Semi-morindae total flavones, preparation method and use thereof

Abstract

The application provides semi total flavonoids of Liquidambar, which is obtained by using stems of Liquidambar cathayensis Hung T.Chang as raw materials and adopting ultrasonic-assisted low eutectic solvent extraction, wherein the yield of the total flavonoids is 71.95%±6.49%. The application also provides a preparation method and application of the semi total flavonoids of Liquidambar. The semi total flavonoids of Liquidambar have significant antioxidant activity, can be used for nerve protection, can be used for treating cerebral ischemia or anti-ischemic stroke, can improve cerebral ischemic nerve function injury, can reduce cerebral infarction volume, can reduce neuron damage degree, can reduce activation of astrocytes, can reduce expression level of apoptotic proteins, can protect neurons and synapses, and can protect vascular endothelial cells. The drug efficacy is clear and controllable, is equivalent to that of positive drug nimodipine, can reach significant drug efficacy at a low dose, is safer in clinical use, and provides a new selection for clinic.

Description

Technical Field

[0001] This invention relates to total flavonoids from *Symplocos buergeriana*, their preparation methods, and uses, and belongs to the pharmaceutical field. Background Technology

[0002] The stem of *Semiliquidambar cathayensis* Hung T. Chang, a plant belonging to the genus *Semiliquidambar cathayensis* in the family Hamamelidaceae, is called *Semiliquidambar cathayensis*. Its main active components are flavonoids, polysaccharides, and their glycosides, which possess antioxidant, anti-rheumatoid, and anti-tumor effects. The extraction and separation of its active component, total flavonoids, has become an important way to increase the added value of the *Semiliquidambar cathayensis* industry. Tang Juan et al., in their research progress on the chemical constituents and pharmacological effects of *Semiliquidambar cathayensis*, published in *Guangzhou Chemical Industry*, November 2022, Vol. 50, No. 21, disclosed the isolation of 17 flavonoid components from *Semiliquidambar cathayensis* leaves; Li Yang et al. identified 10 flavonoid components from *Semiliquidambar cathayensis* roots using UHPLC-Q-TOF-MS / MS technology. These 27 flavonoid compounds include flavones, flavonols, dihydroflavones, chalcones, and flavan-3-ols. Liao Na, et al., Optimization of Extraction Process of Total Flavonoids from *Senecio scandens* by Hot Reflux Method, Journal of Guangxi University of Science and Technology, June 2019, Vol. 30, No. 2. This study investigated the optimal extraction process of total flavonoids from *Senecio scandens* using hot reflux extraction. Single-factor and orthogonal experiments were conducted to determine the optimal extraction conditions. The results showed that the optimal extraction conditions for total flavonoids from *Senecio scandens* by hot reflux extraction were: 80% ethanol concentration, a solid-liquid ratio of 1:50 (g / mL), an extraction time of 2 h, and an extraction temperature of 90℃. Under these conditions, the extraction rate of total flavonoids from *Senecio scandens* was 6.08%, and the RSD value was 4.79%. Application No. 202210331183.X, Invention Title: Application of *Symplocos pubescens* or its extract in the preparation of anti-cerebral ischemia drugs, discloses the application of *Symplocos pubescens* or its extract in the preparation of anti-cerebral ischemia drugs. This invention is the first to discover that *Symplocos pubescens* or its extract has a significant effect in reducing cerebral ischemia damage, and has the potential to prepare pharmaceutical formulations for the prevention or treatment of cerebral ischemia and related diseases. Furthermore, the preparation method of the pharmaceutical formulation is simple, low-cost, and has good economic benefits. This patent discloses the use of the water extract of *Symplocos pubescens*, but does not disclose the specific active ingredients.

[0003] In recent years, deep eutectic solvents (DES), as environmentally friendly new green solvents, have gradually replaced organic solvents and gained widespread attention. DES is typically composed of hydrogen bond donors (HBDs) and hydrogen bond acceptors (HBAs) in a certain proportion through non-covalent bonding, offering advantages such as ease of preparation, wide solubility range, environmental friendliness, and stable properties. Choline chloride and betaine, two quaternary ammonium salts, are commonly chosen as HBAs, while alcohols, carboxylic acids, and amines are used as HBDs. However, due to the different extraction targets, not all deep eutectic solvents are suitable for extracting all active ingredients or fractions from medicinal materials. Summary of the Invention

[0004] This invention provides a total flavonoid from *Senecio scandens*, and also provides a method for preparing and using this total flavonoid.

[0005] This invention provides total flavonoids from *Semiliquidambarcathayensis* Hung T. Chang, a plant belonging to the genus *Semiliquidambarcathayensis* of the Hamamelidaceae family. The total flavonoids are obtained by ultrasonic-assisted eutectic solvent extraction, with a yield of 71.95% ± 6.49%.

[0006] This invention provides a method for preparing the total flavonoids of *Symplocos buergeriana* as described in claim 1, comprising the following steps:

[0007] a. Preparation of eutectic solvent DES: Hydrogen bond acceptor HBA and hydrogen bond donor HBD were mixed at a molar ratio of 1:1-5, heated and stirred at 80°C until a stable, homogeneous and transparent liquid was formed.

[0008] b. Preparation of extraction solvent: Add water to the eutectic solvent DES to prepare DES with a water content of 10-50%;

[0009] c. Pre-treatment of raw materials: Take half of the lotus stem, crush and sieve it;

[0010] d. Extraction: Mix the raw material powder treated in step c with the extraction solvent in step b. The ratio of powder to DES is 1:(10-40) g / mL. Perform ultrasonic extraction at room temperature with a power of 100-500W and a frequency of 40KHz for 10-50 min. After extraction, centrifuge at 3000 rpm and collect the supernatant to obtain the crude extract of total flavonoids from *Symplocos buergeriana*. Repeat the extraction step 1-3 times.

[0011] e. Purification: The crude extract of total flavonoids from *Symplocos pubescens* is prepared by purifying with a resistant solvent in step c. The extract is then allowed to stand, centrifuged, and the precipitate is collected and dried to obtain the total flavonoids from *Symplocos pubescens* of this invention. The resistant solvent is water, and the ratio of the crude extract of total flavonoids from *Symplocos pubescens* to the resistant solvent water is 1:(10-40).

[0012] More preferably, in step a, the hydrogen bond acceptor HBA is one of choline chloride, betaine, taurine, and lactic acid; and the hydrogen bond donor HBD is one of acetamide, ethylene glycol, glycerol, 1,4-butanediol, xylitol, glucose, levulinic acid, and D-sorbitol.

[0013] The moisture content described in step b is 30%;

[0014] The sieve mesh size described in step c is 100 mesh;

[0015] In step d, the ratio of the drug powder to the DES liquid is 1:30 g / mL; the ultrasonic power is 400 W; and the ultrasonic time is 40 min.

[0016] The ratio of the crude extract of total flavonoids from *Symplocos pubescens* to the resistant solvent water in step e is 1:30; the centrifugation conditions are: 3000 rpm for 10 min.

[0017] More preferably, the molar ratio of the hydrogen bond acceptor HBA to the hydrogen bond donor HBD is:

[0018] Choline chloride: ethylene glycol (1:2) or

[0019] Betaine: levulinic acid (1:2) or

[0020] Betaine: Glycerin (1:2).

[0021] More preferably, the molar ratio of the hydrogen bond acceptor HBA to the hydrogen bond donor HBD is: choline chloride: ethylene glycol (1:2).

[0022] This invention provides the use of the total flavonoids from *Symplocos rubra* in the preparation of pharmaceuticals or health foods with antioxidant activity.

[0023] This invention provides the use of the total flavonoids from *Symplocos buergeriana* in the preparation of drugs with neuroprotective effects.

[0024] This invention provides the use of the total flavonoids from *Symplocos rubra* in the preparation of medicaments for treating cerebral ischemia or for treating ischemic stroke.

[0025] More preferably, the drug is used to improve neurological function damage caused by cerebral ischemia, reduce the volume of cerebral infarction, alleviate the degree of neuronal damage, reduce the activation of astrocytes, reduce the expression level of apoptotic proteins, protect neurons and synapses, and protect vascular endothelial cells.

[0026] This invention prepared different types of DES (diethylstilbestrol), optimized the extraction method and determined the optimal process by considering their extraction efficiency and antisolvent purification yield of total flavonoids from *Symplocos rubra*. The antioxidant activity was compared using DPPH free radical scavenging capacity, and the morphological changes of the samples were observed using scanning electron microscopy (SEM). Therefore, ultrasound-assisted DES extraction and antisolvent purification technology provides a new green strategy for preparing total flavonoids from *Symplocos rubra*. The total flavonoids from *Symplocos rubra* of this invention exhibit significant antioxidant activity and can also be used for neuroprotective effects, treating cerebral ischemia or preventing ischemic stroke. They improve neurological function damage caused by cerebral ischemia, reduce infarct volume, alleviate neuronal damage, reduce astrocyte activation, decrease apoptosis protein expression levels, protect neurons and synapses, and protect vascular endothelial cells. The efficacy is clear and controllable, comparable to the positive control drug nimodipine, and significant efficacy can be achieved at low doses, making it safer for clinical use and providing a new clinical option. Attached Figure Description

[0027] Figure 1 Comparison chart of extraction and recovery rates of different DES types;

[0028] Figure 2 Effect of different molar ratios of HBA / HBD on the total flavonoid extraction yield (n=3);

[0029] Figure 3 Effect of solid-liquid ratio on total flavonoid extraction yield (n=3);

[0030] Figure 4 Effect of DES water addition on total flavonoid extraction yield (n=3);

[0031] Figure 5 Effect of different ultrasonic powers on total flavonoid extraction yield (n=3);

[0032] Figure 6 Effect of different ultrasound times on total flavonoid extraction yield (n=3);

[0033] Figure 7 Effect of different extraction methods on the yield of total flavonoids from *Symplocos pubescens* (n=3);

[0034] Figure 8 The scavenging effect of total flavonoids from *Senecio scandens* on DPPH free radicals;

[0035] Figure 9 SEM image of untreated dried half maple tissue (A), ultrasonic extraction with ethanol (B), ultrasonic-assisted DES (C);

[0036] Figure 10TTC staining was used to observe the effect of total flavonoids from *Symplocos pubescens* on the infarct volume of MCAO rats (Note: SHAM represents the sham-operated group, MCAO represents the model group, HS-L and HS-H represent the low and high administration groups of total flavonoids from *Symplocos pubescens*, respectively, and NMDP represents the nimodipine administration group).

[0037] Figure 11 Effects of total flavonoids from *Senecio scandens* on pathological changes in the ischemic cortical tissue of rats in the MCAO group (×200) (Note a: SHAM represents the sham-operated group, MCAO represents the model group, HS-L and HS-H represent the low and high administration groups of total flavonoids from *Senecio scandens*, respectively, and NMDP represents the nimodipine administration group).

[0038] Figure 12 Effects of total flavonoids from *Symplocos pubescens* on GFAP protein expression in the ischemic side of the brain of rats in the MCAO group (×400) (Note: SHAM represents the sham-operated group, MCAO represents the model group, HS-L and HS-H represent the low and high dose groups of total flavonoids from *Symplocos pubescens*, respectively, and NMDP represents the nimodipine-treated group).

[0039] Figure 13 Expression of apoptosis proteins in the ischemic cortex of rats in each group (Note a: SHAM represents the sham-operated group, MCAO represents the model group, HS-L and HS-H represent the low and high dose groups of total flavonoids from *Symplocos rubrum*, respectively, and NMDP represents the nimodipine-treated group; Note b: compared with the sham-operated group at the same time point...) # P<0.05, ## P<0.01; compared with the model group at the same time, * P<0.05, ** P<0.01);

[0040] Figure 14 Expression of axon-related proteins in the synaptic neurons of the ischemic side of rats in each group (Note a: SHAM represents the sham-operated group, MCAO represents the model group, HS-L and HS-H represent the low and high dose groups of total flavonoids from *Symplocos rubrum*, respectively, and NMDP represents the nimodipine-treated group; Note b: compared with the sham-operated group at the same time point...) ## P<0.01: Compared with the model group at the same time, * P<0.05, ** P<0.01);

[0041] Figure 15 Occludin protein expression in the ischemic cortex of rats in each group (Note a: SHAM represents the sham-operated group, MCAO represents the model group, HS-L and HS-H represent the low and high total flavonoid administration groups of *Symplocos rubrum*, respectively, and NMDP represents the nimodipine administration group; Note b: Compared with the sham-operated group at the same time point, ##P<0.01; compared with the model group at the same time point, *P<0.05, **P<0.01). Detailed Implementation

[0042] Example 1: Screening test of process parameters for the preparation of total flavonoids from *Senecio scandens* according to the present invention.

[0043] 1 Materials and Instruments

[0044] 1.1 Materials

[0045] The sample of *Semiliquidambarcathayensis* was identified by the Key Laboratory of Research and Development of Medicinal Resources of Fujian University of Traditional Chinese Medicine as the root of *Semiliquidambarcathayensis* Hung T. Chang, a plant of the genus *Semiliquidambarcathayensis* in the family Hamamelidaceae.

[0046] Choline chloride, betaine, lactic acid, taurine, ethylene glycol, acetamide, 1,4-butanediol, xylitol, glucose, acetylpropionylamine, glycerol, D-sorbitol (analytical grade), Aladdin Reagent Company; Sodium carbonate, anhydrous ethanol, methanol (analytical grade), Tianjin Zhiyuan Chemical Reagent Co., Ltd.; Ascorbic acid (analytical grade), Tianjin Ruijinte Chemical Co., Ltd.; Diphenylpicrylamide phenylhydrazine radical (DPPH), Folin-Ciocalteu (analytical grade), Shanghai Yuanye Technology Co., Ltd.

[0047] 1.2 Instruments

[0048] KQ-500DM-22.5 Ultrasonic Cleaner, Kunshan Ultrasonic Instrument Co., Ltd.; UV-2600 Ultraviolet Spectrophotometer, Shimadzu Corporation, Japan; 2500C Multifunctional Grinder, Yongkang Hongtaiyang Electromechanical Co., Ltd.; BSA124S Electronic Analytical Balance, Sartorius, Germany; Neo15 High-Speed ​​Refrigerated Centrifuge, Shanghai Lishen Scientific Instrument Co., Ltd.; SHJ-6AB Magnetic Stirred Water Bath, Changzhou Jintan Liangyou Instrument Co., Ltd.; SU 8010 Scanning Electron Microscope (SEM) Analyzer, Hitachi, Japan; DHG9030A Electric Heating Drying Oven, Shanghai Yiheng Scientific Instrument Co., Ltd.

[0049] 2 methods

[0050] 2.1 Preparation of different types of DES

[0051] In Table 1, HBA and HBD substances were mixed and heated at 80°C with constant stirring at a specified molar ratio to obtain a stable, homogeneous, and transparent liquid.

[0052] Table 1 Different types of eutectic solvents

[0053]

[0054] 2.2 Ultrasonic-assisted DES extraction of total flavonoids from *Symplocos pubescens*

[0055] The *Symplocos lumbricoides* sample was pulverized and passed through a 100-mesh sieve. Accurately weighed *Symplocos lumbricoides* powder (0.1 g) was mixed with 10 mL of LDES containing 30% water. The mixture was ultrasonically extracted at 500 W power and 40 kHz frequency at room temperature for 30 min. After extraction, the mixture was centrifuged at 3000 rpm, and the supernatant was collected to obtain the crude extract of total flavonoids from *Symplocos lumbricoides*. This process was repeated three times.

[0056] 2.3 Screening of processes for purifying total flavonoids from *Symplocos pubescens* using resistant solvents

[0057] Total flavonoids were extracted from *Symplocos pubescens*, and DES was removed. An antisolvent technique was used to separate the total flavonoids from the DES extract. In this study, water was added to the DES extract at a ratio of 1:20, and the mixture was stirred thoroughly and allowed to stand at room temperature for 24 hours. The mixture was then centrifuged at 3000 rpm for 10 minutes, the precipitate was collected, dried to a constant weight, and the yields of total flavonoids were compared under the same conditions after purification using different water addition ratios of 1:10, 1:20, 1:30, and 1:40.

[0058] 2.4 Optimization of Ultrasonic-Assisted DES Extraction Process for Total Flavonoids from *Symplocos pubescens*

[0059] Accurately weigh 1g of *Symplocos buergeriana* powder and place it in a 100mL Erlenmeyer flask. Investigate the effects of the HBA / HBD molar ratio in DES (1:1, 1:2, 1:3, 1:4, 1:5), the amount of water added to DES (10, 20, 30, 40, 50%), the ratio of *Symplocos buergeriana* powder to DES (1:10, 1:20, 1:30, 1:40 g / mL), the ultrasonic extraction time (10, 20, 30, 40, 50 min), and the ultrasonic power (100, 200, 300, 400, 500 W) on the total flavonoid content of *Symplocos buergeriana*.

[0060] 2.5 Comparison of different extraction methods

[0061] To investigate the efficiency of different extraction methods, three different extraction methods were designed for comparative experiments with the ultrasound-assisted DES extraction method screened above. The methods used were: water extraction (solid-liquid ratio 1:30, extraction for 8 h, boiling); alcohol extraction (solid-liquid ratio 1:30, 70% ethanol, extraction for 8 h, 80℃); and ultrasonic extraction (liquid-solid ratio 30:1, extraction for 40 min, ultrasonic power 500W).

[0062] 2.6 Construction of standard curve and determination of total flavonoid content of *Symplocos pubescens* (Liao Na, Liang Qiuling, Gao Xingling, et al. Optimization of extraction process of total flavonoids of *Symplocos pubescens* by hot reflux method [J]. Journal of Guangxi University of Science and Technology, 2019, 30(02): 93-96+128.)

[0063] Accurately weigh rutin as a reference standard and prepare stock solutions of 1.91, 3.82, 7.64, 11.46, 15.28, and 19.10 mg / mL with 80% methanol. Store these solutions at 4°C for later use. Add 0.7 mL of 5% NaNO₂ solution to each solution, shake well, let stand for 6 min, then add 0.7 mL of 10% Al(NO₃)₃ solution. After 6 min, add 10 mL of 1 mol / L NaOH solution and dilute to the mark. Prepare blank controls using the same method. Measure the absorbance at 509 nm using a UV spectrophotometer. Plot the standard curve as Y = 0.04075X + 0.0076, with coefficient R... 2 =0.9999. Weigh out half-maple powder, add DES solvent, extract with ultrasonic assistance, and filter; add water and stir thoroughly, let stand at room temperature for 24 hours to precipitate, and then centrifuge and dry. Then, determine the yield according to the method of the standard curve above.

[0064] 2.7 In vitro antioxidant activity assay

[0065] The crude extract of total flavonoids from *Symplocos pubescens* was purified with an antisolvent, centrifuged, precipitated, and dried to obtain the total flavonoid extract, which was then used for in vitro antioxidant experiments with DPPH. 1.0 mL of 0.1 mmol / L DPPH solution was added to six test tubes, followed by 2.0 mL of different concentrations (0.02, 0.04, 0.06, 0.08, 0.10, 0.12 mg / mL) of *Symplocos pubescens* total flavonoid test solution. After mixing, the mixture was reacted in the dark for 30 min, and the absorbance (A1) was measured at 519 nm. Under the same conditions, 1.0 mL of anhydrous ethanol was used instead of DPPH solution to measure the absorbance (A2), and the blank test was A0. Ascorbic acid of the same concentration was used as a positive control, and the scavenging rate was calculated according to the following formula.

[0066] DPPH free radical scavenging rate (%) = [1 - (A1 - A2) / A0] × 100%

[0067] 2.8 Scanning Electron Microscopy (SEM) Analysis

[0068] This section provides testing and analysis for the Key Laboratory of Traditional Chinese Medicine Resources Research and Development at the School of Pharmacy, Fujian University of Traditional Chinese Medicine. The microstructure of the total flavonoids powder from *Symplocos buergeriana* before and after extraction was observed using scanning electron microscopy to better understand the extraction process and possible mechanisms. Untreated *Symplocos buergeriana* powder, samples extracted with ethanol via ultrasonic extraction, and dried samples extracted with ultrasonic-assisted DES were mounted on aluminum needles with a thin gold layer and observed on a SU 8010 SEM analyzer under an accelerating voltage of 3.0 kV and an emission current of 10.5 μA.

[0069] 3 Results and Analysis

[0070] 3.1 Filtering of different types of DES

[0071] Fifteen different types of DES were selected for screening, and their ability to extract total flavonoids from *Symplocos buergeriana* was evaluated using ultrasonic extraction, compared with reference solvents (methanol and 75% ethanol). Extraction was performed at a solid / liquid ratio of 1:20 g / 100 mL, with ultrasonication at room temperature for 30 min. Additionally, 30% (v / v) water was added to the DES solution to reduce viscosity and facilitate handling. Extraction rates are shown in Table 2.

[0072] Table 2. Effects of different DES on the extraction of total flavonoids from *Senecio scandens* (n=3)

[0073]

[0074] Note: Compared to traditional solvents 75% ethanol and methanol. # P<0.05, ## P<0.01.

[0075] Table 2 shows that the type of DES has a significant impact on the extraction rate of total flavonoids from *Symplocos pubescens*. Compared with traditional solvents such as 75% ethanol and methanol, different types of DES significantly improved the extraction yield of total flavonoids (p<0.05). The total flavonoids extracted by DES-2, DES-7, and DES-10 (>27 mg / g) were significantly higher than those extracted by traditional methods. The hydrogen donor system, consisting of polycarboxylic acids, contains a certain amount of free hydrogen ions. These ions combine with the phenolic hydroxyl groups in flavonoids to form strong hydrogen bonds, promoting the dissolution of flavonoids into the solvent medium.

[0076] 3.2 Investigation of Antisolvent Purification and Recovery Process

[0077] To address the water solubility of DES and the slight water solubility of total flavonoids from *Symplocos rubra*, an antisolvent method was employed to extract total flavonoids from DES extract. The principle of this method is to add a large amount of water to break the bonds between HBA and HBD, thereby eliminating the properties of DES and precipitating the desired compound.

[0078] See Table 2 for extraction rate and antisolvent purification results. Figure 1 The results indicate that the type of DES significantly affects the purity and recovery rate of *Mallotus philippinensis*, which may be related to the physical properties of DES, such as acidity, alkalinity, and polarity. Furthermore, the extraction and purification recovery rates of total flavonoids from *Mallotus philippinensis* do not correlate with the type of DES. Although the extraction rate of DES-2 composed of choline chloride and ethylene glycol was not the highest, the addition of an antisolvent resulted in a higher purity of total flavonoids from *Mallotus philippinensis*. Therefore, subsequent experiments selected DES-2 composed of choline chloride and ethylene glycol as the extraction solvent. Using this as a condition, the yields of total flavonoids purified with antisolvent water at ratios of 1:10, 1:20, 1:30, and 1:40 were compared. The results showed that a ratio of 1:30 resulted in the most complete precipitation; excessively high ratios increased the workload of centrifugation.

[0079] 3.3 Optimization of Extraction Process

[0080] Investigate the effect of the HBA / HBD molar ratio on extraction, such as Figure 2 As shown, the extraction yield of total flavonoids from *Symplocos rubra* initially increases and then decreases with increasing proportion of hydrogen donors in the system. The molar ratio of hydrogen bond donors to hydrogen bond acceptors is a key factor in the physicochemical properties of the solvent, and the extraction yield of flavonoids is greatly affected by the molar ratio of the solvent system. When the proportion of hydrogen bond donors in the system increases, the solubility is enhanced, but an excessively strong environment can affect the dissolution of flavonoids, leading to a decrease in extraction yield. Simultaneously, increasing the proportion of hydrogen donors in the system can cause DES to become too viscous, resulting in insufficient solubility space for flavonoids in the system, thus reducing the extraction yield.

[0081] The effect of the material-to-liquid ratio on the extraction yield of total flavonoids from *Symplocos pubescens* was investigated. Figure 3 It was found that the extraction yield of total flavonoids from *Symplocos pubescens* showed a trend of first significantly increasing and then significantly decreasing (P<0.05), with the highest extraction yield observed at a solid-liquid ratio of 1:30 g / mL. This may be because the increased concentration gradient between the target compound and the solvent increases the diffusion space of the total flavonoid particles, thus increasing the contact area with the solvent and increasing the mass transfer driving force, accelerating the material transport process and promoting flavonoid extraction. However, as the solvent volume continues to increase, the DES system becomes too viscous, leading to a decrease in the radiative energy acting on the raw material, resulting in reduced flavonoid dissolution. This may promote the dissolution of some lipid-soluble and alcohol-soluble impurities, not only reducing the extraction yield of the target compound but also wasting solvent. Therefore, a solid-liquid ratio of 1:30 g / mL was chosen for subsequent experiments.

[0082] The effect of water addition on the total flavonoid extraction yield of *Scutellaria baicalensis* was investigated, such as... Figure 4 It was found that by taking water contents of 10%, 20%, 30%, 40%, and 50% as variables, the effect of water content on flavonoid extraction rate was compared. As the water content of the DES solvent increased, the extraction rate of total flavonoids also increased continuously. When the water content increased to 30%, the extraction rate of total flavonoids reached its maximum value. Further increasing the water content of the DES solvent resulted in a decrease in the extraction rate. Therefore, 30% was selected as the optimal water content level.

[0083] The effect of different ultrasonic powers on the extraction yield of total flavonoids from *Symplocos pubescens* was investigated, such as... Figure 5It is observed that when the ultrasonic power is between 100-400W, the flavonoid extraction yield gradually increases with increasing power. The total flavonoid extraction yield reaches its maximum at 400W and then decreases. This may be because, in the initial extraction stage, the ultrasound causes the raw material particles to vibrate in the solvent space, resulting in cavitation and partially damaging plant cells, thus increasing the diffusion of the target substances. When the ultrasonic power exceeds 300W, the excessive power may cause thermal and mechanical effects that damage flavonoids, leading to partial flavonoid decomposition and a decrease in content.

[0084] The effect of different ultrasound times on the extraction yield of total flavonoids from *Symplocos pubescens* was investigated. Figure 6 It was found that the extraction yield of total flavonoids from *Symplocos rubra* initially increased and then decreased with increasing ultrasonic time, reaching a peak at 40 min. Ultrasonic time is a crucial parameter affecting flavonoid extraction yield. Shorter extraction times make it difficult to separate flavonoids from other active substances. Increased ultrasonic time leads to more thorough cell wall disruption, facilitating the release and dissolution of flavonoids, thus increasing the extracted flavonoid yield. However, after 40 min, the flavonoids are oxidized or even decomposed due to the influence of light and heat, reducing the extraction yield. Furthermore, excessively long ultrasonic times increase energy consumption, leading to increased extraction costs. Therefore, an ultrasonic time of 40 min was chosen for subsequent experiments.

[0085] 3.4 Verification Experiment of Optimal Process Conditions

[0086] Based on the screening of DES types and optimization of extraction and purification processes, the optimal extraction conditions were as follows: A DES solution was prepared by stirring choline chloride and ethylene glycol at a 1:2 molar ratio at 80℃; *Hemiberlesia argyi* was pulverized and passed through a 100-mesh sieve; and then set aside. The extraction conditions were: DES water addition rate 30% (w / w), solid-liquid ratio 1:30, ultrasonic power 400W, and ultrasonic extraction time 40 min. The obtained *Hemiberlesia argyi* total flavonoid DES extract was mixed with water at a 1:30 ratio and stirred thoroughly, then allowed to stand at room temperature for 24 h. The mixture was centrifuged at 3000 rpm for 10 min, the precipitate was collected, and dried to a constant weight.

[0087] Three verification experiments were conducted under the above conditions: the total flavonoid content was 28.65 ± 3.85%, and the total flavonoid yield after purification using the antisolvent method was 71.95% ± 6.49%. This invention provides a highly efficient and rapid method for extracting total flavonoids.

[0088] 3.5 Comparative Analysis of Different Extraction Methods

[0089] like Figure 7It can be seen that ultrasonic extraction is superior to other extraction methods. Alcohol extraction is superior to water extraction, and ultrasonic-assisted DES extraction is superior to the other three single extraction methods. Moreover, it yields the highest total flavonoids from *Symplocos rubra*, with the best extraction effect and a total flavonoid content of 28.59% ± 0.75%.

[0090] 3.6 Determination of antioxidant activity based on DPPH free radical scavenging capacity

[0091] like Figure 8 It was found that the total flavonoid extract of *Symplocos pubescens* exhibited a certain scavenging effect on DPPH free radicals within the concentration range of 0.1 mg / mL to 3.2 mg / mL, and this effect increased rapidly with increasing concentration, showing a positive correlation with concentration. However, at the same concentration, vitamin C's free radical scavenging ability was consistently higher than that of the total flavonoid extract of *Symplocos pubescens*. At a concentration of 3.2 mg / mL, the total flavonoid extract of *Symplocos pubescens* showed a DPPH scavenging ability of 77.52%, indicating its strong ability to inhibit DPPH free radicals.

[0092] 3.7 Microstructure Analysis

[0093] The mechanism of action of UAE-DES and the microstructure of untreated and dried EH after extraction were observed using scanning electron microscopy.

[0094] like Figure 9 As shown in Figure A, the untreated sample powder has a relatively smooth and flat outer surface, and the cell structure is intact without obvious broken structures. In contrast, after ultrasonic extraction with 70% ethanol ( Figure 9 B) The outer surface of the sample powder showed slight cracks, and the rough surface exhibited obvious cracks, indicating that the cells and cell walls were disrupted. Ultrasonic waves exposed the target compound to the extraction solution. The cavitation, mechanical, and thermal effects generated during ultrasound are widely considered to be the main reasons for improved extraction rates. Furthermore, Figure 9 C shows that after ultrasound-assisted DES (choline chloride / ethylene glycol) extraction, the sample powder became coarser and significantly cracked, indicating that DES has a stronger penetrating and damaging ability than 70% ethanol solution, resulting in a higher flavonoid extraction rate.

[0095] Because DES more readily forms hydrogen bonds with the polyhydroxyl groups of flavonoids, its affinity for dissolving flavonoids is significantly greater than that of 50% ethanol, which is considered one of the main factors contributing to the high extraction efficiency of DES. Simultaneously, the low vapor pressure of choline chloride and the fact that ethylene glycol facilitates the formation of high-intensity cavitation further enhance extraction efficiency. Ultrasound plays a crucial role in the extraction process; the high degree of cell wall disruption caused by ultrasound promotes the release of target compounds from the plant matrix, improving extraction efficiency and thus increasing the extraction rate.

[0096] 4. Conclusion

[0097] In this study, taking advantage of the low melting point of DES, ultrasonic-assisted extraction with DES solvent was used to extract total flavonoids from Semiliquidambar cathayensis Hance with the best effect. Moreover, the obtained total flavonoids from Semiliquidambar cathayensis Hance had high antioxidant activity and showed a dose-effect relationship in terms of concentration. This method has many advantages. First, as a green solvent, DES will not cause environmental pollution. Second, the efficient extraction and separation of total flavonoids from Semiliquidambar cathayensis Hance are controlled with the help of hydrogen bond theory.

[0098] This method is expected to become a new alternative for the preparation of total flavonoids from Semiliquidambar cathayensis Hance. However, there may still be some deficiencies, such as low extraction efficiency and many by-products. Through further optimization, the extraction efficiency can be improved, the by-products can be reduced, and the cost can be lowered.

[0099] The beneficial effects of the present invention are demonstrated by the following pharmacodynamic experiments.

[0100] Experimental Example 1 Neuroprotective effect of total flavonoids from Semiliquidambar cathayensis Hance of the present invention on rats with cerebral ischemia-reperfusion injury

[0101] This chapter intends to explore whether total flavonoids from Semiliquidambar cathayensis Hance act on MCAO rats by observing the effects of total flavonoids from Semiliquidambar cathayensis Hance on the neurological function and the components of NVU in MCAO rats.

[0102] 1 Experimental materials

[0103] 1.1 Experimental animals

[0104] A total of 120 specific pathogen-free (SPF) healthy male rats with a body weight of (260±10) g were used in the experiment. All rats were purchased from Hangzhou Medical College, and the production license number was SCXK(Zhe) 2019-0002. They were provided by the Animal Experiment Center of Fujian University of Traditional Chinese Medicine, and the animal certificate number was 20210112Aazz0100000682. The rats were housed in a specific sterile environment with strictly controlled humidity and temperature (22°C) under a 12-hour light-dark cycle and were allowed to eat and drink freely. The animal experiment procedures were carried out strictly in accordance with international ethical guidelines and the guidelines for the care and use of laboratory animals formulated by the National Institutes of Health and were approved by the Animal Management and Use Committee of Fujian University of Traditional Chinese Medicine, with the number FJTCMIACUC 2020042.

[0105] 1.2 Experimental drugs

[0106] The research group previously studied the pharmacodynamic substance basis of the total flavonoid extract from Semiliquidambar cathayensis Hance, and the total flavonoid content was greater than 20%.

[0107] Nimodipine tablets: 20mg specification, manufactured by Yabao Pharmaceutical Group Co., Ltd., with a production date of November 11, 2020, and an expiration date of October 2023. Product batch number: 201101 National Approval Number H14022821.

[0108] 2 Experimental Methods

[0109] 2.1 Group administration

[0110] Eighty-four rats that successfully developed the model were randomly divided into four groups: the model group (MCAO), the low-dose group of total flavonoids from *Symplocos pubescens* (HS-L), the high-dose group of total flavonoids from *Symplocos pubescens* (HS-H), and the nimodipine group (NMDP), with 21 rats in each group. After modeling, all groups were administered the drugs by gavage on the same day after the animals recovered from surgery. The SHAM group and the MCAO group were given normal saline once a day for 7 consecutive days.

[0111] Based on the literature and preliminary experimental results, the preparation process for low-dose total flavonoids of *Symplocos lucida* in this experiment was as follows: 105g of total flavonoids of *Symplocos lucida* was dissolved in 150ml of physiological saline to obtain a total flavonoid solution with a concentration of 0.7g / ml; the dosage per rat was 5ml / kg / day, meaning that each rat in the low-dose group was administered 3.5g / kg of raw drug by gavage daily. The preparation process for high-dose total flavonoids of *Symplocos lucida* was as follows: 210g of total flavonoids of *Symplocos lucida* was dissolved in 150ml of physiological saline to obtain a total flavonoid solution with a concentration of 1.4g / ml; the dosage per rat was 5ml / kg / day, meaning that each rat in the high-dose group was administered 7.0g / kg of raw drug by gavage daily. Based on the conversion of clinically equivalent doses between humans and rats, the dosage of nimodipine was 6.0 mg / kg. The preparation process involved crushing 126 mg nimodipine tablets and dissolving them in 26.25 ml of physiological saline. The dosage for each rat was 5 ml / kg / day.

[0112] 3 Experimental Results

[0113] 3.1 Effects of total flavonoids from *Senecio scandens* on general physical signs in MCAO rats

[0114] Rats in the sham-operated group had a rounded body shape, smooth and shiny fur, and normal water and food intake. Their stool volume was normal. Rats in the model group had frizzy and dull fur, were emaciated, and had significantly reduced water and food intake, as well as significantly reduced and soft stool volume. Rats in the drug-treated group did not continue to lose weight after 3 days and slowly recovered. Their fur was dull but smoother than before, and their water and food intake gradually increased. Their stool volume increased and became slightly drier.

[0115] The changes in body weight of rats in each group are shown in Table 3. On day 3 post-surgery, compared with the sham-operated group, the body weight of the model group decreased significantly (P<0.01). Subsequently, the body weight of the model group showed a continuous and significant downward trend, while the body weight of each treatment group increased slowly. On day 5, compared with the sham-operated group, the body weight of rats in the model group was significantly reduced (P<0.01); compared with the model group, the body weight of the low-dose total flavonoids group and the nimodipine group was significantly increased (P<0.05). On day 7, compared with the model group, the body weight of the total flavonoids group was significantly increased (P<0.05), and the body weight of the nimodipine group was significantly increased (P<0.01).

[0116] Table 3. Changes in rat body weight in each group.

[0117]

[0118] Note: Compared with the sham surgery group at the same time point. ## P<0.01: Compared with the model group at the same time, * P<0.05, ** P<0.01.

[0119] 3.2 Effects of total flavonoids from *Senecio scandens* on the degree of neurological deficit in MCAO rats

[0120] 3.2.1mNSS score

[0121] The neurological deficit scores of rats in each group are shown in Table 4. Compared with the sham-operated group, the neurological function scores of the model group were significantly increased on days 3, 5, and 7 after administration (P<0.01); compared with the model group, the neurological function scores of each administration group were significantly decreased on days 5 and 7 (P<0.01). This indicates that the total flavonoids of *Symplocos buergeriana* and nimodipine can improve neurological function impairment.

[0122] Table 4. Neurological deficit scores of rats in each group

[0123]

[0124] Note: Compared with the sham surgery group at the same time point. ## P<0.01: Compared with the model group at the same time, ** P<0.01.

[0125] 3.2.2 Foot tension test

[0126] The neurological deficit scores of rats in each group are shown in Table 5. Compared with the SHAM group, the paw tension of rats in the MCAO group was significantly reduced (P<0.01); compared with the MCAO group, the paw tension of rats in the drug-treated group was significantly increased (P<0.01).

[0127] Table 5 Results of rat paw tension tests in each group

[0128]

[0129] Note: Compared with the sham surgery group at the same time point. ## P<0.01: Compared with the model group at the same time, ** P<0.01.

[0130] 2.3 Corner Experiment

[0131] The results of the turning test in each group of rats are shown in Table 6. On days 3, 5, and 7 after administration, the right-turning frequency in the MCAO group was significantly higher than that in the SHAM group (P<0.01); there was no significant difference in the administration groups on day 3 compared with MCAO; on days 5 and 7, the right-turning frequency in the total flavonoids-treated group was significantly lower (P<0.05), and the frequency in the nimodipine-treated group was significantly lower (P<0.01). This indicates that total flavonoids from *Scutellaria baicalensis* significantly improve the motor function of MCAO rats.

[0132] Table 6 Results of the rat turning experiment in each group

[0133]

[0134] Note: Compared with the sham surgery group at the same time point. ## P<0.01: Compared with the model group at the same time, * P<0.05, ** P<0.01.

[0135] 3.3 Effect of total flavonoids from *Senecio scandens* on cerebral infarction volume in MCAO rats

[0136] The cerebral infarction volume of rats in each group is shown in Table 7. Figure 10 As shown, red represents normal brain tissue, and white represents the site of cerebral infarction. In the sham-operated group, the brain tissue stained red, indicating no cerebral infarction. In the model group, rats underwent 2 hours of ischemia followed by 7 days of reperfusion in the left middle cerebral artery, resulting in significant infarction in the area supplied by the middle cerebral artery. The area of ​​the cerebral infarction zone was significantly smaller in all treatment groups with total flavonoids from *Scutellaria baicalensis* compared to the MCAO group. This suggests that administration of total flavonoids from *Scutellaria baicalensis* can reduce the volume of cerebral infarction in rats with cerebral ischemia-reperfusion injury.

[0137] Table 7. Effects of total flavonoids from *Senecio scandens* on cerebral infarction volume in MCAO rats.

[0138]

[0139] Note: Compared with the sham surgery group at the same time point. ## P<0.01: Compared with the model group at the same time, **P<0.01.

[0140] 3.4 Effects of total flavonoids from *Senecio scandens* on pathological changes in the ischemic cerebral cortex of MCAO rats

[0141] HE staining results are shown in […]. Figure 11 As shown, in the sham-operated group, the nuclei of nerve cells in the cerebral cortex of rats were round, stained light blue, located in the center of the cell, and arranged neatly and tightly. The cell structure was intact and the outline was clear, with no obvious pathological changes. In the model group, the ischemic side cortical tissue of rats showed nuclear condensation, stained dark blue, with the nuclei compressed to one side of the cell, and even some cells ruptured, arranged in a disordered manner, with large pericellular gaps and blurred outlines, indicating cell apoptosis or necrosis. After 7 days of intervention with total flavonoids from *Symplocos rubrum*, the above phenomena in the ischemic side cortical tissue of rats at all doses were improved to a certain extent, and the degree of neuronal damage was reduced.

[0142] Note b: Solid red arrows represent normal neurons, and dashed red arrows represent degenerated neurons.

[0143] 3.6 Effects of total flavonoids from *Senecio scandens* on GFAP protein expression in MCAO rats

[0144] Immunofluorescence staining results as follows Figure 12 As shown, compared with the sham-operated group, the number, areal density, and optical density of GFAP-positive cells on the ischemic side of MCAO rats were significantly increased, indicating that cerebral ischemia-reperfusion can stimulate astrocyte activation. After 7 days of administration of total flavonoids from *Symplocos pubescens* and nimodipine, the number, areal density, and optical density of GFAP-positive cells in each treatment group were significantly reduced, indicating that total flavonoids from *Symplocos pubescens* can reduce astrocyte activation.

[0145] 3.7.2 Effects of total flavonoids from *Senecio scandens* on the expression level of apoptosis proteins in the cortex of MCAO model rats

[0146] Western blot analysis was used to detect the expression levels of Bad, Bcl2, Bax, Caspase9, Caspase3, and Cleaved caspase3 proteins. Figure 13As shown: Compared with the sham-operated group, the expression of Bad, Caspase9, Caspase3, and Cleaved caspase3 proteins and the Cleaved caspase3 / Caspase3 ratio were significantly increased in the model group (P<0.01), while Bcl2 protein expression was significantly decreased (P<0.01), Bax protein expression was significantly increased (P<0.05), and the Bcl2 / Bax ratio was significantly decreased (P<0.01). Compared with the model group, the expression of Bad protein was significantly decreased (P<0.01), while Bcl2 protein expression was significantly increased (P<0.05), Bax protein expression was significantly decreased (P<0.05), and the Bcl2 / Bax ratio was significantly increased (P<0.01) in each drug-treated group. The expression of Caspase9, Caspase3, and Cleaved caspase3 proteins was significantly decreased (P<0.01), and the Cleaved caspase3 / Caspase3 ratio was significantly decreased (P<0.01) in each drug-treated group. The above results indicate that total flavonoids from *Symplocos rubra* can increase the expression level of anti-apoptotic proteins and decrease the expression level of apoptotic proteins in rats in the MCAO group.

[0147] 3.8 Effects of total flavonoids from *Senecio scandens* on the expression levels of axonal proteins in cortical neurons of MCAO rats

[0148] Western blot analysis was used to detect the expression of NeuN, MAP2, Synuclein-1, and α-synuclein proteins. Figure 14 As shown, compared with the sham-operated group, the expression of NeuN, MAP2, Synuclein-1, and α-synuclein proteins in the model group was significantly decreased (P<0.01). Compared with the model group, the expression of NeuN, Synapsin-1, and α-synuclein proteins in each treatment group was significantly increased (P<0.01); the expression of MAP2 protein was significantly increased in each dose group of total flavonoids from *Scutellaria baicalensis* (P<0.05). This suggests that total flavonoids from *Scutellaria baicalensis* have a protective effect on neurons and synapses.

[0149] 3.9 Effect of total flavonoids from *Liriope muscari* on the expression level of tight junction protein in the cortex of MCAO model rats. The expression of the tight junction protein ocludin was detected by Western blotting. Figure 15 As shown, compared with the SHAM group, the expression of Occludin protein in the MCAO group was significantly decreased (P<0.01). Compared with the MCAO group, the expression of Occludin protein was significantly increased in all doses of total flavonoids from *Scutellaria baicalensis* (P<0.01), and significantly increased in the nimodipine group (P<0.05). This indicates that total flavonoids from *Scutellaria baicalensis* have a protective effect on the tight junctions between vascular endothelial cells in MCAO rats.

[0150] summary

[0151] This invention elucidates from behavioral, morphological, and molecular biological perspectives that total flavonoids from *Symplocos rubrum* may exert their anti-ischemic stroke effect through anti-apoptosis and protection of various components of the neurovascular unit.

Claims

1. Total flavonoids from *Symplocos pubescens*, characterized in that: It is a species of the genus *Senecio scandens* in the family Hamamelidaceae. Semiliquidambarcathayensis The total flavonoids were obtained from the roots of Hung T. Chang using ultrasonic-assisted eutectic solvent extraction, with a yield of 71.95% ± 6.49%. It was prepared by the following steps: a. Preparation of eutectic solvent DES: Hydrogen bond acceptor HBA and hydrogen bond donor HBD were mixed at a molar ratio of 1:1-5, heated and stirred at 80°C until a stable, homogeneous and transparent liquid was formed. b. Preparation of extraction solvent: Add water to the eutectic solvent DES to prepare DES with a water content of 10-50%; c. Pre-treatment of raw materials: Take half of the lotus stem, crush and sieve it; d. Extraction: Mix the raw material powder processed in step c with the extraction solvent in step b. The ratio of powder to DES is 1:(10-40) g / mL. Perform ultrasonic extraction at room temperature with a power of 100-500W and a frequency of 40KHz for 10-50 min. After extraction, centrifuge at 3000 rpm and collect the supernatant to obtain the crude extract of total flavonoids from *Symplocos buergeriana*. Repeat the extraction step 1-3 times. e. Purification: The crude extract of total flavonoids from *Symplocos pubescens* is prepared by purifying with a resistant solvent in step d. After standing and centrifugation, the precipitate is collected and dried to obtain total flavonoids from *Symplocos pubescens*. The resistant solvent is water, and the ratio of the crude extract of total flavonoids from *Symplocos pubescens* to the resistant solvent water is 1:(10-40). The hydrogen bond acceptor HBA is one of choline chloride, betaine, and lactic acid; the hydrogen bond donor HBD is one of acetamide, ethylene glycol, glycerol, 1,4-butanediol, xylitol, glucose, levulinic acid, and D-sorbitol.

2. A method for preparing total flavonoids from *Symplocos buergeriana* as described in claim 1, characterized in that: It includes the following steps: a. Preparation of eutectic solvent DES: Hydrogen bond acceptor HBA and hydrogen bond donor HBD were mixed at a molar ratio of 1:1-5, heated and stirred at 80°C until a stable, homogeneous and transparent liquid was formed. b. Preparation of extraction solvent: Add water to the eutectic solvent DES to prepare DES with a water content of 10-50%; c. Pre-treatment of raw materials: Take half of the lotus stem, crush and sieve it; d. Extraction: Mix the raw material powder processed in step c with the extraction solvent in step b. The ratio of powder to DES is 1:(10-40) g / mL. Perform ultrasonic extraction at room temperature with a power of 100-500W and a frequency of 40KHz for 10-50 min. After extraction, centrifuge at 3000 rpm and collect the supernatant to obtain the crude extract of total flavonoids from *Symplocos buergeriana*. Repeat the extraction step 1-3 times. e. Purification: The crude extract of total flavonoids from *Symplocos pubescens* is prepared by purifying with a resistant solvent in step d. After standing and centrifugation, the precipitate is collected and dried to obtain total flavonoids from *Symplocos pubescens*. The resistant solvent is water, and the ratio of the crude extract of total flavonoids from *Symplocos pubescens* to the resistant solvent water is 1:(10-40).

3. The method for preparing total flavonoids from *Symplocos pubescens* according to claim 2, characterized in that: The water content of the extraction solvent in step b is 30%; The sieve mesh size described in step c is 100 mesh; In step d, the ratio of the drug powder to the DES liquid is 1:30 g / mL; the ultrasonic power is 400 W; and the ultrasonic time is 40 min. The ratio of the crude extract of total flavonoids from *Symplocos pubescens* to the resistant solvent water in step e is 1:30; the centrifugation conditions are: 3000 rpm for 10 min.

4. The method for preparing total flavonoids from *Symplocos pubescens* according to claim 3, characterized in that: The molar ratio of the hydrogen bond acceptor HBA to the hydrogen bond donor HBD is: Choline chloride:ethylene glycol = 1:2; or, Betaine: levulinic acid = 1:2; or, Betaine: Glycerol = 1:

2.

5. The method for preparing total flavonoids from *Symplocos pubescens* according to claim 4, characterized in that: The molar ratio of the hydrogen bond acceptor HBA to the hydrogen bond donor HBD is: choline chloride: ethylene glycol = 1:

2.

6. Use of the total flavonoids of *Liriope muscari* according to claim 1 in the preparation of a medicament for treating cerebral ischemia or anti-ischemic stroke.

7. The use according to claim 6, characterized in that: The drug described is used to improve neurological function damage caused by cerebral ischemia, reduce the volume of cerebral infarction, alleviate the degree of neuronal damage, reduce the activation of astrocytes, reduce the expression level of apoptotic proteins, protect neurons and synapses, and protect vascular endothelial cells.

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