Application of senecio scandens extract in preparation of medicine for preventing and treating depression and anxiety behaviors

Abstract

The invention relates to the technical field of natural medicines, and particularly provides application of a senecio scandens extract in preparation of medicines for preventing and treating depression and anxiety behaviors. The climbing groundsel herb ethyl acetate extract has a remarkable improvement effect on depression and anxiety behaviors of depression mice. Animal experiments prove that the senecio scandens ethyl acetate extract can remarkably increase the staying time of depression mice in a central area and the number of times of entering the central area in an open field experiment; in an elevated cross maze experiment, the number of times that a depression mouse enters an open arm and the staying time of the depression mouse in a target area can be obviously increased, the depression and anxiety behaviors of the mouse are well improved, and the exploration behavior and interestingness of the mouse in a new environment are improved. The climbing groundsel herb ethyl acetate extract provides a new thought for treating depression and anxiety behaviors by adopting natural medicines, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of natural medicine technology, specifically providing the application of Senecio scandens extract in the preparation of drugs for the prevention and treatment of depression and anxiety. Background Technology

[0002] Depression (DEP) is a serious mental disorder that severely impairs health and quality of life, typically manifesting as anhedonia, depressed mood, fatigue, sleep disturbances, appetite disorders, and cognitive decline. The pathological causes of depression are related to multiple factors, such as neuronal atrophy, synapse loss, and decreased neurotransmitter activity in the central nervous system (CNS) due to inflammatory responses. The theory of the monoamine neurotransmitter system (such as norepinephrine, serotonin, and dopamine) has dominated research on its pathogenesis, leading to the development of various antidepressants, including SSRIs, SNRIs, tricyclic antidepressants, and monoamine oxidase inhibitors. However, these drugs suffer from delayed onset of action, numerous side effects, and even ineffectiveness in some patients.

[0003] Anxiety disorders (AD) are mental disorders characterized by excessive and persistent worry, tension, and anxiety, often accompanied by autonomic hyperactivity, such as palpitations, chest tightness, and motor restlessness, leading to impaired social functioning, poor concentration, memory loss, and other cognitive problems. It is generally believed that anxiety disorders result from the interaction of genetic and environmental factors, leading to disorders in neurotransmitter metabolism, endocrine function, and the immune system. Clinical treatment primarily involves psychotherapy, physical therapy, and medication. Medication is often the first-line treatment due to its low cost and rapid effectiveness. However, existing medications have drawbacks such as tolerance, high addictive potential, and potential side effects, significantly impacting efficacy and patient recovery. Therefore, the search for safe, effective, and low-side-effect natural remedies for depression and anxiety has become a research hotspot in this field.

[0004] Senecio scandens Buch-.Ham., a perennial herb belonging to the genus Senecio in the family Asteraceae, is mainly distributed in Qinghai Province, Tibet Autonomous Region, and Gansu Province. Its dried aerial parts are used medicinally; they are bitter and cold in nature. Modern pharmacological studies have shown that extracts and some monomeric compounds of Senecio scandens possess various pharmacological effects, including antibacterial, antiviral, antitumor, and analgesic properties. Clinically, Senecio scandens is often used to treat carbuncles, boils, colds, eczema, and other acute inflammatory diseases. However, its effects on antidepressant and / or anxiety behaviors have not been reported. Summary of the Invention

[0005] To address the problems in the technical background, this invention provides a safe and effective new pharmaceutical application of Senecio scandens. The ethyl acetate extract of Senecio scandens has a significant effect on preventing and treating depression and anxiety behaviors, with no obvious toxic side effects, and can be prepared into any dosage form of capsules, tablets, granules, powders, oral liquids, or pills.

[0006] To achieve the above objectives, the present invention provides the following technical solution: The extraction method for the ethyl acetate extract of *Senecio scandens* according to this invention is as follows: Place sliced ​​*Senecio scandens* into a three-necked flask, add an ethanol-water solution, and heat under reflux three times. Filter while hot, combine the filtrates, and concentrate under reduced pressure. Stop rotary evaporation when the alcohol extract reaches 10% of the ethanol-water solution. Add the alcohol extract, petroleum ether, dichloromethane, and ethyl acetate sequentially to a separatory funnel, extract three times respectively, and collect the ethyl acetate layer after rotary evaporation. Dry the ethyl acetate layer solution to obtain the ethyl acetate extract of *Senecio scandens*.

[0007] The components of the ethyl acetate extract of Senecio scandens obtained by this invention include flavonoids and flavonoid glycosides such as quercetin, isoquercetin, and afrodextrin, as well as chlorogenic acid, a phenolic acid.

[0008] The ethyl acetate extract of Senecio scandens obtained by this invention can significantly increase the number of times depressed mice enter the open arm in the elevated cross maze experiment and increase the time the mice stay in the target area of ​​the open arm; it can also significantly increase the frequency of depressed mice entering the central area in the open field experiment and prolong the time the depressed mice stay in the central area of ​​the open field without affecting the total movement distance of the depressed mice.

[0009] Compared with existing technologies, the present invention has the following advantages: (1) This invention takes the ethyl acetate extract of Senecio scandens as the research object and rationally uses the methods of medicinal chemistry, pharmacology and biology to prove that it can be developed as a new substance for antidepressant and anti-anxiety behavior, thus expanding the application scope of Senecio scandens.

[0010] (2) The natural drug extract of the present invention contains a variety of bioactive components, including flavonoids and flavonoid glycosides such as quercetin, isoquercetin, afrodextrin, and chlorogenic acid, which have the characteristics of multiple components and multiple targets.

[0011] (3) The natural drug extract of the present invention can significantly increase the number of times and the time spent in the open arm of the elevated cross maze of depressed mice, and increase the number of times and the time spent in the central area of ​​the open field. This demonstrates the significant effect of the ethyl acetate extract of Senecio scandens in preventing and treating depression and anxiety. It has no obvious toxic side effects and its efficacy is better than that of the antidepressant fluoxetine at the same dose.

[0012] (4) The natural drug extraction process of the present invention is simple and easy to operate, suitable for standardized scale-up production, which can significantly improve production efficiency and economy, and avoid complex separation and purification processes and high costs.

[0013] (5) In H&E staining pathological tests, the natural drug extract of the present invention had no significant effect on the liver and kidneys of mice and had no toxic side effects. Attached Figure Description

[0014] Figure 1 This is the total ion chromatogram of the ethyl acetate extract of Senecio scandens in positive ion mode.

[0015] Figure 2 This is the total ion chromatogram of the ethyl acetate extract of Senecio scandens in negative ion mode.

[0016] Figure 3 The effect of ethyl acetate extract of Senecio scandens on the number of times a depressed mouse model entered the open arm of the elevated cross maze experiment.

[0017] Figure 4 To investigate the effect of ethyl acetate extract of Senecio scandens on the time spent in the target area of ​​the elevated cross maze in a depressed mouse model.

[0018] Figure 5 The effect of ethyl acetate extract of Senecio scandens on the dwell time in the central region of an open field in a depressed mouse model.

[0019] Figure 6 The effect of ethyl acetate extract of Senecio scandens on the number of times a depressed mouse model enters the central region of an open field experiment.

[0020] Figure 7 The effects of the ethyl acetate extract of Senecio scandens on the liver and kidneys of mice were evaluated using H&E staining. Detailed Implementation

[0021] The present invention is further described in detail through the following embodiments, but it should be noted that the scope of the present invention is not limited by these embodiments.

[0022] The detection method used in this invention: Liquid chromatography-mass spectrometry (LC-MS) analysis was performed using an Agilent Technologies AB SCIEX Instruments 6600 triple quadrupole mass spectrometer and a Shimadzu Prominence-I LC-2030C 3D high-performance liquid chromatography system. Chromatographic separation was performed on an Excsep™ C18 column (5 μm, 4.6 mm × 250 mm). The mobile phase was water and acetonitrile, with a flow rate of 0.3 mL / min and an injection volume of 2 μL, at 40°C. Gradient elution was performed as follows: 0–55 min, from acetonitrile:water = 20:80 to = 70:30; 55–60 min, returning to column equilibration at 20:80. The detection wavelength was 254 nm. The mass spectrometer parameters were set to scan in both positive and negative ion modes. Curtain gas (CUR) 35 psi, atomizing gas (GS1) 50 psi, auxiliary gas (GS2) 50 psi; ion source temperature 550°C; electrospray voltage 5500 V in positive mode and -4500 V in negative mode; declusivation voltage and collision energy set to ±60 V and ±35 V respectively, with a collision energy error range of 15 eV.

[0023] Example 1: Preparation process of ethyl acetate extract of Senecio scandens

[0024] First, place 100 g of *Senecio scandens* slices into a three-necked flask, add 1 L of 70% ethanol-water solution, and then place the flask in a heating mantle. Heat under reflux and condense at 80 ℃. After a slight boil, control the temperature at around 60 ℃. After 2 hours, collect the first batch of filtrate and filter while hot. Retain the residue and continue adding 1 L of 70% ethanol-water solution to the three-necked flask. Reflux and condense for 1.5 hours, then collect the second batch of filtrate and filter while hot. Retain the residue and continue adding 1 L of 70% ethanol-water solution to the three-necked flask. Reflux and condense for 1 hour, then collect the third batch of filtrate. Discard the residue, filter while hot, and combine the three batches of filtrate. Concentrate the filtrate under reduced pressure using a rotary evaporator. After removing 100 mL of the remaining alcohol extract, rotary evaporation was stopped. 5 mL of the extract was then freeze-dried to obtain the alcohol extract of *Senecio scandens*. The separatory funnel was checked for airtightness before extraction began. The remaining alcohol extract was added to the separatory funnel, and 100 mL of petroleum ether was measured and added to the funnel. Extraction was performed three times. 100 mL of dichloromethane was added to the lower layer (after petroleum ether extraction), and extraction was performed three times. The lower layer was collected. 100 mL of ethyl acetate was added to the upper layer (after dichloromethane extraction), and extraction was performed three times. The upper ethyl acetate layer was collected. The upper layer was evaporated to dryness to obtain the ethyl acetate extract of *Senecio scandens* (EESS). The components of the ethyl acetate extract were analyzed using LC-MS / MS, obtaining full-scan total ion chromatograms in both positive and negative ion modes. Figure 1 , Figure 2 As shown in Table 1, the components of the ethyl acetate extract of Senecio scandens include flavonoids and flavonoid glycosides such as quercetin, isoquercetin, and afrodextrin, as well as chlorogenic acid, a phenolic acid.

[0025] Table 1. LC-MS / MS analysis results of ethyl acetate extract of Senecio scandens

[0026] Example 2: Elevated Cross Maze Experiment

[0027] 1. Animal grouping and administration

[0028] Mice were SPF-grade male C57BL / 6 (7 weeks old), provided by the Experimental Animal Center of Nantong University. All mice were of similar age and weight, with an initial weight of approximately 20-25 g. All mice were acclimatized for 7 days at a room temperature of 20±2 ℃ and humidity of 40-60%, with a 12-hour light-out cycle from 7:00 to 19:00. Water and food were freely available. All mice were randomly assigned to four groups: a normal control group (Control); a corticosterone model group (Model, 20 mg / kg); a corticosterone + positive control group (Fluoxetine, 20 mg / kg); and a corticosterone + Senecio scandens ethyl acetate extract group (QLG, 20 mg / kg). Behavioral assessments were performed on the depressed model mice after model establishment and drug administration.

[0029] 2. Elevated Cross Maze (EPM)

[0030] 2.1 Equipment Preparation

[0031] The elevated cross maze utilizes the conflicting behaviors of animals' exploratory nature towards novel environments and their fear of suspended open arms to examine their anxiety states. This device consists of one pair of open arms and one pair of closed arms, each arm measuring 25 × 5 cm, extending outwards from a central 5 × 5 cm cross-shaped platform. The two opposing arms are enclosed by a 20 cm opaque wall, forming the two closed arms. The entire cross maze is 40 cm above the ground. Mice, due to their preference for darkness, tend to move within the closed arms, but their exploratory nature leads them to want to enter the open arms, thus triggering a conflicting behavior of exploratory impulse and fear, resulting in anxiety.

[0032] 2.2 Experimental Methods

[0033] Mice were placed at the central intersection of an elevated cross maze, head to the open arm area. The mice's free movement trajectory was recorded over 5 minutes using the ANY-maze analysis system. The number of times mice entered the open and closed arms and the duration of their stay in each arm were analyzed. The time spent in the open arm and the number of entries were calculated. Specifically, the percentage of time spent in the open arm (%) = (Open arm stay time / (Open arm stay time + Closed arm stay time) × 100%), and the percentage of times mice stayed in the open arm (%) = (Number of times mice stayed in the open arm / (Number of times mice stayed in the open arm + Number of times mice stayed in the closed arm) × 100%. The number of entries into the open arm and the duration of stay were negatively correlated with depression and anxiety in the model mice. Fewer entries into the open arm and shorter stay times indicated more severe depression and anxiety; increased entries into the open arm and longer stay times indicated that the drug had antidepressant and anti-anxiety effects.

[0034] 3. Experimental Results

[0035] Figure 3 , Figure 4 As shown, the number of times the model group of depressed mice entered the open arm was significantly lower than that of the normal group (P < 0.001). Compared with the model group, 20 mg / kg of Senecio scandens ethyl acetate extract significantly increased the number of times the depressed model mice entered the open arm (P < 0.001), and its effect was even better than that of the positive control drug at the same dose (P < 0.001). The model group of depressed mice had the shortest stay in the open arm (P < 0.001), indicating that their depression and anxiety were severe, while 20 mg / kg of Senecio scandens ethyl acetate extract increased the stay time of the depressed mice (P < 0.001). The results confirm that 20 mg / kg of Senecio scandens ethyl acetate extract can significantly improve the exploratory nature of depressed mice in novel environments and alleviate their anxiety.

[0036] 4. Statistical Analysis

[0037] Data were analyzed using GraphPad Prism 10.0 software and expressed as mean ± standard deviation (SD). P < 0.05 was considered statistically significant.

[0038] Example 3: Open Field Experiment

[0039] The grouping and administration regimens of the experimental animals were the same as in Example 2.

[0040] Open Field Experiment (OFT)

[0041] (1) The open field test, also known as the open box test, is a method for evaluating the spontaneous movement behavior, exploratory behavior, and tension of rodent laboratory animals to observe their anxiety levels. It is now widely used in research on mental illnesses such as depression and anxiety disorders. The open field box consists of four 50 cm high white acrylic panels forming a 50 cm wide enclosure. A 50 cm square was used, with a camera positioned directly above to track the mice's movements. Data such as the time the mice spent in the central area and the number of times they entered the central area directly reflected their anxiety levels. Generally, mice with lower anxiety levels tended to spend more time in the center of the open field, i.e., the open area.

[0042] (2) Experimental methods

[0043] The experimental mice were removed from their cages and placed facing away from the experimenter in the central area of ​​the prepared open field. After placement, the experimenter quickly left, allowing the mice to move freely within the experimental chamber. The ANY-maze data acquisition and analysis system was used to record and analyze the mice's spontaneous activity, dwell time in the central and peripheral areas, and number of entries over 15 minutes to evaluate the amount of spontaneous activity and anxiety behavior. After each monitoring session, the mice were removed and returned to their cages. Subsequently, the bottom, placement materials, and side walls of the open field reaction chamber were thoroughly cleaned with ethanol to prevent mouse residue from affecting the results of subsequent tests. The experiment was repeated after the ethanol had completely evaporated.

[0044] 2. Experimental Results

[0045] Figure 5 , Figure 6 As shown, the depressed mice in the model group spent the shortest time in the central area of ​​the open field (P < 0.001), while 20 mg / kg of Senecio scandens ethyl acetate extract prolonged the time they spent in the central area (P < 0.001). The number of times the depressed mice in the model group entered the central area of ​​the open field was significantly lower than that in the normal group (P < 0.001). However, compared with the model group, 20 mg / kg of Senecio scandens ethyl acetate extract significantly increased the number of times the depressed model mice entered the central area (P < 0.001), showing a better effect than the same dose of the positive control drug fluoxetine (P < 0.001). The results indicate that 20 mg / kg of Senecio scandens ethyl acetate extract can improve the interest and exploratory behavior of depressed mice in novel environments.

[0046] 3. Statistical Analysis

[0047] Data were analyzed using GraphPad Prism 10.0 software and expressed as mean ± standard deviation (SD). P < 0.05 was considered statistically significant.

[0048] Example 4 Pathological staining

[0049] The grouping and administration regimens of the experimental animals were the same as in Example 2.

[0050] 1. Hematoxylin-Eosin staining (HE)

[0051] Hematoxylin-eosin staining is one of the commonly used pathological staining methods. Hematoxylin is an alkaline staining solution, which makes the chromatin in the cell nucleus and the nucleic acids in the cytoplasm appear purple-blue; eosin is an acidic dye, which makes the components in the cytoplasm and extracellular matrix appear red, thereby identifying the general morphological and structural characteristics of various tissue or cell components and lesions.

[0052] 2. Experimental Methods

[0053] The tissues were rinsed with pre-cooled physiological saline and blotted dry with filter paper, then fixed in 4% formaldehyde solution for 24 h. After fixation, they were dehydrated using a gradient of ethanol. Subsequently, they were infiltrated with xylene solution and embedded in paraffin. Paraffin blocks were cut to a thickness of 5 μm and stained with H&E staining solution for 10 min. The stained tissue sections were observed and evaluated under a microscope.

[0054] 3. Experimental Results

[0055] As shown in Figure 7, the hepatocytes in each group were structurally intact with clearly visible nuclei, and the hepatic cords were neatly arranged without obvious inflammatory cell infiltration. The renal tubular epithelial cells were regularly morphologically regular, and the glomeruli were clearly visible. These results indicate that no significant lesions occurred in the liver or kidneys of the normal group, the model group, and the group treated with the ethyl acetate extract of *Senecio scandens*. This confirms that the ethyl acetate extract of *Senecio scandens* has no significant toxicity to the liver or kidneys and has a high safety profile.

Claims

1. Use of an extract of Senecio jacobaea, characterized in that, The Senecio scandens Buch.-Ham extract is used for preparing a medicine for preventing and treating depression and anxiety behavior, and the Senecio scandens Buch.-Ham extract is an ethyl acetate extract of Senecio scandens Buch.-Ham.

2. Use of an extract of Senecio according to claim 1, characterized in that, Components of the Senecio scandens Buch.-Ham extract include flavones and flavonoid glycosides such as quercetin, quercitrin, isoquercitrin, acacetin, and phenolic acids such as chlorogenic acid.

3. The use of a extract of Senecio scandens according to claim 1, characterized in that, The Senecio scandens Buch.-Ham extract is used for preventing and treating depression and anxiety behavior, and the dosage is 20 mg / kg.

4. The use of an extract of Senecio according to claim 1, characterized in that The extraction method of the Senecio scandens Buch.-Ham extract is as follows: Senecio scandens Buch.-Ham decoction pieces are loaded into a three-necked flask, and an ethanol-water solution is added, and heated to reflux three times, and then filtered under heat, and the filtrates are combined and concentrated under reduced pressure; when the alcohol extract is 10% of the mass of the ethanol-water solution, the rotary evaporation is stopped, and the alcohol extract, petroleum ether, dichloromethane, and ethyl acetate are sequentially added into a separatory funnel, and extracted three times, and then dried, and the ethyl acetate layer is collected; the ethyl acetate layer solution is dried to obtain the ethyl acetate extract of Senecio scandens Buch.-Ham.

5. The use of an extract of Senecio according to claim 1, characterized in that, The depression and anxiety behavior includes any one or more of self-closing, reduced social activity ability, low activity exploration ability, and reduced will activity.

6. The use of an extract of Senecio according to claim 1, characterized in that The Senecio scandens Buch.-Ham extract can be prepared into any one of a capsule, a tablet, a granule, a powder, an oral liquid, and a pill.

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