.05; 50 mg / kg, p>0.05), and NCPE (10 mg / kg, p>0.05; 100 mg / kg, p<0.0001) to the depressed mice (see Figure 1 ), among which, the NCEA low-dose group (10 mg / kg) and the NCPE high-dose group (100 mg / kg) significantly increased the number of central zone crossings in mice (see Figure 1 ); FLX (20 mg / kg, p<0.0001), NCEA (10 mg / kg, p<0.0001; 50 mg / kg, p<0.01), NCPE (10 mg / kg, p<0.01; 100 mg / kg, p<0.0001) treatment significantly improved the reduction in the total distance of mice moving (see Figure 2 In the OFT experiment, the main focus was on the total distance that mice moved in the open field. From the test results of the total distance that mice moved in the open field, NCEA showed a more significant improvement effect than NCPE (compared between the NCEA10 group and the NCPE10 group, p<0.0001; compared between the NCEA50 group and the NCPE10 group, p<0.01; compared between the NCEA10 group and the NCPE100 group, p<0.001).

[0075] In the EPM experiment, compared with the Sham group, the number of times (p<0.01) and time (p<0.0001) that mice in the LPS group entered the open arm were significantly reduced, indicating that LPS successfully induced depression and anxiety in mice. Compared with the LPS group, the administration of FLX (20 mg / kg), NCEA (10 mg / kg and 50 mg / kg), and NCPE (10 mg / kg and 100 mg / kg) to the depressed and anxious mice inhibited this phenomenon to varying degrees. The number of times and time that mice entered the open arm increased to varying degrees. The FLX (20 mg / kg), NCEA (10 mg / kg and 50 mg / kg), and NCPE (10 mg / kg and 100 mg / kg) groups significantly increased the number of times mice entered the open arm (FLX20, p<0.05; NCEA10, p<0.05; NCEA5 0, p<0.01; NCPE10, p<0.001; NCPE100, p<0.05); FLX (20 mg / kg), NCEA (10 mg / kg and 50 mg / kg) and NCPE (10 mg / kg) groups significantly increased the time mice spent in the open arms (FLX20, p<0.05; NCEA10, p<0.01; NCEA50, p<0.001; NCPE10, p<0.05), NCPE (100 mg / kg) group increased the time mice spent in the open arms to some extent, but the increase was not significant (NCPE100, p>0.05) (see Figure 3 and Figure 4 ). This indicates that NCEA and NCPE have anti-anxiety and anti-depressant effects. In the elevated plus maze experiment, the main focus was on the movement time of mice in the open arms. From the experimental results of the movement time of mice in the open arms, NCEA showed more significant anti-anxiety and anti-depressant effects than NCPE (compared with the NCPE10 group, the NCEA10 group p<0.05, the NCEA50 group p<0.01; compared with the NCPE100 group, the NCEA10 group p<0.01, the NCEA50 group p<0.001, see Figure 4 ). It is suggested that NCEA can be used to prepare medicines, health products or foods for treating, alleviating or preventing anxiety and depression.

[0076] In the TST and FST experiments, the results are shown in Figure 5 and Figure 6:Compared with the Sham group, the immobility time of the LPS group was significantly increased (TST: p<0.01; FST: p<0.01), indicating that LPS successfully induced depressive behavior in mice. Compared with the LPS group, the administration of FLX (20 mg / kg; TST: p<0.05; FST: p<0.05) and NCEA (10 mg / kg and 50 mg / kg; TST: NCEA10, p<0.05, NCEA50, p<0.0001; FST: NCEA10, p<0.001, NCEA50, p<0.0001) to the depressed mice reversed these changes and significantly reduced the immobility time of the depressed mice, among which the NCEA (50 mg / kg) group showed a very significant antidepressant effect (see Figure 5 and Figure 6 Compared with the LPS group, the administration of NCPE to depressed mice (10 mg / kg and 100 mg / kg; TST: NCPE10, p>0.05, NCPE100, p>0.05; FST: NCPE10, p>0.05, NCPE100, p>0.05) indicated that the petroleum ether extract of the ethanol aqueous solution extract of snow white water lily (NCPE) had a lower degree of relief in reducing the immobility time of depressed mice (10 mg / kg and 100 mg / kg had equivalent effects and could not effectively reduce the immobility time of depressed mice). The results were not significant, not as good as the ethyl acetate extract (NCEA) of the ethanol-water extract of snow white water lily, and there was a significant difference between NCEA and NCPE (TST: NCEA50 group compared with NCPE10 group, p<0.01; NCEA50 group compared with NCPE100 group, p<0.001; FST: compared with NCPE10 group, NCEA10 group p<0.05, NCEA50 group p<0.0001; compared with NCPE100 group, NCEA10 group p<0.01, NCEA50 group p<0.0001). This finding shows that the efficacy of the organic solvent extracts of snow white water lily is not equivalent or the difference is small, and it is valuable to conduct further research on extracts with different solvents.

[0077] The above experimental results collectively indicate that NCEA can effectively alleviate LPS-induced depression and anxiety behaviors in mice, indicating that NCEA can be used to prepare drugs, health products or foods for treating, preventing or improving depression and anxiety.

[0078] Example 3 Effects of Snow Lily Extract on LPS-induced Tissues and Organs in Mice

[0079] Pathological sections of mouse tissues and organs in each experimental group are shown in the figure below. Figure 7 and Figure 8As shown. In the LPS group, the epithelial cells of the small intestinal villus mucosa were destroyed and lost, the lungs showed mild capillary congestion and inflammatory cell infiltration in the interstitium, the liver showed disordered tissue structure, hepatocyte swelling, lipid vacuolar changes and mild inflammatory cell infiltration, and the other organs did not show obvious pathological changes. However, similar organ damage did not occur in the Sham group and the drug-treated group. It shows that the treatment of FLX (20 mg / kg), NCEA (10 mg / kg and 50 mg / kg), and NCPE (10 mg / kg and 100 mg / kg) prevented the damage of tissues and organs of depressed mice, protected various tissues and organs, and no obvious toxicity was observed at any dose, indicating that NCEA and NCPE have basically no toxic side effects on mouse organs and can be used as safe, long-term drugs, health products or foods.

[0080] From the results of HE staining sections, NCEA (10mg / kg and 50mg / kg) and NCPE (10mg / kg and 100mg / kg) had comparable protective effects on various organs of depressed mice, and showed protective effects on LPS-induced liver, lung and intestinal damage, without LPS-like damage.

[0081] Example 4 Effect of Snow Lily Extract on the Expression of Microglial Marker Iba-1 and Brain BDNF in Hippocampus of Depressed Mice

[0082] Microglial activation in the hippocampus is one of the causes of depression, and Iba-1 is a specific marker for microglial activation. Increased levels of proinflammatory cytokines in the brain can lead to impaired BDNF expression. The results of the effects of each experimental group on the expression of microglial markers Iba-1 and BDNF in the hippocampus of mice are shown in Figure 2. Fig. 9 and Fig.10 Compared with the Sham group, LPS injection induced an increase in the immunoreactivity of Iba-1 in the dentate gyrus (DG) subregion of the hippocampus ( Fig.10 A), the relative fluorescence intensity was also higher (p<0.01, Fig.10 B); however, treatment with FLX and NCEA (10 mg / kg, 50 mg / kg) significantly suppressed this change ( Fig.10 A), compared with the LPS group, the relative fluorescence intensity of the FLX group and the NCEA (10 mg / kg, 50 mg / kg) group was significantly decreased (FLX: p < 0.01; NCEA10: p < 0.01; NCEA50: p < 0.05; Fig.10 B). Fig. 9 and Fig.10 C shows that compared with the Sham group, LPS stimulation led to the decrease of BDNF content (p<0.0001) and expression level ( Fig.10C) was significantly decreased, and the relative fluorescence intensity was also significantly decreased (p<0.001, Fig.10 D); Compared with the LPS group, the FLX group and the NCEA group (10 mg / kg, 50 mg / kg) significantly upregulated the content of BDNF (FLX: p<0.01; NCEA10: p<0.01; NCEA50: p<0.05; Fig. 9 ) and expression levels ( Fig.10 C), significantly increased the relative fluorescence intensity of BDNF (FLX: p < 0.05; NCEA10: p < 0.01; NCEA50: p < 0.05; Fig.10 D). Compared with the LPS group, the NCPE10 (10 mg / kg) group significantly upregulated the BDNF content (NCPE10: p<0.05; Fig. 9 ), but the effect of NCPE100 (100 mg / kg) group in upregulating BDNF content was weaker (NCPE100: p > 0.05; Fig. 9 ). It can be seen that multiple intraventricular injections of LPS can induce microglial activation in the hippocampus and reduce the expression of BDNF; NCEA (10mg / kg, 50mg / kg) significantly inhibited this change; compared with the FLX group, both low-dose and high-dose NCEA showed similar effects to FLX. This shows that the NCEA of the present invention has a significant effect in inhibiting the expression of Iba-1 in the dentate gyrus (DG) subregion of the hippocampus and upregulating the level of BDNF in the hippocampus.

[0083] Example 5 Effect of Snow Lily Extract on Dendritic Spine Density in Hippocampus of Depressed Mice

[0084] Dendritic spines are formed by the protrusions of the dendritic membrane on the branches of neuronal dendrites. Their morphology and number are variable and play an important role in transmitting information between neurons and forming neural circuits. Damage to synaptic structure, changes in plasticity, atrophy of dendritic spines in the hippocampus, and loss of dendritic spines may all be one of the causes of depression.

[0085] Effects of each experimental group on dendritic spine density in the mouse hippocampus Fig.11As shown. Compared with the Sham group, the density of hippocampal dendritic spines and the number of intersections in the LPS group were significantly reduced (p<0.0001). Compared with the LPS group, the density of hippocampal dendritic spines and the number of intersections in the depressed mice treated with FLX and NCEA (10mg / kg, 50mg / kg) were significantly increased (FLX: p<0.0001; NCEA10: p<0.0001; NCEA50: p<0.01). Among them, the treatment effect of NCEA10mg / kg was more significant. This shows that NCEA can protect brain neurons or repair brain neuron damage, such as brain neuron damage caused by depression, and can be used to prepare drugs, health products and / or foods for protecting brain neurons or repairing brain neuron damage.

[0086] Example 6 Effect of Snow Lily Extract on Proinflammatory Cytokine Levels in Hippocampus of Depressed Mice

[0087] Increased levels of TNF-α and IL-1β can destroy the integrity of the animal blood-brain barrier, reduce neurogenesis in hippocampal cells, inhibit the action pathway of antidepressants, and greatly increase the depression rate. Hippocampal inflammation analysis: Use an ELISA kit to detect inflammatory factors IL-1β and TNF-α in the hippocampus. Add 10μL of sample and 40μL of sample diluent to a 96-well plate, incubate at 37°C for 30 minutes, wash the plate, then add enzyme-labeled reagent, incubate at 37°C for 30 minutes, then add color developer A and color developer B respectively, shake and mix, color at 37°C in the dark for 10 minutes, add stop solution to stop the reaction, and then measure the OD value at 450nm. See the test results. Fig.12 and Fig.13 .

[0088] Compared with the Sham group, the levels of inflammatory factors IL-1β and TNF-α in the hippocampus of the LPS group were significantly increased (IL-1β: p<0.0001; TNF-α: p<0.001). Compared with the LPS group, the levels of inflammatory factors IL-1β and TNF-α were significantly reduced after treatment with FLX and NCEA (10 mg / kg, 50 mg / kg) in the depressed mice (IL-1β: FLX, p<0.001; NCEA10, p<0.05; NCEA50, p<0.05; TNF-α: FLX, p<0.01; NCEA10, p<0.01; NCEA50, p<0.05). After the depressed mice were treated with NCPE (10mg / kg, 100mg / kg), the levels of inflammatory factors IL-1β and TNF-α were reduced to varying degrees. Among them, the levels of inflammatory factors IL-1β and TNF-α were significantly reduced after NCPE10 treatment (IL-1β: NCPE10, p<0.05; TNF-α: NCPE10, p<0.05), and the level of inflammatory factor TNF-α was significantly reduced after NCPE100 treatment (p<0.05), but the level of inflammatory factor IL-1β was not significantly reduced after NCPE100 treatment (p>0.05). The results showed that NCEA can significantly reduce the expression of inflammatory factors 1L-1β and / or TNF-α.

[0089] Example 7 Effect of Snow Lily Extract on Norepinephrine Levels in the Hippocampus of Depressed Mice

[0090] The disorder of central neurotransmitters such as norepinephrine, serotonin and dopamine is one of the pathogenesis of depression. Norepinephrine can induce "fight or flight" behavior and "anger and fear" emotions. Use an ELISA kit to detect the NA content in the hippocampus. Add 25μL of sample and 25μL of sample diluent to a 96-well plate, incubate at 37°C for 30 minutes, wash the plate, then add enzyme labeling reagent, incubate at 37°C for 30 minutes, then add color developer A and color developer B respectively, shake to mix, color at 37°C in the dark for 10 minutes, add stop solution to stop the reaction, and then measure the OD value at 450nm. See the test results. Fig.14 .

[0091] Compared with the Sham group, the NA content in the LPS group was significantly reduced (p<0.0001). Compared with the LPS group, the NA content in the hippocampus of the depressed mice treated with FLX, NCEA (10mg / kg, 50mg / kg) and NCPE (10mg / kg, 100mg / kg) was significantly increased (FLX, p<0.01; NCEA10, p<0.01; NCEA50, p<0.01; NCPE10, p<0.05; NCPE100, p<0.05). The test results showed that NCEA and NCPE could significantly reduce the NA content; the effect of NCEA was closer to FLX and better than NCPE.

[0092] Example 8

[0093] Serum biochemical indicators include liver function, kidney function and myocardial enzymes and other biochemical indicators: ALT, TBIL, ALB, TP, BUN, CREA, TG, HDL, LDH in the serum of mice in each experimental group were detected to determine the physiological toxicity of oral drugs in each experimental group to mice. The results are shown in Fig.15 .

[0094] Compared with the control group, the LPS group had decreased ALT and BUN levels, and increased ALB and TG levels ( Fig.15 A, C, E, G), but the changes were not significant, and the differences were not statistically significant; there were no significant changes in other biochemical indicators. The FLX group, NCEA group (10mg / kg, 50mg / kg) and NCPE (10mg / kg, 100mg / kg) treatment did not show toxicity to the liver, kidney and myocardial enzymes of mice. Referring to the range of biochemical indicators in serum, it was found that the biochemical indicators of each group were within the normal range. Tip: After the treatment of depressed mice with FLX, NCEA (10mg / kg, 50mg / kg) and NCPE (10mg / kg, 100mg / kg), the levels of ALT, TBIL, ALB, TP, BUN, CREA, TG, HDL and LDH in serum did not exceed the normal range, indicating that oral FLX, NCEA (10mg / kg, 50mg / kg) and NCPE (10mg / kg, 100mg / kg) have no physiological toxicity and are safe for oral use.

[0095] Comprehensive examples 2-8, the ethyl acetate extract of the ethanol aqueous solution extract of the snow-white water lily of the present invention has significant antidepressant and antianxiety effects at low and high doses, and its efficacy is comparable to or better than the antidepressant fluoxetine, and is better than the petroleum ether extract of the ethanol aqueous solution extract of the snow-white water lily, and can significantly improve depressive behavior and anxiety behavior, protect or repair brain neurons, reduce the expression of inflammatory factors, effectively inhibit the activation of microglial marker Iba-1 in the dentate gyrus subregion of the hippocampus, significantly upregulate the expression of BDNF and NA content in the hippocampus, increase the density of dendritic spines, and show basically no toxic side effects, high safety, can be taken for a long time, and has a potential protective effect on LPS-induced damaged tissues and organs. These results further confirm that NCEA has significant antidepressant activity and antianxiety activity, has the potential to become a new antidepressant and antianxiety drug, and can also be used for the adjustment and treatment of abnormal inflammatory factor expression, Iba-1, BDNF expression and NA content.

[0096] In the present invention, since the ethyl acetate extract of the ethanol aqueous solution extract of the snow-white water lily is extracted with the snow-white water lily as the raw material, and the ethanol aqueous solution with a mass fraction of 90%±5% as the extraction solvent in a state capable of reflux, the extract is extracted by ethyl acetate extraction, and the existing technology, such as changing the pressure to cause the reflux temperature to change, will not affect the effective extraction of the ethyl acetate extract of the ethanol aqueous solution extract of the snow-white water lily of the present invention. The change of parameters in the preparation method of the present invention does not affect the preparation of the ethyl acetate extract of the ethanol aqueous solution extract of the snow-white water lily, so any combination of parameters in the preparation method of the present invention can achieve the effective extraction of the ethyl acetate extract of the ethanol aqueous solution extract of the snow-white water lily, for example, the milliliter of the ethanol aqueous solution with a mass fraction of 90%±5% is adjusted to 20 times the gram of the snow-white water lily, the time of each reflux extraction is adjusted to 1.5 hours, the number of extractions is adjusted to 2 times, the number of ethyl acetate extractions is adjusted to 5 times, etc., the effective extraction of the ethyl acetate extract of the ethanol aqueous solution extract of the snow-white water lily can still be achieved, showing the efficacy of the ethyl acetate extract of the ethanol aqueous solution extract of the snow-white water lily of the present invention. They are not listed one by one here.

[0097] The above embodiments describe the technical solutions of the present invention in detail to help technicians understand and apply them. However, they are only specific embodiments and do not limit the scope of the present invention. Any modification, supplement or replacement made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of a snow-white water lily extract in the preparation of a drug for treating, preventing or improving depression and anxiety.

2. Use of a snow-white water lily extract in the preparation of a drug for protecting brain neurons or repairing brain neuron damage, a drug for protecting tissues and organs induced by LPS, a drug for reducing the expression of inflammatory factors 1L-1β and / or TNF-α, a drug for reducing the level of hippocampal microglial marker Iba-1, a drug for up-regulating the expression of brain-derived neurotrophic factor in the hippocampus, and / or a drug for up-regulating the content of norepinephrine in the hippocampus.

3. The use according to claim 2, characterized in that: The tissues and organs include one or more of the liver, lungs, and intestines.

4. The use according to claim 1 or 2, characterized in that: The snow white water lily extract is the ethyl acetate extract of the snow white water lily ethanol aqueous solution extract.

5. The use according to claim 4, characterized in that: The snow-white water lily extract is the ethyl acetate extract of the snow-white water lily ethanol aqueous solution extract.

6. The use according to claim 5, characterized in that: The preparation method of the snow-white water lily extract comprises: crushing dried snow-white water lily flowers, refluxing extraction with an ethanol aqueous solution for multiple times, combining the extracts, removing the solvent and suspending in water, extracting with ethyl acetate for multiple times, combining the ethyl acetate extracts, removing the solvent, and obtaining the ethyl acetate extract of the snow-white water lily ethanol aqueous solution extract.

7. The use according to claim 4, characterized in that: The mass fraction of ethanol in the ethanol aqueous solution is 85%-95%.

8. The use according to claim 6, characterized in that: In each reflux extraction step, the ratio of milliliters of the ethanol aqueous solution to grams of the snow-white water lily is 10-20:

1.

9. The use according to claim 6, characterized in that: The time of each reflux extraction is 1 hour to 1.5 hours, and the number of reflux extractions is 2 to 3 times.

10. The use according to claim 6, characterized in that: The number of times of ethyl acetate extraction is 3-5 times.

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