CN111358826A - Total flavone extract with antidepressant effect, and preparation method and application thereof - Google Patents
Total flavone extract with antidepressant effect, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a total flavone extract with an anti-depression effect, a preparation method and an application thereof, wherein the total flavone extract is prepared by the following preparation method: adding an extraction solvent into semen Cuscutae powder, heating and reflux-extracting, mixing filtrates, and concentrating to obtain extract; and (3) respectively and sequentially adding the extract into a polyamide column and a macroporous resin column for chromatography, and concentrating the obtained eluent to obtain the total flavone extract of the semen cuscutae. The total flavone extract prepared by the invention has high purity, and experimental results show that the semen cuscutae total flavone can obviously shorten the immobilization time of TST and FST of CUMS mice without influencing the autonomous activity of the TST and FST, and the semen cuscutae total flavone extract has stronger antidepressant action and no central excitation or inhibition action. And the dodder total flavonoids can activate a TXINIP/NLRP 3 inflammation corpuscle signal pathway, so that the dodder total flavonoids are proved to play an anti-depression role by regulating the TXINIP/NLRP 3 inflammation corpuscle pathway.
Description
Technical Field
The invention relates to the technical field of medicines, and particularly relates to a total flavone extract with an anti-depression effect, and a preparation method and application thereof.
Background
Depression is one of the most common mental diseases, is mainly characterized by remarkable and persistent emotional depression, is often accompanied by serious psychological and physiological disorders, has great harm to human health, and has huge investment on research, development and treatment every year in the world, thereby causing serious burden to society and families. In addition, depression patients often accompany changes in eating patterns associated with insomnia, lethargy, binge eating, weight loss or anorexia, and more seriously, some extreme manifestations of symptoms including circadian rhythms, endocrine disorders, and the like. At present, at least 9000 tens of thousands of depression patients in China are treated; meanwhile, in the suicide and suicide near-adolescence population in China, the height of depression patients is 50% -70%, which is second to ischemic heart disease, and brings serious harm to the health and social development of the people in China. The depression patients often need to take medicines for a long time, and the treatment period is long, so the research at present focuses on providing a medicine and a method for safely and efficiently treating the depression.
Currently, clinically commonly used antidepressant drugs can be classified into 5-hydroxytryptamine-norepinephrine reuptake inhibitors (SNRIs), selective 5-hydroxytryptamine reuptake inhibitors (SSRIs), monoamine oxidase A Reversible Inhibitors (RIMAs), NA reuptake inhibitors, tricyclic antidepressant drugs (TCAs), tetracyclic antidepressant drugs, specific 5-hydroxytryptamine antidepressant drugs, and monoamine oxidase inhibitors (MAOIs) according to their action mechanisms. Although these drugs can play a certain role in resisting depression, a series of side effects are generated along with the drug effect; moreover, investigations have shown that about one third of patients who are administered antidepressant drugs are forced to stop taking the drug due to intolerable side effects of the drug. The side effects such as serotonin toxicity (also called serotonin syndrome) are the main causes of mania, uneasiness, insomnia and the like; side effects also include hypertensive crisis, exacerbation of symptoms of dysphoria and the resulting loss of libido in the patient.
Therefore, how to develop a novel antidepressant with better curative effect and lower side effect from natural products is a problem which needs to be solved urgently by the technical personnel in the field.
Disclosure of Invention
The invention aims to solve the defects of the prior art, and provides a semen cuscutae total flavone extract with an antidepressant effect, a preparation method and application thereof through deep system research on chemical components of semen cuscutae.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for preparing a total flavone extract with an anti-depression effect comprises the following steps:
(1) adding extracting solvent into semen Cuscutae, heating and reflux extracting, filtering, mixing filtrates, and concentrating to obtain extract;
(2) loading the extract on polyamide column, performing column chromatography, eluting to obtain eluate, concentrating, diluting with water, standing, and centrifuging to obtain precipitate;
(3) separating the precipitate with macroporous adsorbent resin column, and eluting to obtain semen Cuscutae total flavone extract.
Preferably, the extraction solvent in the step (1) is ethanol with a volume concentration of 95%, wherein the volume ratio of the extraction solvent to the dodder is (5-8): 1.
preferably, the extraction times in the step (1) are 1-3 times, the heating reflux temperature is 80 ℃, and the time is 2 hours; the concentration is carried out at 65 ℃ under reduced pressure until no alcohol smell is produced.
Preferably, the rotation speed of the centrifugation in the step (2) is 3000-5000rpm, and the time is 30 min.
Preferably, the polyamide column in the step (2) is 60 meshes, and is eluted by 30% and 70% ethanol solutions respectively, the 30% and 70% ethanol eluates are collected, added with water, stirred and diluted, and then kept stand for 6-8h, and precipitates are obtained after centrifugation.
Preferably, in the step (3), the ratio of the medicinal materials to the resin by mass is 3: 1. loading the D-101 macroporous resin into a column at the pH of 4-6, dissolving the precipitate by using ethanol, and loading the precipitate at the volume flow of 3.0BV/h at room temperature for adsorption saturation; in the desorption process, eluting with 4BV of water, discarding the eluent, eluting with 50% ethanol solution for multiple times, collecting and combining the 5BV of eluent, and vacuum drying to obtain the semen Cuscutae total flavone extract.
The beneficial effects of the preferred technical scheme are as follows: the invention combines a polyamide column and a macroporous resin column, screens out ethanol eluent with optimal volume concentration, and adopts 50% ethanol for elution, so that the flavone compound can be eluted to the maximum extent, and the obtained product has low impurity content. In addition, the semen cuscutae total flavone extract prepared by the method has a good anti-depression effect, and can be used for preparing a medicine for preventing and treating depression.
It should be noted that the semen cuscutae total Flavonoids (FSC) can be extracted and separated from the mature seeds of semen cuscutae. Semen Cuscutae (Cuscuta chinensis Lam.), alias Zen Zhen, herba Taxilli, Hayawang, etc., is an annual parasitic plant of semen Cuscutae of Convolvulaceae (Convolvulaceae Juss.), and has the effects of tonifying liver and kidney, arresting seminal emission, reducing urination, etc., and its seed is pungent, sweet, neutral in flavor. Modern pharmacological studies show that the dodder has the effects of protecting liver and kidney, resisting osteoporosis, resisting an antioxidant, resisting aging, improving sexual function and the like.
The invention also provides an antidepressant total flavone extract which is prepared by adopting the method.
The total flavone extract disclosed by the invention has an anti-depression effect, and can be used for preparing a medicament or food for preventing or treating depression.
The invention also provides application of the total flavone extract prepared by the method in medicines or foods for preventing or treating depression.
Has the advantages that:
the invention discloses a preparation method of a traditional Chinese medicine composition, which is characterized in that the activity of the semen cuscutae total flavonoids is greatly screened, and experimental results show that the semen cuscutae total flavonoids can obviously shorten the immobilization time of TST and FST of CUMS mice without influencing the autonomous activity of the TST and FST, and show that the semen cuscutae total flavonoids extract has a strong antidepressant effect and has no central excitation or inhibition effect. The semen cuscutae total flavonoids can activate a TXINIP/NLRP 3 inflammation corpuscle signal passage, and prove that the semen cuscutae total flavonoids play an anti-depression role by adjusting the TXINIP/NLRP 3 inflammation corpuscle passage, the action intensity of the semen cuscutae total flavonoids is similar to that of fluoxetine, and the semen cuscutae total flavonoids can be used for preparing a medicament for treating depression. The medicine is prepared from semen cuscutae, belongs to nontoxic and harmless natural ingredients, does not cause serious side effects, is safe to take, and can solve the problem of treating depression.
According to the technical scheme, compared with the prior art, the invention discloses and provides an antidepressant total flavone extract, and a preparation method and application thereof, and the antidepressant total flavone extract has the following beneficial effects:
(1) the preparation method of the total flavone extract disclosed by the invention has the advantages of simple steps and high extraction efficiency, can save raw materials, and can obtain the high-purity total flavone extract with the antidepressant effect. Through comparison of experimental screening and preparation processes, the invention discovers that the purity of the total flavone can be greatly improved to more than 82% by combining polyamide chromatography (30% and 70% ethanol elution) and macroporous resin (50% ethanol solution elution).
(2) The total flavone extract extracted by the invention has obvious antidepressant activity, can be used for preparing antidepressant medicaments and foods, screens out an efficient low-toxicity effective component for treating depression, and provides a new source for seeking new antidepressant medicaments.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a bar graph of the effect of total flavonoids of dodder on SPT carbohydrate preference, TST, FST test immobility time and OFT autonomous activity in mice;
in fig. 1: (A) SPT sugar water preference level; (B) the total OFT distance; (C) the TST experiment immobility time; (D) the FST experiment is not moved for time; # P <0.01, # 0.05, # P, compared to the placebo group; p <0.05, P <0.01 compared to model group.
Figure 2 is a bar graph of the effect of protein expression on TXNIP/NLRP3 inflammatory-body signaling 14 days after administration of total flavonoids of dodder.
In FIG. 2, (A) TXNIP protein expression level, (B) NLRP3 protein expression level, (C) ASC protein expression level, (D) clear-caspase 1 protein expression level, (E) clear-IL-1 β protein expression level, # <0.05, # # P <0.01 as compared to the blank control group, P <0.05, # P <0.01 as compared to the model group.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 a method for preparing a total flavone extract having an antidepressant effect, comprising the steps of:
(1) the semen cuscutae medicinal materials are mixed according to the solid-liquid ratio of 1: 8, adding ethanol with the volume concentration of 95%, heating and refluxing for extraction for 3 times, 2 hours each time, filtering and combining the extracting solutions, and concentrating under reduced pressure at 65 ℃ until no alcohol smell exists;
(2) separating and purifying the concentrate in the step (1) by using a polyamide chromatographic column, eluting by using 30% and 70% ethanol solutions respectively, collecting 30% and 70% ethanol eluates, mixing, and concentrating to obtain a concentrated solution.
(3) Adding water into the concentrated solution obtained in the step (2), stirring and diluting, standing for 6-8h, and centrifuging (3000-.
(4) According to the mass ratio of the medicinal materials to the resin of 3: 1, loading D-101 macroporous resin into a column, adjusting the pH to 4-6, then taking the precipitate obtained by separation in the step (3), dissolving the precipitate with ethanol, loading the precipitate at the volume flow of 3.0BV/h at room temperature, and carrying out adsorption saturation; in the desorption process, eluting with 4BV of water, discarding the eluent, eluting with 50% ethanol solution with volume concentration, collecting and combining the 5BV of eluent, and vacuum drying to obtain the semen Cuscutae total flavonoids. The content of total flavonoids was found to be 82%.
Example 2 a method for preparing a total flavone extract having an antidepressant effect, comprising the steps of:
(1) the semen cuscutae medicinal materials are mixed according to the solid-liquid ratio of 1: 8, adding ethanol with the volume concentration of 95%, heating and refluxing for extraction for 3 times, 2 hours each time, filtering and combining the extracting solutions, and concentrating under reduced pressure at 65 ℃ until no alcohol smell exists;
(2) separating and purifying the concentrate in the step (1) by using a polyamide chromatographic column, eluting by using ethanol solutions with volume concentrations of 40% and 80%, respectively, collecting ethanol eluates with volume concentrations of 40% and 80%, mixing, and concentrating to obtain a concentrated solution.
(3) Adding water into the concentrated solution obtained in the step (2), stirring and diluting, standing for 6-8h, and centrifuging (3000-.
(4) According to the mass ratio of the medicinal materials to the resin of 3: 1, loading D-101 macroporous resin into a column, adjusting the pH to 4-6, then taking the precipitate obtained by separation in the step (3), dissolving the precipitate with ethanol, loading the precipitate at the volume flow of 3.0BV/h at room temperature, and carrying out adsorption saturation; in the desorption process, eluting with 4BV of water, discarding the eluent, eluting with 60% ethanol solution with volume concentration, collecting and combining the 5BV of eluent, and vacuum drying to obtain the semen Cuscutae total flavonoids. The content of total flavonoids is 60 percent.
Example 3 antidepressant test
1. The modeling method of the chronic stress stimulation depressed mouse comprises the following steps:
the experimental mice were given 2% sugar water acclimation for 3 days prior to the depression molding. After 3 days, the normal group of mice was kept separately from the other groups of mice, and water was fasted and deprived when the sugar water preference experiment was performed, and free food was taken at other times. Making animal model by 3 weeks of chronic mild stimulation including cold water swimming (15 deg.C, 6 min); tail clamping (1 min); water is forbidden (24 h); fasting (24 h); raising in an inclined cage (taking out the padding, inclining the cage by 45 degrees); wet rearing (100 ml of water added to the bottom of the cage with sawdust); lighting and stimulating all night. I.e. mice received one stimulus per day, ensuring that each stimulus was performed randomly but not consecutively, at least twice a week with overnight lighting.
2. Research of mouse SPT experiment by semen cuscutae total flavonoids
(1) Experimental methods
Animals were randomly divided into a control group (con), a chronic stress model group (mod), a semen cuscutae total flavone administration low dose group (L-AST,50mg/kg) prepared in example 1, a semen cuscutae total flavone administration high dose group (H-AST,100mg/kg) prepared in example 1, and a fluoxetine group (positive drug control group, Flu,20mg/kg) in advance, and 10 mice per group. The administration is carried out continuously for 14 days, and the behavioral test is carried out after the administration by gavage once a day. All mice were housed in a single cage with a 2% sugar solution and a 2% pure water bottle administered to each mouse, prior to the sugar solution test, with prior fasting and water deprivation for 24 hours. The sugar water bottle and the pure water bottle were exchanged once after one hour. And after two hours the consumption of sugar water and pure water was weighed and recorded. According to the syrup preference formula: sugar water consumption was calculated as% sugar water preference ═ sugar water consumption (g)/total consumption (sugar water consumption (g) + pure water consumption (g) multiplied by 100%.
(2) Results of the experiment
As shown in fig. 1, intragastric administration of semen cuscutae total flavonoids can significantly reverse the decrease of sugar water preference in SPT in depressed mice caused by 3 weeks of chronic stress stimulation. The positive control drug fluoxetine also shows the same effect, and the result shows that the sugar water preference of depressed mice is obviously improved by intragastric administration of the semen cuscutae total flavonoids, and the semen cuscutae total flavonoids have a certain anti-depression effect.
3. Research of total flavonoids of semen cuscutae on mouse TST (mouse TST) experiment
(1) Experimental methods
Animals were randomly divided into a control group (con), a chronic stress model group (mod), a dodder total flavonoids administered low dose group (L-AST,50mg/kg) prepared in example 1, a dodder total flavonoids administered high dose group (H-AST,100mg/kg) prepared in example 1, and a fluoxetine group (positive drug control group, Flu,20mg/kg), each group consisting of 10 mice, and subjected to a behavioral test after intragastric administration once a day for 14 days continuously, the tail box was 25 × 25 × 25cm, the center of the top plate was connected to a small clip, the adhesive tape was attached to the tail end of the mouse at 2cm, the adhesive tape was clipped to allow the mouse to assume an inverted suspended position with the head about 5cm from the bottom of the tail box, and the immobility time of 4min after the test period of 6min was recorded using ANY behavioral analysis software.
(2) Results of the experiment
As shown in fig. 1, gavage administration of semen cuscutae total flavonoids can significantly reduce immobility time of depressed mice in the TST experiment. The positive control drug fluoxetine also shows the same effect, and the result shows that the gastric administration of the semen cuscutae total flavonoids obviously shortens the immobility time of a depressed mouse in a TST (time of onset test) experiment and has better anti-depression effect.
4. Research of mouse FST experiment by semen cuscutae total flavonoids
(1) Experimental methods
Animals were randomly divided into a control group (con), a chronic stress model group (mod), a semen cuscutae total flavone administration low dose group (L-AST,50mg/kg) prepared in example 1, a semen cuscutae total flavone administration high dose group (H-AST,100mg/kg) prepared in example 1, and a fluoxetine group (positive control group, Flu,20mg/kg) in advance, and 10 mice per group. The behavioral tests were performed after a single daily gavage administration for 14 consecutive days. The mice were placed in a round glass container 20cm high, 12cm in diameter, 10cm in water depth, at 25 ℃ and the immobility time 4min after the mice in the 6min test period was recorded using ANY ANY-maze behavioural analysis software. The standard for determining immobility is that the animal stops struggling in water, is in a floating state, and only has small limb movement to keep the head floating on the water.
(2) Results of the experiment
As shown in fig. 1, gavage administration of semen cuscutae total flavonoids can significantly reduce immobility time of depressed mice in FST experiments. The positive control drug fluoxetine also shows the same effect, and the result shows that the gastric administration of the semen cuscutae total flavonoids obviously shortens the immobility time of a depressed mouse in an FST (free space time) experiment and has a certain antidepressant effect.
5. Research of semen cuscutae total flavonoids on mouse OFT experiment
(1) Experimental methods
Animals were randomly divided into a control group (con), a chronic stress model group (mod), a doddor total flavonoids administration low dose group (L-AST,50mg/kg) prepared in example 1, a doddor total flavonoids administration high dose group (H-AST,100mg/kg) prepared in example 1, and a fluoxetine group (positive control group, Flu,20mg/kg), 10 mice per group, and behavioral testing was performed after gastric gavage once a day for 14 days continuously, and the activity of each mouse in a well-transparent acrylic box (40 × 40 × 15cm) was recorded using ANY ANY-maze behavioral collection analysis software, and the total course of the mouse in the central area was calculated within 5 minutes.
(2) Results of the experiment
As shown in figure 1, compared with the total movement path of the mice with the gastric lavage of the semen cuscutae total flavonoids, the mice have no obvious difference, and the positive control drug fluoxetine also shows the same effect. The results show that the gastric administration of the semen cuscutae total flavonoids has no obvious influence on the autonomous activity of mice, and the administration of the medicine is safe without central excitation or inhibition.
6. Research on regulation and control of mouse TXNIP/NLRP3 inflammatory corpuscle signal pathway by semen cuscutae total flavonoids
(1) Experiment grouping
Male BALB/C mice, body weight between 20-22 g. The mice are raised in the center of the experimental animal barrier, the temperature and the relative humidity are respectively controlled at 22 +/-1 ℃ and 60% +/-10%, 12 hours are taken as the light-dark period, and the mice can freely eat and drink water. Mice were randomly divided into a control group (con), a chronic stress model group (mod), a semen cuscutae total flavone administration low dose group (L-AST,50mg/kg) prepared in example 1, a semen cuscutae total flavone administration high dose group (H-AST,100mg/kg) prepared in example 1, and a fluoxetine group (positive control group, Flu,20mg/kg), and 10 mice per group. The feeding conditions are strictly controlled. The administration is performed by intragastric administration before stress every morning.
(2) On the next day after the behavior test experiment, the skull of the mouse is cut off rapidly by using a large-size blunt shear, the mouse is immediately placed on an ice bag to strip off the brain, the left and right hippocampal parts are separated out and are subpackaged in an EP tube, the mouse is placed in liquid nitrogen for rapid freezing, and then the mouse is transferred to an ultra-low temperature refrigerator at minus 80 ℃ for storage and testing.
(3) Sample detection method
Western blot detection: extraction of the protein was performed using lysis buffer on ice, followed by centrifugation of the protein in a high speed centrifuge at 4 ℃ for 5 minutes, 0.5mL of the supernatant was taken in an EP tube using a pipette gun, and the total protein concentration was determined using the BCA kit. The extracted proteins were further subjected to SDS-PAGE gel electrophoresis and rapidly transferred to a PVDF membrane. The appropriate antibody was selected for overnight incubation in a refrigerator at 4 ℃ followed by incubation in the secondary antibody for about 1 hour at room temperature. Finally, the protein concentration was quantified using an imaging system to detect the expression of TXNIP/NLRP3 inflammatory corpuscle signaling pathway key node proteins (TXNIP, NLRP3, ASC, clear-caspase-1, clear-IL-1 beta) in the hippocampus.
(4) Results of the experiment
As shown in figure 2, the semen cuscutae total flavonoids can activate the inflammatory corpuscle signal pathway of TXINIP/NLRP 3, and further regulate related node proteins to play an antidepressant role. The action intensity of the compound is similar to that of fluoxetine, and the compound can be used for preparing a medicament for treating depression.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (8)
1. A preparation method of a total flavone extract with an anti-depression effect is characterized by comprising the following steps:
(1) adding an extraction solvent into semen cuscutae, heating and refluxing for 1-3 times, filtering, combining filtrates, and concentrating to obtain an extract;
(2) loading the extract on polyamide column, performing column chromatography, eluting to obtain eluate, concentrating, diluting with water, standing, and centrifuging to obtain precipitate;
(3) separating the precipitate with macroporous adsorbent resin column, and eluting to obtain semen Cuscutae total flavone extract.
2. The preparation method of a total flavone extract with an antidepressant effect according to claim 1, characterized in that, the extracting solvent in step (1) is 95% ethanol by volume concentration, wherein the volume weight ratio of the extracting solvent to the dodder is (5-8): 1; heating and refluxing at 80 deg.C for 2 hr; the concentration is carried out at 65 ℃ under reduced pressure until no alcohol smell is produced.
3. The method for preparing a total flavone extract having an antidepressant effect according to claim 1, characterized in that the polyamide column in step (2) is 60 mesh, the eluent is 30% and 70% by volume ethanol solution, and 30% and 70% by volume ethanol eluent is collected.
4. The method for preparing total flavone extract having antidepressant effect as claimed in claim 1, wherein the rotation speed of the centrifugation in step (2) is 3000-5000rpm for 30 min.
5. The preparation method of total flavonoids with antidepressant effect according to claim 1, characterized in that in step (3) the ratio of the mass of the medicinal materials to the mass of the macroporous adsorbent resin is 3: 1, loading the mixture on a D-101 macroporous adsorption resin column, dissolving and precipitating the mixture by adopting ethanol, and loading the mixture at the volume flow of 3.0BV/h at room temperature for adsorption saturation; in the desorption process, eluting with 4BV of water, discarding the eluent, eluting with 50% ethanol solution with volume concentration, collecting and combining the 5BV of eluent, and vacuum drying to obtain the semen Cuscutae total flavone extract.
6. A total flavone extract having an antidepressant effect, characterized in that it is prepared by the method of any one of claims 1 to 6.
7. Use of the total flavone extract with antidepressant effect according to claim 7 in the preparation of medicaments or foods for preventing or treating depression.
8. The use of claim 7, wherein the medicament is in the form of capsule, pill, tablet, granule, oral liquid, mixture or powder.
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