Albizia julibrissin extract, preparation method and application thereof

Abstract

The application provides a kind of Albizia julibrissin extract and its preparation method and application, belong to the field of biological medicine technology.The application uses Albizia julibrissin as raw material, is extracted by refluxing with methanol, and active part is obtained by ethyl acetate extraction;In vitro cell experiment proves that the extract can significantly improve the SH-SY5Y nerve cell injury induced by glutamic acid and H2O2;Mechanism research shows that it plays an antidepressant effect by activating cAMP signal pathway, up-regulating pCREB / CREB protein expression and promoting BDNF synthesis.Animal experiment shows that the extract can effectively improve the symptoms such as loss of pleasure, behavior despair, activity reduction and hippocampal neuron injury of depressive model mice, and the effect is dose-dependent.The preparation process of the application is simple and stable, the activity of the extract is clear, and the safety is good, which provides a reliable material basis and scientific basis for developing new antidepressant traditional Chinese medicine with multiple targets and low side effects, and has important clinical application value.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to an extract of Albizia julibrissin flowers, its preparation method, and its application. Background Technology

[0002] Depression is an affective disorder characterized by persistent low mood and loss of interest. Its global incidence continues to rise, making it a major public health problem. Its pathological mechanisms are complex, involving multiple factors such as monoamine neurotransmitter imbalance, hypothalamic-pituitary-adrenal axis hyperfunction, neuroinflammation, and neurotrophic disorders. Currently used antidepressants have limitations such as single-target therapy, numerous side effects, and slow onset of action, necessitating the development of alternative drugs with novel mechanisms of action and higher safety profiles. Against this backdrop, traditional Chinese medicine, with its advantages of multi-component and multi-target synergistic regulation, demonstrates enormous application potential.

[0003] Albizia julibrissin flower is the dried inflorescence of the legume Albizia julibrissin. It is sweet and neutral in nature, and enters the heart and liver meridians, possessing the effects of "relieving depression and calming the mind." The *Shennong Bencao Jing* records that Albizia julibrissin "treats the five internal organs, harmonizes the mind, and brings joy and peace of mind," while the *Zhonghua Bencao* also clearly states its use for "restlessness, depression, and insomnia," making it an important medicinal and edible ingredient in traditional Chinese medicine for treating "depression." Modern pharmacological studies have preliminarily confirmed that Albizia julibrissin flower extract has antidepressant activity; however, its specific pharmacodynamic material basis and molecular mechanism of action remain unclear, hindering its modern development and clinical application. Therefore, this study established glutamate-induced and H2O2-induced SH-SY5Y cell damage models to screen the antidepressant activity of different polarity extracts of Albizia julibrissin flower. UPLC-Q-TOF-MS / MS and UPLC-ESI-MS / MS techniques were used for qualitative and quantitative analysis of the active ingredients to determine the material basis for the antidepressant activity of these extracts. This study employs network pharmacology combined with in vitro cell experiments to explore the key antidepressant targets and related mechanisms of action of the ethyl acetate fraction of Albizia julibrissin flower, aiming to provide a reference for further research on the antidepressant effects of Albizia julibrissin flower. Summary of the Invention

[0004] In view of this, the present invention provides an extract of Albizia julibrissin flowers, its preparation method and application, in order to solve the above problems.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides an extract of Albizia julibrissin flowers, which is obtained by extraction and extraction of Albizia julibrissin flowers, and its active ingredients include flavonoids.

[0007] Preferably, the flavonoids include quercetin, quercetin, afrocin, euryptoside, myricetin, and hyperoside.

[0008] The present invention also provides a method for preparing the mimosa flower extract, comprising the following steps:

[0009] S1. Take Albizia julibrissin flower powder, add extractant, heat and reflux for 50-70 min, filter to obtain residue and first extract;

[0010] S2. Add the dregs to the extractant and heat under reflux for 50-70 minutes. Filter to obtain the second extract and combine the two extracts.

[0011] S3. Add the extractant to the extract and extract the extract 2-3 times. Combine the extracts to obtain the final product.

[0012] Preferably, the extractant is methanol, and the extraction agent is ethyl acetate.

[0013] Preferably, in step S1, the volume-to-mass ratio of the extractant to the mimosa flower powder is 15-17 mL:1 g; in step S2, the volume-to-mass ratio of the extractant to the mimosa flower powder is 11-13 mL:1 g.

[0014] The present invention also provides an extract of Albizia julibrissin flowers prepared according to the preparation method described herein.

[0015] The present invention also provides the application of the aforementioned Albizia julibrissin flower extract in the preparation of antidepressant drugs.

[0016] The present invention also provides an antidepressant pharmaceutical composition comprising the aforementioned Albizia julibrissin flower extract and pharmaceutically acceptable excipients; the excipients include one or more of a carrier, a diluent, and a prostaglandin.

[0017] Preferably, the dosage form of the pharmaceutical composition is a tablet, capsule, granule, oral liquid, or injection.

[0018] By adopting the above technical solution, this invention has the following beneficial effects: Using Albizia julibrissin flowers as raw material, the active fraction is obtained through methanol reflux extraction and ethyl acetate extraction. In vitro cell experiments have confirmed that the extract can significantly improve glutamate and H2O2-induced SH-SY5Y neuronal damage. Mechanistic studies show that it exerts its antidepressant effect by activating the cAMP signaling pathway, upregulating pCREB / CREB protein expression, and promoting BDNF synthesis. Animal experiments show that the extract can effectively improve symptoms such as anhedonia, behavioral hopelessness, reduced activity, and hippocampal neuronal damage in depressed model mice, with a dose-dependent effect. The preparation process of this invention is simple and stable, the extract has clear activity, and good safety, providing a reliable material basis and scientific evidence for the development of novel antidepressant traditional Chinese medicines with multiple targets and low side effects, and has important clinical application value. Attached Figure Description

[0019] Figure 1 The results of the behavioral experiments on mice include body weight (A), sucrose preference rate (B), tail suspension immobility time (C), and forced swimming immobility time (D).

[0020] Figure 2 The effect of ethyl acetate fraction of Albizia julibrissin flowers on the central region dwell time and total distance of movement in depressed mice is shown in Figure A, where A represents the open field trajectory of each group of mice; B represents the total distance of movement of each group of mice within 5 minutes; and C represents the central region dwell time of each group of mice within 5 minutes.

[0021] Figure 3 The effect of ethyl acetate fraction of Albizia julibrissin flower on neuronal damage in the DG, CA1, and CA3 regions of depressed mice was investigated. A represents the percentage of Nissl body-positive cells in the DG region; B represents the percentage of Nissl body-positive cells in the CA1 region; and C represents the percentage of Nissl body-positive cells in the CA3 region.

[0022] Figure 4 Effects of ethyl acetate fraction of Albizia julibrissin flower on neuronal damage in the DG, CA1, and CA3 regions of depressed mice (Nissl staining, scale bar: 20 μM). Detailed Implementation

[0023] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0024] The experimental materials used in the embodiments of the present invention are as follows:

[0025] Medicinal material: Albizia Flos, produced in Handan, Hebei Province, batch number 033240601, was identified by the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, using DNA barcoding technology as the dried inflorescence or flower bud of Albizia julibrissin Durazz., a legume.

[0026] Example 1. Preparation of different polarity fractions from Albizia julibrissin flowers

[0027] Two kg of dried Albizia julibrissin flower powder was extracted twice by heating and reflux. The first extraction was with 16 times the volume of methanol and heated and refluxed for 1 hour. The second extraction was with 12 times the volume of methanol and heated and refluxed for 1 hour. The extracts were combined and concentrated under reduced pressure to recover the methanol, yielding 340 g of extract (17%). The extract was diluted with water and then extracted sequentially with petroleum ether, ethyl acetate, and n-butanol, three times with each solvent. The extracts were combined and concentrated under reduced pressure to dryness, yielding 68.7 g of petroleum ether fraction, 73.6 g of ethyl acetate fraction, and 85.6 g of n-butanol fraction.

[0028] Example 2. Cellular experiments to screen the in vitro antidepressant activity of multiple extracts from Albizia julibrissin flowers.

[0029] 1. Cell Culture

[0030] SH-SY5Y cells were cultured in DMEM medium containing 10% FBS and 1% streptomycin / penicillin, and then placed in a constant temperature incubator containing 5% CO2 at 37°C. When the cell confluence reached 80-90%, the cells were passaged and used for seeding plates and drug experiments.

[0031] 2. Construction of H2O2 and glutamate cell injury models

[0032] Log-phase SH-SY5Y cells were digested and then distributed at 1×10⁻⁶ cells per well. 4 Cells were seeded in 96-well plates. After 12 h of stable cell adhesion, the cells were treated with H2O2 (31.25–1000 μM) or glutamate (5.2–32 mM), respectively. After 24 h, 10 µL of CCK-8 reagent was added to each well, and the cells were incubated for another 2 h. The absorbance was then measured at 450 nm to evaluate the degree of cell damage. The results are shown in Tables 1 and 2.

[0033] Table 1 Effects of H2O2 on the viability of SH-SY5Y cells ( ±s, n=5)

[0034]

[0035] Table 2. Effects of glutamate on the viability of SH-SY5Y cells ( ±s, n=5)

[0036]

[0037] Note: Compared with the control group, 1) P<0.05, 2) P<0.01, 3) P < 0.001 (Tables 1a and 1b).

[0038] The CCK-8 results (Table 1) showed that the cell viability of SH-SY5Y cells decreased significantly with increasing H2O2 or glutamate concentrations after 24 h of treatment with different concentrations of H2O2 or glutamate. Cell survival rates were 40%-60% when H2O2 was 250 μM and glutamate was 16 mM; therefore, these concentrations were selected for subsequent experiments.

[0039] 3. Screening of effective antidepressant sites

[0040] Toxicity test of different extracts of Albizia julibrissin: The cell seeding method was the same as before. Samples of different extracts of Albizia julibrissin were added to the cells at final concentrations of 12.5, 25, 50 and 100 μg / mL and co-incubated with SH-SY5Y cells for 24 h. Cell viability was detected by CCK-8 assay to determine the safe concentration range of the samples. The results are shown in Table 3.

[0041] Table 3. Effects of different extracts from Albizia julibrissin flowers on the viability of SH-Y5Y cells ( ±s, n=4)

[0042] control group - 100.19±1.13 n-Butanol group 12.5 101.95±1.02 25 <![CDATA[77.93±5.63 1) ]]> 50 <![CDATA[73.49±5.15 1) ]]> 100 <![CDATA[65.98±8.7 1) ]]> Ethyl acetate group 12.5 101.79±6.04 25 104.2±4.09 50 105.96±4.17 100 105.82±2.75 Petroleum ether group 12.5 100.41±3.57 25 100.91±4.22 50 105.74±5.64 100 106.33±6.36

[0043] Table 3 shows that at a concentration of 12.5 μg / mL, the n-butanol fraction did not significantly change cell viability, while at concentrations above 25 μg / mL, cell viability decreased significantly (P<0.01). The ethyl acetate and petroleum ether fractions did not significantly change SH-SY5Y cell viability within the concentration range of 12.5–100 μg / mL. Therefore, subsequent experiments will investigate its in vitro antidepressant activity at safe concentrations of n-butanol, ethyl acetate, and petroleum ether.

[0044] In vitro antidepressant activity assay: Petroleum ether, ethyl acetate, and n-butanol fractions were selected for activity verification, with sample concentrations of 25 μg / mL and 50 μg / mL, respectively. SH-SY5Y cells were treated with 250 μM H2O2 and 16 mM glutamate for 24 h, respectively, with 1 μM fluoxetine as a positive control. Cell viability was assessed using the CCK-8 assay after the experiment to evaluate the in vitro antidepressant activity of each extract fraction. The results are shown in Table 4.

[0045] Table 4. Protective effects of different extracts from Albizia julibrissin flowers on SH-Y5Y cell damage ( ±s, n=6)

[0046] control group - 100±2.84 100±2.17 Model group <![CDATA[46.93±0.9 1) ]]> <![CDATA[57.18±3.1 1) ]]> Fluoxetine group 1 <![CDATA[64.8±5.05 4) ]]> <![CDATA[75.17±2.71 4) ]]> n-Butanol group 3.125 49.05±1.6 60.99±6.61 6.25 51.57±1.05 <![CDATA[69.32±2.09 3) ]]> 12.5 52.02±1.64 <![CDATA[70.22±3.75 3) ]]> Ethyl acetate group 25 <![CDATA[55.55±2.94 2) ]]> <![CDATA[84.5±6.37 4) ]]> 50 <![CDATA[67.57±7.74 4) ]]> <![CDATA[83.43±2.8 4) ]]> 100 <![CDATA[57.15±3.84 3) ]]> <![CDATA[81.72±5.38 4) ]]> Petroleum ether group 25 47.27±2.66 56.58±1.76 50 46.68±1.32 55.5±2.83 100 39.51±2.98 55.17±2.95

[0047] Note: Compared with the control group, 1) P<0.001; compared with the model group, 2) P<0.05, 3) P<0.01, 4) P<0.001

[0048] Example 3. Detection of chemical components in the ethyl acetate fraction of Albizia julibrissin flowers using UPLC-Q-TOF-MS / MS.

[0049] Sample preparation: Dissolve 100 mg of the vacuum freeze-dried sample in 4 ml of 80% methanol solution, sonicate for 30 min, centrifuge at 4°C (12,000 r / min, 5 min), and filter the supernatant through a 0.22 μm filter membrane for instrumental analysis.

[0050] Chromatographic conditions: An Agilent 1290 Infinity II ultra-high performance liquid chromatography system and an Agilent InfinityLab Poroshell 120 SB-C18 (3.0 × 150 mm, 2.7 μm) column were used; column temperature was 25℃; flow rate was 0.5 mL / min; injection volume was 5 μL; mobile phase composition was 0.1% formic acid water (A) - acetonitrile (B); gradient elution program was as follows: 0~20 min, 1%~5% B; 20~35 min, 5%~30% B; 35~55 min, 30%~100% B.

[0051] Mass spectrometry conditions: An Agilent 6546 LC / Q-TOF mass spectrometer system was used for analysis. Raw data acquisition was performed in both positive and negative ion modes. In positive ion mode, the capillary voltage was 3500 V and the nozzle voltage was 500 V; in negative ion mode, the capillary voltage was 4000 V and the nozzle voltage was 1000 V. The sheath gas flow rate was 11 L / min and the sheath gas temperature was 350 °C in both modes; the drying gas flow rate was 9 L / min and the capillary temperature was 320 °C. The mass scan range was 50–1500 m / z. The detection results are shown in Table 5.

[0052] Table 5. Component analysis results of ethyl acetate fraction of Albizia julibrissin flowers

[0053] 1 2.901 <![CDATA[C7H6O5]]> 169.0149 169.0143 3.5 <![CDATA[[M-H] - ]]> 124.0162，79.0188，51.0239 gallic acid 2 5.624 <![CDATA[C7H6O4]]> 153.0199 153.0193 3.9 <![CDATA[[M-H] - ]]> 108.0216，53.0397 Protocatechuic acid 3 12.934 <![CDATA[C7H6O2]]> 121.0300 121.0295 4.1 <![CDATA[[M-H] - ]]> 92.0267，65.0395 4-Hydroxybenzaldehyde 4 14.636 <![CDATA[C9H 10 O4]]> 181.0512 181.0506 3.3 <![CDATA[[M-H] - ]]> 146.9737，61.9882 Syringaldehyde 5 26.432 <![CDATA[C 10 H8O4]]> 193.0482 193.0495 -6.7 <![CDATA[[M+H] + ]]> 133.0284 Scopolamine 6 27.312 <![CDATA[C 15 H 12 O8]]> 319.0466 319.0459 2.2 <![CDATA[[M-H] - ]]> 183.0301，139.0402，97.0294 Dihydromyricetin 7 28.595 <![CDATA[C 21 H 20 O 12 ]]> 463.0893 463.0882 2.4 <![CDATA[[M-H] - ]]> 316.0220，271.0240，178.9981 Myrica rubra 8 28.755 <![CDATA[C 27 H 30 O 16 ]]> 609.1475 609.1461 2.3 <![CDATA[[M-H] - ]]> 300.0273，271.0245，178.9982 Rutin 9 28.895 <![CDATA[C 27 H 30 O 15 ]]> 593.1523 593.1512 1.9 <![CDATA[[M-H] - ]]> 284.0326，255.0300，178.9989 Nicotiflorin 10 29.045 <![CDATA[C 21 H 20 O 12 ]]> 465.1017 465.1028 -2.3 <![CDATA[[M+H] + ]]> 319.0446，71.0490 Hyperoside 11 29.456 <![CDATA[C 20 H 18 O 11 ]]> 433.0786 433.0776 2.3 <![CDATA[[M-H] - ]]> 300.0272，271.0248，151.0036 Polygonum hydropiper 12 30.297 <![CDATA[C 21 H 20 O 11 ]]> 447.0942 447.0933 2.0 <![CDATA[[M-H] - ]]> 300.0274，271.0246，151.0035 Quercetin 13 30.337 <![CDATA[C 21 H 20 O 11 ]]> 447.0941 447.0933 1.8 <![CDATA[[M-H ]- ]]> 300.0273，271.0246，151.0035 Astragaloside 14 30.939 <![CDATA[C 27 H 30 O 15 ]]> 593.1523 593.1512 1.9 <![CDATA[[M-H] - ]]> 300.0271，271.0234，178.9984 Kanfel 3-Neohesperidin 15 31.779 <![CDATA[C 21 H 20 O 10 ]]> 431.0993 431.0984 2.1 <![CDATA[[M-H] - ]]> 284.0321，255.0293 Aftobin 16 33.703 <![CDATA[C 15 H 10 O7]]> 301.0361 301.0354 2.3 <![CDATA[[M-H] - ]]> 151.0035，107.0136，65.0029 Quercetin 17 35.325 <![CDATA[C 15 H 12 O5]]> 272.0612 271.0619 2.6 <![CDATA[[M-H] - ]]> 151.0036， 119.0520， 65.0032 Naringin chalcone 18 35.866 <![CDATA[C 15 H 10 O5]]> 269.0460 269.0456 1.5 <![CDATA[[M-H] - ]]> 151.0027，117.0342，65.0042 Celery 19 36.266 <![CDATA[C 15 H 10 O6]]> 285.0409 285.0405 1.4 <![CDATA[[M-H] - ]]> 285.0405，229.0509，93.0345 Kaempferol 20 36.346 <![CDATA[C 16 H 12 O6]]> 299.0565 299.0561 1.3 <![CDATA[[M-H] - ]]> 199.0408，61.9884 Geranin 21 39.852 <![CDATA[C 15 H 12 O4]]> 255.0667 255.0663 1.6 <![CDATA[[M-H] - ]]> 178.0701，108.0208，63.0231 Qiao Songsu

[0054] Based on precise molecular weight and fragment ion information, and comparison with database and literature data, 24 compounds were preliminarily identified (see Table 3), including 18 flavonoids, 3 phenolic acids, 2 triterpenes, and 1 coumarin.

[0055] Example 4. Detection of flavonoid content by UPLC-ESI-MS / MS

[0056] Sample preparation: Take 20 mg of the vacuum freeze-dried sample and add 10 μL of 4000 nmol / L internal standard mixed working solution and 500 μL of 70% methanol solution. After sonication for 30 min, centrifuge at 4°C (12000 r / min, 5 min). Filter the supernatant through a 0.22 μm filter membrane for instrumental analysis.

[0057] Chromatographic conditions: A Waters ACQUITY UPLC HSS T3 C18 column (100 mm × 2.1 mm, 1.8 µm) was used; the flow rate was 0.35 mL / min, the column temperature was 40°C, and the injection volume was 2 μL. The mobile phase consisted of 0.05% formic acid water (A) and 0.05% formic acid acetonitrile (B), with the following gradient elution program: 0–1 min, 10%–20% B; 1–9 min, 20%–70% B; 9–12.5 min, 70%–95% B; 12.5–13.5 min, 95% B; 13.5–13.6 min, 95%–10% B; 13.6–15 min, 10% B.

[0058] ESI-MS / MS: Electrospray ionization (ESI) source, temperature 550℃, mass spectrometry voltage 5500V in positive ion mode, mass spectrometry voltage -4500V in negative ion mode, curtain gas (CUR) 35 psi. In a QTRAP 6500+, each ion pair was scanned and detected based on its declustering potential (DP) and collision energy (CE). The results are shown in Table 6.

[0059] Table 6. Analysis of flavonoid content in the ethyl acetate fraction of Albizia julibrissin flowers.

[0060] 1 Quercetin 791.642510 y = 8986.28991x - 112.49262 0.99791 1-20000 1 2 Quercetin 192.011050 y = 5642.85318x - 17756.78871 0.99823 0.5-20000 5 3 Aftobin 104.716510 y = 21591.68931x + 734.00182 0.99917 0.5-5000 1 4 Polygonum aviculare 65.733855 y = 20418.18615x + 1035.37542 0.99931 5-5000 1 5 Myrica rubra 55.658695 y = 906.91112x - 4054.73266 0.99806 20-2000 10 6 Hyperoside 37.538297 y = 3.80316x + 2006.11966 0.99788 0.5-1000 1 7 Rutin 10.369711 y = 19959.20010x + 4.47813 0.99952 0.5-1000 1 8 Kanfel 3-Neohesperidin 8.848564 y = 6922.48926x + 4454.95872 0.99903 0.5-1000 5 9 Kaempferol 6.527459 y = 1156.98946x + 4447.84106 0.99643 5-5000 20 10 Astragaloside 6.417775 y = 13932.82425x + 1836.02122 0.99612 0.5-2000 1 11 Naringin chalcone 4.985145 y = 21990.70242x + 2952.34213 0.99863 1-1000 5 12 Qiao Songsu 3.516536 y = 3039.25892x + 2302.33025 0.99864 5-5000 1 13 Nicotiflorin 2.467609 y = 14607.79458x + 26459.77573 0.99680 0.5-2000 1 14 Syringaldehyde 1.372516 y = 214.95369x + 1152.83295 0.99768 100-10000 50 15 Dihydromyricetin 1.093315 y = 3443.27979x - 4024.44338 0.99416 1-1000 5 16 galangin 0.999347 y = 1324.95923x + 2010.27908 0.99745 20-2000 10 17 Celery 0.673278 y = 9470.65948x + 2031.94009 0.99814 1-1000 5 18 Geranin 0.615921 y = 4.60981x + 9913.96943 0.99887 5-5000 1

[0061] Note: A: Standard; B: Sample.

[0062] The content of flavonoids in the ethyl acetate fraction of Albizia julibrissin flowers was determined by UPLC-ESI-MS / MS. The relative contents were calculated by peak area and standard curve, as shown in Table 6. The top 12 components were: quercetin, quercetin, afusin, eurygnetin, myricetin, hyperoside, rutin, calciferol 3-neohesperidin, kaempferol, astragalin, naringenin chalcone, and styracin.

[0063] Example 5. Effect of the ethyl acetate fraction of Albizia julibrissin flowers on intravenous cAMP levels in glutamate-damaged SH-SY5Y cells.

[0064] Logarithmic growth phase SH-SY5Y cells were seeded at 5 × 10⁵ cells / well in 6-well plates. Fluoxetine (1 μM) or ethyl acetate (25, 50 μg / ml) and glutamate (250 μM) were added and incubated for 24 h. After washing with pre-cooled PBS, the cells were digested with trypsin, centrifuged, and the supernatant was collected. The cAMP content in the cells was detected according to the ELISA kit instructions. The results are shown in Table 7.

[0065] Table 7. Effects of ethyl acetate fraction of Albizia julibrissin flowers on intracellular cAMP levels in glutamate-damaged SH-SY5Y cells ( ±s, n=6)

[0066] control group 9.02±0.49 Model group <![CDATA[5.36±0.39 1) ]]> Fluoxetine group 1 <![CDATA[7.89±0.53 3) ]]> Low-dose group of ethyl acetate from Albizia julibrissin flowers 25 <![CDATA[7.46±0.88 3) ]]> High-dose group of ethyl acetate from Albizia julibrissin flowers 50 <![CDATA[8.38±0.75 3) ]]>

[0067] Note: Compared with the control group, 1) P<0.01; compared with the model group, 3) P<0.01.

[0068] ELISA results showed that, compared with the control group, the intracellular cAMP level in the glutamate-damaged SH-SY5Y cell model group was significantly reduced (P<0.01); compared with the model group, the intracellular cAMP level in the low-dose and high-dose ethyl acetate groups was significantly increased (P<0.01). The results suggest that the protective effect of ethyl acetate on the glutamate-damaged SH-SY5Y cell model may be related to the increase in intracellular cAMP content.

[0069] Example 6. Effects of ethyl acetate fraction of Albizia julibrissin flower on the expression of CREB, pCREB and BDNF proteins in glutamate-damaged SH-SY5Y cells.

[0070] Cells were plated as described in Example 2. After 24 hours of combined drug treatment, the cells were washed three times with pre-cooled PBS. 100 μL of RIPA lysis buffer (RIPA:PMSF = 100:1) was added to each well for lysis for 30 min. After centrifugation at 13000 rpm for 15 min, the supernatant was collected, and protein quantification was performed using a BCA kit. Protein samples were separated by SDS-PAGE electrophoresis and transferred to PVDF membranes using a wet transfer method. After blocking with 5% BSA, primary antibodies against BDNF (1:1000), CREB (1:1000), PCREB (1:1000), and GAPDH (1:3000) were added, and the membranes were incubated overnight at 4°C. After washing three times with TBST (15 min each time), secondary antibody was added, and the membranes were incubated at room temperature for 1 h. After washing three times with TBST, ECL chromogenic agent was added, and the membranes were developed using a developer. The grayscale values ​​were analyzed using ImageJ software.

[0071] Table 8. Effects of ethyl acetate fraction of Albizia julibrissin flowers on the expression of CREB, pCREB and BDNF proteins in glutamate-damaged SH-SY5Y cells. ±s, n=3)

[0072] control group -- 1.03±0.03 1±0.04 0.96±0.01 Model group -- <![CDATA[0.38±0.05 1) ]]> <![CDATA[0.42±0.06 1) ]]> <![CDATA[0.55±0.08 1) ]]> Fluoxetine group 1 <![CDATA[0.89±0.17 3) ]]> <![CDATA[0.91±0.07 3) ]]> <![CDATA[0.85±0.01 3) ]]> Low-dose group of ethyl acetate from Albizia julibrissin flowers 25 <![CDATA[0.76±0.13 2) ]]> <![CDATA[0.75±0.06 3) ]]> <![CDATA[0.7±0.05 2) ]]> High-dose group of ethyl acetate from Albizia julibrissin flowers 50 <![CDATA[0.98±0.09 3) ]]> <![CDATA[0.93±0.04 3) ]]> <![CDATA[0.84±0.09 3) ]]>

[0073] Note: Compared with the control group, 1) P<0.01; compared with the model group, 2) P<0.05,3) P<0.01.

[0074] Western blot results (see Table 8) showed that, compared with the control group, the expression levels of pCREB / CREB and BDNF proteins in SH-SY5Y cells of the model group (glutamate-damaged group) were significantly decreased (P<0.01); compared with the model group, the expression levels of pCREB / CREB and BDNF proteins in the fluoxetine group and the low- and high-dose groups of ethyl acetate fraction of Albizia julibrissin were significantly increased, and the differences were statistically significant (P<0.05, P<0.01). These results suggest that the protective effect of ethyl acetate on the glutamate-damaged SH-SY5Y cell model may be related to the upregulation of the expression of key proteins pCREB / CREB and BDNF in the cAMP signaling pathway.

[0075] Example 7. Animal antidepressant experiment

[0076] 1. Animal grouping, model establishment, and drug administration methods

[0077] After being acclimatized in the animal room for 7 days, the mice were randomly divided into a control group, a model group, a positive drug (fluoxetine group), a low-dose group of ethyl acetate fraction of Albizia julibrissin flower, a medium-dose group, and a high-dose group, with 12 mice in each group.

[0078] The control group mice were fed normally without any stimulation. The other groups were treated with: (1) reversed day and night (12h); (2) water deprivation (24h); (3) tail clamping (1.5-2 cm from the tail root, 5min); (4) cold water swimming (10℃, 5min); (5) hot water swimming (40℃, 5min); (6) tubular restraint (3h); (7) damp mouse bedding (12h); (8) cage tilting (at a 45° angle to the horizontal plane, 12h). One or two types of stimulation were used each day, and the stimulation methods were not repeated on adjacent days. This was continued for 28 days to establish the CUMS model.

[0079] Both the control and model groups were administered an equal volume of physiological saline containing 0.1% dimethyl sulfoxide daily via gavage. The positive control group received fluoxetine (10 mg / kg), and the low-dose, medium-dose, and high-dose groups received ethyl acetate from Albizia julibrissin (100 mg / kg). All drugs were dissolved in physiological saline containing 0.1% dimethyl sulfoxide. Administration was performed via gavage one hour after the daily modeling stimulation ended, for 28 consecutive days.

[0080] 2. Changes in mouse body weight

[0081] Body weight change is a key indicator for assessing depressive status and the efficacy of antidepressants in mice. Body weight changes in mice were monitored and recorded weekly. Results... Figure 1As shown in Figure A, compared with the control group, CUMS mice had a significantly lower body weight in week four (P < 0.001), while compared with the model group, mice in different doses of ethyl acetate from Albizia julibrissin flowers had significantly higher body weight. These experimental results suggest that ethyl acetate from Albizia julibrissin flowers may improve the clinical manifestation of weight loss in patients with depression.

[0082] 3. Sugar Water Preference Experiment (SPT)

[0083] This experiment assesses the degree of anhedonia in mice by detecting their preference for sucrose water. This test is often used as a key indicator in evaluating the efficacy of antidepressants. The specific steps of the sucrose water adaptation test are as follows:

[0084] On the last day of the 7-day acclimatization period in the animal facility, mice were placed in their cages with one bottle of 1% sucrose solution and one bottle of purified water, respectively, for 12 hours of free access to water. Afterward, the locations were changed, and mice continued free access to water for another 12 hours. This was to ensure the mice became familiar with the taste of the sugar water and to eliminate any interference from the placement. Formal Experiment: Before the formal test, mice were separated into individual cages. After 12 hours of water and food restriction, a weighed bottle of 1% sucrose solution was placed on the left side of each cage, and a weighed bottle of purified water was placed on the right side. After 12 hours of free access to water, the positions of the bottles were swapped, and mice continued free access to water for another 12 hours. The remaining weight of both bottles was then measured, and the sucrose solution preference rate was calculated using the following formula: Sucrose Solution Preference Rate (100%) = (Sucrose Solution Consumption / (Sucrose Solution Consumption + Purified Water Consumption)) × 100%

[0085] Experimental results are as follows Figure 1 As shown in Figure B, compared with the control group, the CUMS model group mice showed a significantly reduced preference for sucrose water (P < 0.001); compared with the model group, the fluoxetine group (positive control) and different doses of ethyl acetate fraction of Albizia julibrissin flowers significantly increased the sucrose water preference of CUMS mice (P < 0.001). Furthermore, low, medium, and high doses of ethyl acetate fraction of Albizia julibrissin flowers showed a dose-dependent improvement in sucrose water preference.

[0086] 4. Suspended Tail Test (TST)

[0087] The behavioral despair level of depressed mice is assessed by measuring the time they remain still while their tails are suspended in mid-air. This test is one of the key indicators used to evaluate the efficacy of antidepressants. The specific steps of the tail suspension test are as follows:

[0088] Gently remove the mouse from its cage and attach one end of a small piece of tape (approximately 2-3 cm) about 1-2 cm from the tip of its tail, ensuring it is secure enough so the mouse cannot pull it off with its hind legs; however, it should not be too tight to avoid affecting blood circulation to the tail or causing injury. Hang the other end of the tape on a hook at the top of the cage, with the mouse head down, about 15 cm from the bottom. Ensure the mouse's body is suspended and cannot touch the cage walls or any surface. The total experiment time is 6 minutes; record and analyze the mouse's stillness time for the last 4 minutes. After the experiment, gently remove the mouse from the hook and carefully remove the tape from its tail.

[0089] Experimental results are as follows Figure 1 As shown in Figure C, compared with the control group, the tail immobility time of mice in the CUMS model group was significantly increased (P < 0.001); while compared with the model group, the fluoxetine group (positive control) and different doses of ethyl acetate fraction of Albizia julibrissin flower significantly reduced the tail immobility time of CUMS mice (P < 0.01, P < 0.001). In addition, the low, medium and high doses of ethyl acetate fraction of Albizia julibrissin flower showed a dose-dependent improvement in tail immobility time in mice.

[0090] 5. Forced swimming test (FST)

[0091] The forced swimming test assesses the level of behavioral despair in mice under extreme conditions by measuring their immobility time in water. This test is also a key indicator for evaluating the efficacy of antidepressants. The specific steps of the forced swimming test are as follows:

[0092] The mice were gently placed into a cylindrical transparent tube (10 cm in diameter and 25 cm in height) filled with water at (24±2)℃, with the water depth such that the mouse's tail could not touch the bottom. Timing was started when the mice were placed in the water, and the total experimental time was 6 minutes. The time the mice spent floating on the surface and struggling motionless in the water was observed and recorded for the last 4 minutes.

[0093] Experimental results are as follows Figure 1 As shown in Figure D, compared with the control group, the immobility time of CUMS model mice in the forced swimming test was significantly increased (P < 0.001); compared with the model group, the fluoxetine group (positive control) and different doses of ethyl acetate fraction of Albizia julibrissin flower significantly reduced the immobility time of CUMS mice (P < 0.001). Low, medium, and high doses of ethyl acetate fraction of Albizia julibrissin flower showed a dose-dependent reduction in the immobility time of mice.

[0094] 6. Open Field Test (OFT)

[0095] The activity level and anxiety level of mice were reflected by measuring the total distance they moved and the time they spent in the center of an open field. The steps are as follows:

[0096] Mice were gently placed in a fixed corner of a thoroughly cleaned (using 75% ethanol) white cube-shaped open field test chamber (40cm x 40cm). The person placing the mouse quickly left to avoid interference. A camera on top continuously recorded the test mice for 5 minutes, and analysis software was used to record the time the mice spent in the center and the total distance they moved. After each test, the mouse was immediately returned to its cage, and the entire chamber was thoroughly cleaned with 75% ethanol to eliminate odors and excrement, preventing any impact on the next test mouse. The experimental environment must be kept quiet and evenly lit throughout the experiment.

[0097] Experimental results are as follows Figure 2 As shown, the average total distance traveled by mice in the control group was 3471 cm, and the average time spent at the center was 27.7 s. The average total distance traveled by mice in the model group was 1030 cm, and the average time spent at the center was 4.5 s, both significantly lower than those in the control group (P < 0.001). The average total distance traveled by mice in the fluoxetine group was 2746 cm, and the average time spent at the center was 18.2 s, both significantly higher than those in the model group (P < 0.001). The average total distance traveled by mice in the low-dose group of ethyl acetate fraction of Albizia julibrissin was 1675 cm, and the average time spent at the center was 10.2 s, showing no significant difference compared to the model group. The average total distance traveled by mice in the medium- and high-dose groups of ethyl acetate fraction of Albizia julibrissin was 2257 cm and 2878 cm, respectively, with average times spent at the center of gravity of 16.3 s and 21.9 s, respectively, significantly higher than those in the model group (P < 0.001).

[0098] 7. Nissl body staining (Nissl)

[0099] Nissl bodies are structures formed by the aggregation of rough endoplasmic reticulum and free ribosomes, and can serve as histological markers of neuronal function. To further elucidate the effects of drug treatment on hippocampal neurons, this study performed Nissl staining on mouse hippocampal tissue.

[0100] After behavioral testing, mice were euthanized using cervical dislocation. The whole brain of the mice was cleaned with physiological saline, fixed in 4% paraformaldehyde fixative, dehydrated, cleared, embedded in paraffin, and sectioned. Before staining, the sections were dewaxed, dissolved in ethanol, rinsed with water, stained with Nissl stain for 5 minutes, cleared with xylene, and mounted with neutral resin. The structure and pathological changes of neurons in the CA1, CA3, and DG regions of the mouse hippocampus were observed under a microscope.

[0101] Nissl staining results showed that in the normal group mice, the neurons in the CA1, CA3, and DG regions of the hippocampus had intact structures, were neatly arranged, had uniform cell size, and abundant Nissl bodies. Compared with the control group, the model group mice showed significant pathological changes in the DG, CA1, and CA3 regions of the hippocampus, mainly manifested as shrunken and deeply stained neurons, unclear nuclei and cytoplasm decomposition, irregular morphology, and a significantly reduced number of Nissl bodies (p < 0.001). Compared with the model group, the neurons in the low, medium, and high dose groups of ethyl acetate from Albizia julibrissin flower and the fluoxetine group had normal structures and morphology in the DG, CA1, and CA3 regions of the hippocampus, with obvious nuclei, rounded edges, abundant Nissl bodies, and a significantly increased number of Nissl bodies (p < 0.05, p < 0.01, p < 0.001). Figure 3 and Figure 4 .

[0102] As can be seen from the above embodiments, the present invention provides an extract of Albizia julibrissin flowers, its preparation method and application, providing a material basis and scientific evidence for the development of novel antidepressant traditional Chinese medicines.

[0103] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An extract of Albizia julibrissin flowers, characterized in that, The extract is obtained by extracting and extracting from Albizia julibrissin flowers, and its active ingredients include flavonoids.

2. The mimosa flower extract according to claim 1, characterized in that, The flavonoids include quercetin, quercetin, apocytosine, euryptoside, myricetin, and hyperoside.

3. The method for preparing the Albizia julibrissin flower extract according to claim 1, characterized in that, Includes the following steps: S1. Take Albizia julibrissin flower powder, add extractant, heat and reflux for 50-70 min, filter to obtain residue and first extract; S2. Add the dregs to the extractant and heat under reflux for 50-70 minutes. Filter to obtain the second extract and combine the two extracts. S3. Add the extractant to the extract and extract the extract 2-3 times. Combine the extracts to obtain the final product.

4. The preparation method according to claim 3, characterized in that, The extractant is methanol, and the extraction agent is ethyl acetate.

5. The preparation method according to claim 4, characterized in that, In step S1, the volume-to-mass ratio of the extractant to the mimosa flower powder is 15-17 mL: 1 g; in step S2, the volume-to-mass ratio of the extractant to the mimosa flower powder is 11-13 mL: 1 g.

6. The mimosa flower extract prepared by the preparation method according to claim 4 or 5.

7. The use of the mimosa flower extract according to claim 6 in the preparation of antidepressant drugs.

8. An antidepressant drug composition, characterized in that, It comprises the mimosa flower extract of claim 3 and pharmaceutically acceptable excipients; the excipients include one or more of a carrier, a diluent, and a prostaglandin.

9. The pharmaceutical composition according to claim 8, characterized in that, The dosage form of the pharmaceutical composition is tablets, capsules, granules, oral liquid, or injection.

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