Anti-asthma inflammation traditional Chinese medicine extract as well as extraction method and application thereof

Abstract

The invention belongs to the field of medicinal plant development, and particularly relates to an anti-asthma inflammation traditional Chinese medicine extract as well as an extraction method and application thereof. Millettia specisoa champ is used as a raw material, and Millettia specisoa champ total flavonoids and Millettia specisoa champ total The two extracts are subjected to pharmacological experiments, and researches show that the beautiful millettia root extract containing the total flavonoids or the total polysaccharides can remarkably reduce the levels of Th2 inflammatory factors such as IL-4, IL-5, IL-9, IL-13 and GATA-3 in lung tissues, improve the levels of Th1 anti-inflammatory factors such as IFN-gamma, IL-12 and IL-27, regulate the proportion balance of Th1 / Th2 and inhibit airway inflammatory response; in addition, GATA-3 expression can be blocked, Th2 cell activation hyperfunction is inhibited, pathological characteristic change of asthma is relieved, and great development and utilization potential in asthma treatment is achieved.

Description

Technical Field

[0001] This invention belongs to the field of medicinal plant development, specifically relating to an anti-asthmatic and anti-inflammatory traditional Chinese medicine extract, its extraction method, and its application. Background Technology

[0002] Asthma, also known as bronchial asthma, is a chronic inflammatory disease of the airways involving multiple cells. Clinical manifestations often include increased airway mucus secretion, airway hyperresponsiveness, and reversible airflow limitation. This is a global public health problem; studies indicate that by 2025, there are projected 400 million asthma patients worldwide, with approximately 180,000 deaths annually. Furthermore, the incidence and mortality rates of asthma continue to increase annually. Current clinical medications for asthma mainly include glucocorticoids, β2 agonists, and leukotriene receptor antagonists; however, these drugs only temporarily suppress airway inflammation and cannot completely cure asthma, and they also have serious adverse reactions. Therefore, finding and developing safe and effective asthma treatments holds great promise.

[0003] Numerous studies have shown that the balance of immune T cell differentiation plays a crucial role in the development and progression of asthma. Th2 cells secrete cytokines such as IL-4 and IL-5, inducing B lymphocytes to produce IgE, eosinophils to infiltrate the airways, and goblet cell proliferation and excessive mucus production. Th1 cells secrete cytokines such as IFN-γ and IL-12, antagonizing Th2 cytokines and thus suppressing the inflammatory response, reducing the incidence of asthma. Dendritic cells (DCs) induce CD4+... + T cells differentiate into Th2 cells and are inhibited from differentiating into Th1 cells, while most asthma patients exhibit a Th2 / Th1 imbalance with Th2 dominance. Further research has revealed that the transcription factor GATA3 can specifically regulate Th2 cell differentiation through its own mechanisms, promoting the transformation of Th2 cells into Th1 cells to some extent. Therefore, investigating the molecular mechanisms by which Th1 / Th2 cell balance is regulated could be a major breakthrough in asthma immunotherapy; furthermore, detecting GATA3 protein expression levels can better elucidate these mechanisms.

[0004] In recent years, Traditional Chinese Medicine (TCM) has received widespread attention due to its long and rich history in China, and has demonstrated its advantages as a supplement or alternative to other treatments for diseases. Therefore, there is an urgent need to develop a TCM extract for treating asthma and inflammation. *Millettia dielsiana*, a popular medicinal and edible plant in the Lingnan region of my country, belongs to the genus *Millettia* of the Fabaceae family. Millettiaspeciosa The dried root of *Champ.*. Its main producing areas include Guangxi, Hainan, and Guangdong provinces. Due to its inexpensive availability, this application proposes an anti-asthmatic and anti-inflammatory extract based on *Champ.*. Summary of the Invention

[0005] The purpose of this invention is to provide an anti-asthmatic inflammatory traditional Chinese medicine extract, its extraction method and application. The operation is simple and convenient, and the obtained anti-asthmatic inflammatory extract can inhibit the inflammatory response of asthma and reduce the pathological changes of asthma.

[0006] Another objective of this invention is to provide the mechanism of action of the above-mentioned anti-asthmatic inflammatory extract in the treatment of asthma.

[0007] To achieve the above objectives, the technical solution adopted in this application is as follows: An anti-asthmatic and anti-inflammatory traditional Chinese medicine extract is prepared using *Smilax glabra* as a raw material, specifically a total flavonoid extract or a total polysaccharide extract of *Smilax glabra*.

[0008] The method for extracting the anti-asthmatic and anti-inflammatory traditional Chinese medicine extract involves using alcohol extraction to extract the total flavonoid extract of *Smilax glabra* and using water extraction to extract the total polysaccharide extract of *Smilax glabra*.

[0009] The specific steps for extracting total flavonoids from *Smilax glabra* using alcohol extraction are as follows: (1) After crushing the ox-duck-duck, mix the crushed material with 70% ethanol at a mass-volume ratio of 1:30~100 (w / v) and heat under reflux for 0.5-2 hours; (2) Take the product obtained in step (1), filter it, take the filtrate, place it in a refrigerated centrifuge and centrifuge at 3000 rpm for 10 min to separate it by centrifugation; (3) Take the supernatant obtained in step (2) and concentrate it under reduced pressure at 50~65℃ for 2~4 h to obtain the concentrate; (4) After the concentrate from step (3) is frozen at -80℃ for 12-24h, it is then placed in a vacuum freeze dryer at -50~-65℃ for 32-36h to obtain the total flavonoid extract of *Smilax glabra*.

[0010] Preferably, in step (1), the mass-to-volume ratio of the pulverized material to 70% ethanol is 1:60; and the mixture is heated under reflux for 1 hour.

[0011] The method for extracting the anti-asthmatic and anti-inflammatory traditional Chinese medicine extract, using water extraction to extract the total polysaccharide extract of *Achyranthes bidentata*, involves the following steps: (1) After crushing the powder, mix it with ultrapure water at a mass-volume ratio of 1:60~100 (w / v) and heat extract at 95-100℃ for 1-2 hours; (2) After filtering the product obtained in step (1), the filtrate is concentrated under reduced pressure at 50~70℃ for 4~6 h until it becomes viscous; (3) Add 80% ethanol to the concentrate obtained in step (2), place it in a refrigerator at 4°C and let it stand for 5 hours to precipitate alcohol. (4) After removing the upper liquid from the product obtained in step (3), place the remaining mixture in a centrifuge and centrifuge at 3000 rpm for 10 min. (5) After removing the supernatant from the product obtained in step (4), the flocculent precipitate is frozen at -80℃ for 12-24h, and then freeze-dried at -50~-65℃ in a freeze dryer for 32-36h to obtain the total polysaccharide extract of *Smilax glabra*.

[0012] Preferably, in step (1), the mass-to-volume ratio of *Euphorbia lactea* powder to ultrapure water is 1:80; and the hot extraction time in step (1) is 1.5 h.

[0013] Preferably, in step (3), 80% ethanol is added at a volume three times that of the concentrate.

[0014] Application of an anti-asthmatic and anti-inflammatory traditional Chinese medicine extract in the preparation of anti-asthmatic and anti-inflammatory drugs.

[0015] The application of the aforementioned anti-asthmatic and anti-inflammatory traditional Chinese medicine extract in the preparation of anti-asthmatic and anti-inflammatory drugs is characterized in that the specific effective components are total flavonoid extract of *Smilax glabra* and / or total polysaccharide extract of *Smilax glabra*.

[0016] The application of the aforementioned anti-asthmatic and anti-inflammatory traditional Chinese medicine extract in the preparation of anti-asthmatic and anti-inflammatory drugs is that the drug dosage form is any oral preparation.

[0017] Extracts of *Achyranthes bidentata* containing total flavonoids or total polysaccharides can regulate the Th1 / Th2 immune balance by reducing the levels of Th2 cytokines such as IL-4, IL-5, IL-9, IL-13, and GATA-3, and increasing the levels of Th1 cytokines such as IFN-γ, IL-12, and IL-27. In addition, the extracts can inhibit the expression level of GATA-3, a key transcription factor for Th2 differentiation, and weaken the Th2-mediated immune inflammatory response, thereby playing a role in inhibiting airway inflammation and remodeling, and significantly improving the pathological changes in lung tissue of OVA-induced asthmatic mice. Attached Figure Description

[0018] Figure 1 The pathological effects of FMSCs on lung tissue in OVA-induced asthmatic mice; A) HE staining of lung tissue sections; B) PAS staining of lung tissue sections (100×). Figure 2 Effects of FMSCs on the levels of pro-inflammatory cytokines in lung tissue of OVA-induced asthmatic mice; a) Relative expression level of IL-4; b) Relative expression level of IL-5; c) Relative expression level of IL-9; d) Relative expression level of IL-13; e) Relative expression level of GATA-3; Note: Data are expressed as mean ± standard deviation (n≥3); * indicates a significant difference between the normal control group and other groups (p<0.05); # indicates a significant difference between the model group and the treatment group (p<0.05).

[0019] Figure 3 Effects of FMSCs on the levels of anti-inflammatory cytokines in lung tissue of OVA-induced asthmatic mice; a) Relative expression level of IFN-γ; b) Relative expression level of IL-12; c) Relative expression level of IL-27; Note: Data are expressed as mean ± standard deviation (n≥3). * indicates a significant difference between the normal control group and other groups (p<0.05); # indicates a significant difference between the model group and the treatment group (p<0.05).

[0020] Figure 4 The effect of FMSCs on GATA-3 expression levels (200×); Figure 5 The pathological effects of PMSC on lung tissue in OVA-induced asthmatic mice; A) Hematoxylin and eosin (HE) staining of lung tissue sections; B) PAS staining of lung tissue sections (100×). Figure 6 Effects of PMSCs on the levels of pro-inflammatory cytokines in lung tissue of OVA-induced asthmatic mice; a) Relative expression level of IL-4; b) Relative expression level of IL-5; c) Relative expression level of IL-9; d) Relative expression level of IL-13; e) Relative expression level of GATA-3; Note: Data are expressed as mean ± standard deviation (n≥3); * indicates a significant difference between the normal control group and other groups (p<0.05); # indicates a significant difference between the model group and the treatment group (p<0.05).

[0021] Figure 7 Effects of PMSCs on the levels of anti-inflammatory cytokines in lung tissue of OVA-induced asthmatic mice; a) Relative expression level of IFN-γ; b) Relative expression level of IL-12; c) Relative expression level of IL-27; Note: Data are expressed as mean ± standard deviation (n≥3). * indicates a significant difference between the normal control group and other groups (p<0.05); # indicates a significant difference between the model group and the treatment group (p<0.05).

[0022] Figure 8 Effect of FMSCs on GATA-3 expression levels (200×) Detailed Implementation

[0023] The raw material for this application, *Niu Dali*, was purchased from Guangdong Xiaoyang Ecological Agriculture Co., Ltd., and the variety is Yunyang *Niu Dali*.

[0024] The present invention will be further described below with reference to embodiments.

[0025] Example 1: After crushing the *Achyranthes bidentata*, extract it by heating and reflux at 78-80℃ for 1 hour with a material-to-liquid ratio of 1:60 for the powdered herb and 70% ethanol. After filtering with double-layer gauze, centrifuge the extract at 3000 rpm for 10 minutes in a large refrigerated centrifuge. Take the supernatant and concentrate it under reduced pressure in a rotary evaporator until it becomes viscous. Then freeze the concentrate at -80℃ for 12-24 hours and freeze-dry it in a vacuum freeze dryer for 32-36 hours to obtain *Achyranthes bidentata* extract powder containing total flavonoids.

[0026] Example 2: After crushing the *Achyranthes bidentata*, extract it by heating and reflux at 78°C with a material-to-liquid ratio of 1:40 (powdered medicinal material to 70% ethanol). After filtering with double-layer gauze, the extract was centrifuged at 3000 rpm for 10 min in a large refrigerated centrifuge. The supernatant was concentrated under reduced pressure in a rotary evaporator until it reached a viscous state. The concentrate was then frozen at -80°C for 15 h and then freeze-dried in a vacuum freeze dryer for 32 h to obtain *Achyranthes bidentata* extract powder containing total flavonoids.

[0027] Example 3: After crushing the *Achyranthes bidentata*, extract it by heating and reflux at 80°C for 1 hour with a material-to-liquid ratio of 1:100 for the powdered herb and 70% ethanol. After filtering with double-layer gauze, the extract was centrifuged at 3000 rpm for 10 minutes in a large refrigerated centrifuge. The supernatant was then concentrated under reduced pressure in a rotary evaporator until it reached a viscous state. The concentrate was then frozen at -80°C for 24 hours and then freeze-dried in a vacuum freeze dryer for 33 hours to obtain *Achyranthes bidentata* extract powder containing total flavonoids.

[0028] Example 4: The *Achyranthes bidentata* powder and ultrapure water were extracted at a ratio of 1:80 at 95-100℃ for 1.5 hours. After filtration through double-layered gauze, the filtrate was concentrated under reduced pressure in a rotary evaporator until it reached a viscous state. Three times the volume of 80% ethanol was slowly added to the concentrate, and the mixture was placed in a 4℃ refrigerator and allowed to stand for 5 hours for alcohol precipitation. Most of the upper liquid was discarded, and the remaining mixture was centrifuged at 3000 rpm for 10 minutes. After discarding the supernatant, the flocculent precipitate was frozen at -80℃ for 12-24 hours, and then freeze-dried in a vacuum freeze dryer for 32-36 hours to obtain *Achyranthes bidentata* extract powder containing total polysaccharides.

[0029] Example 5: The *Achyranthes bidentata* powder and ultrapure water were extracted at 100°C for 2 hours. After filtration through double-layered gauze, the filtrate was concentrated under reduced pressure in a rotary evaporator until it reached a viscous state. Three times the volume of 80% ethanol was slowly added to the concentrate, and the mixture was placed in a 4°C freezer for 5 hours to allow for alcohol precipitation. Most of the upper liquid was discarded, and the remaining mixture was centrifuged at 3000 rpm for 10 minutes. After discarding the supernatant, the flocculent precipitate was frozen at -80°C for 20 hours, and then freeze-dried in a vacuum freeze dryer for 32 hours to obtain *Achyranthes bidentata* extract powder containing total polysaccharides.

[0030] Example 6: The mixture of crushed *Achyranthes bidentata* powder and ultrapure water was heated at 98°C for 3 hours. After filtration through double-layered gauze, the filtrate was concentrated under reduced pressure in a rotary evaporator until it reached a viscous state. Three times the volume of 80% ethanol was slowly added to the concentrate, and the mixture was placed in a 4°C freezer for 5 hours to allow for alcohol precipitation. Most of the supernatant was discarded, and the remaining mixture was centrifuged at 3000 rpm for 10 minutes. After discarding the supernatant, the flocculent precipitate was frozen at -80°C for 24 hours, and then freeze-dried in a vacuum freeze dryer for 36 hours to obtain *Achyranthes bidentata* extract powder containing total polysaccharides.

[0031] The extracts of *Achyranthes bidentata* containing total flavonoids and extracts of *Achyranthes bidentata* containing total polysaccharides prepared in Examples 1 and 4 respectively were subjected to the following experiments: Establishment of a BALB / c mouse asthma model: Sixty-four 3-4 week old female BALB / c mice were acclimatized to the following environment for one week: temperature 22-25℃, relative humidity 50%-60%, regular 12 / 12-hour light / dark cycles, and provided with sufficient standard feed and drinking water. After one week, the mice were randomly divided into 8 groups: normal control group, model group, 20 g / kg of *Achyranthes bidentata* extract containing total flavonoids (FMSC-H group), 10 g / kg of *Achyranthes bidentata* extract containing total flavonoids (FMSC-M group), 5 g / kg of *Achyranthes bidentata* extract containing total flavonoids (FMSC-L group), 0.4 g / kg of *Achyranthes bidentata* extract containing total polysaccharides (PMSC-H group), 0.2 g / kg of *Achyranthes bidentata* extract containing total polysaccharides (PMSC-M group), and 0.1 g / kg of *Achyranthes bidentata* extract containing total polysaccharides (PMSC-L group), with 8 mice in each group.

[0032] On days 0 and 7, mice in the model group and each drug administration group were intraperitoneally injected with 0.2 mL of sensitization solution containing 100 μg OVA powder and 2.25 mg aluminum hydroxide, while the normal control group was intraperitoneally injected with 0.2 mL of phosphate-buffered saline (PBS).

[0033] After two sensitizations, starting from day 15, the mice were placed in a 4L sealed container and challenged with 5% OVA solution for 30 minutes daily for 7 consecutive days.

[0034] The extract of *Achyranthes bidentata* containing total flavonoids was prepared into solutions of 20 g / kg, 10 g / kg, and 5 g / kg using PBS; the extract of *Achyranthes bidentata* containing total polysaccharides was prepared into solutions of 0.4 g / kg, 0.2 g / kg, and 0.1 g / kg using PBS. Starting from day 28, mice in the FMSC-H, FMSC-M, FMSC-L, PMSC-H, PMSC-M, and PMSC-L groups were given oral gavage at a volume of 0.2 mL per mouse; normal and model mice were given PBS solution instead, and the administration continued for 28 days.

[0035] 1. HE staining was used to observe inflammatory infiltration in mouse lung tissue and changes in bronchial epithelium. After washing the right lung tissue with PBS, it was fixed in 10% (v / v) neutral formaldehyde for 24 hours. Then, it was dehydrated with ethanol of different concentrations, embedded in paraffin to form a paraffin block, and cut into 4 μm thick sections using a microtome. The sections were dewaxed and hydrated using a series of xylene and graded ethanol treatments, stained with Hematoxylin and Eosin (HE), and the pathological changes in the lung tissue were observed under bright-field conditions at 40× and 100×.

[0036] 2. PAS staining was used to observe changes in mucus secretion and goblet cells in the bronchial epithelium of mice. After cutting paraffin blocks of lung tissue into 4μm thick sections using a microtome, the sections were dewaxed and hydrated, stained with Periodic-Acid-Schiff (PAS) staining agent, and observed under bright field conditions at 40× and 100× using an inverted fluorescence microscope.

[0037] 3. Real-time quantitative PCR detection of expression levels of inflammatory and anti-inflammatory factors in mouse lung tissue Left lung tissue was removed and washed with PBS, then placed in an RNase-free homogenizing tube. 1 mL of lysis buffer was added, and the lung tissue was homogenized using a tissue homogenizer. Total RNA was extracted from the homogenized lung tissue according to the kit instructions. The concentration and purity of total RNA were determined using a micro spectrophotometer. Subsequently, the RNA was reverse transcribed into cDNA using a reverse transcription kit. Real-time quantitative PCR analysis was performed using SYBR Greenchemistry probes fluorescent dye and a PCR instrument. Primer sequences were obtained from the NCBI database and are shown in Table 1 below. Changes in gene expression levels were analyzed using 2... −ΔΔCT Methodological calculation and analysis.

[0038] The primer sequences used in Table 1

[0039] 4. Immunohistochemical detection of GATA-3 expression in mouse lung tissue Lung tissue paraffin blocks were cut into 4 μm thick sections using a microtome. The sections were dewaxed with xylene, then dehydrated using a gradient of ethanol solutions, immersed in boiling sodium citrate antigen retrieval solution, and rinsed three times with PBS. They were then blocked with 5% goat serum at room temperature for 30 minutes. Next, the sections were incubated overnight at 4°C with anti-GATA-3 primary antibody, followed by incubation with secondary antibody. Finally, the tissue sections were stained with 3,3'-diaminobenzidine tetrahydrochloride (DAB), and the expression level of GATA-3 protein in mouse lung tissue was observed using an inverted fluorescence microscope.

[0040] 5. Data Analysis The obtained data are expressed as mean ± standard deviation. Statistical analysis of all relevant data was performed using IBM SPSS Statistics 27 software. One-way ANOVA was used to analyze the data between groups. If the variances were homogeneous, the minimum significance test was used; otherwise, Tamhane's T² test was used. Statistical charts were created using GraphPad Prism 10 software. p <0.05 indicates a statistically significant difference. p <0.05,** p <0.01, *** p <0.001 indicates a comparison with the control group; # p <0.05, ## p <0.01, ### p <0.001 indicates a comparison with the model group.

[0041] 6. Results 6.1 Effects of *Smilax glabra* extract containing total flavonoids on the pathological characteristics of lung tissue in asthmatic mice To evaluate the pathological changes in mouse lung tissue induced by OVA and the anti-asthmatic efficacy of *Achyranthes bidentata* extract (FMSC) containing total flavonoids, mouse lung tissue sections were stained with hematoxylin and eosin (HE). Figure 1 A). The results showed that OVA-induced lung tissue in mice exhibited significant peribronchial inflammatory cell infiltration. Other manifestations included tracheal wall thickening and hypertrophy, partial airway epithelial cell shedding, alveolar septal destruction, and accompanying alveolar hemorrhage. No abnormal pathological features were observed in the control group. After treatment with FMSCs, these pathological features significantly improved: the degree of peribronchial inflammatory cell infiltration decreased, the tracheal wall and epithelial structure tended to return to normal, and alveolar hemorrhage was significantly alleviated.

[0042] Excessive mucus secretion in the bronchi and airways, goblet cell hyperplasia, and bronchial wall thickening are hallmark pathological features of asthma. To assess the degree of bronchoalveolar mucus secretion, PAS staining was used to stain lung tissue sections. Figure 1 B). Compared with the control group, OVA-induced stimulation significantly increased airway mucus secretion and goblet cell proliferation in mice. In the FMSC treatment group, a dose-dependent decrease in airway mucus secretion was observed in mice.

[0043] 6.2 Effects of *Smilax glabra* extract containing total flavonoids on the levels of pro-inflammatory cytokines in lung tissue of asthmatic mice Studies have shown that various inflammatory cytokines play a crucial role in the occurrence and development of asthma. Real-time quantitative PCR was used to detect the expression levels of pro-inflammatory cytokines in the lung tissues of mice in each group. Figure 2 As shown, compared with the control group, the expression levels of cytokines such as IL-4, IL-5, IL-9, and IL-13, as well as the Th2 polarization transcription factor GATA-3, were significantly increased in the lung tissue of the model group mice. After FMSC treatment, the levels of cytokines IL-4, IL-5, IL-9, IL-13, and GATA-3 in mouse lung tissue were significantly reduced. This indicates that FMSC can reduce the levels of pro-inflammatory factors in mouse lung tissue after ovalbumin-induced stimulation.

[0044] 6.3 Effects of *Smilax glabra* extract containing total flavonoids on the levels of anti-inflammatory cytokines in lung tissue of asthmatic mice The results are as follows Figure 3 As shown, the levels of Th2-related anti-inflammatory cytokines (IFN-γ, IL-12, and IL-27) were low in the lung tissue of the model group mice. However, in the FMSC treatment group, the levels of anti-inflammatory cytokines in the mouse lung tissue showed a significant increase in a dose-dependent manner. It is speculated that FMSCs may exert their therapeutic effect by promoting the secretion of Th1 cytokines and regulating the Th1 / Th2 cytokine balance.

[0045] 6.4 Effects of *Smilax glabra* extract containing total flavonoids on GATA-3 expression levels in lung tissue of asthmatic mice GATA-3 is a key transcription factor for Th2 cell differentiation and can promote CD4+ differentiation. + T cells differentiate into Th2 cells. Meanwhile, under the regulation of GATA-3, activated Th2 cells increase the secretion of pro-inflammatory cytokines such as IL-4, IL-5, and IL-13, thereby inducing and aggravating asthma. Figure 4 The results showed that GATA-3 expression was significantly increased in the lung tissue of mice in the model group. Conversely, GATA-3 expression in the lung tissue of mice treated with FMSC drugs showed a significant dose-dependent decrease.

[0046] 6.5 Effects of *Smilax glabra* extract containing total polysaccharides on the pathological characteristics of lung tissue in asthmatic mice To evaluate the therapeutic effect of *P. mongolica* extract containing total polysaccharides (PMSC) on asthma, lung tissue sections from mice in the PMSC treatment group were stained with hematoxylin and eosin (HE). Figure 5 A). Comparative analysis of the results showed that, compared with the model group, the asthma-related pathological features in the lung tissue of mice treated with PMSC were significantly improved.

[0047] In addition, the degree of airway mucus secretion in mouse lung tissue was analyzed by PAS staining. Figure 5 B). The results showed that the degree of mucus secretion was significantly reduced in the PMSC-H group and also reduced to some extent in the PMSC-M group, while there was no significant difference between the PMSC-L group and the model group.

[0048] 6.6 Effects of *Smilax glabra* extract containing total polysaccharides on the levels of pro-inflammatory cytokines in lung tissue of asthmatic mice The results are as follows Figure 6 As shown, the levels of Th2 pro-inflammatory cytokines in the lung tissue of asthmatic mice decreased with increasing PMSC dosage. Among them, the levels of IL-4, IL-5, IL-13, and GATA-3 were significantly reduced compared with the model group.

[0049] 6.7 Effects of *Smilax glabra* extract containing total polysaccharides on the levels of anti-inflammatory cytokines in lung tissue of asthmatic mice Analysis results Figure 7 It can be seen that, compared with the normal control group and the model group, the level of Th1 anti-inflammatory cytokines in the lung tissue of mice in the PMSC treatment group was significantly increased.

[0050] 6.8 Effect of *Smilax glabra* extract containing total polysaccharides on GATA-3 expression levels in lung tissue of asthmatic mice Immunohistochemical results showed that, compared with the model group, GATA-3 expression was reduced to varying degrees in both the PMSC-H and PMSC-M groups. The decrease was particularly significant in the PMSC-H group, with positive expression levels approaching those of the control group.

Claims

1. A traditional Chinese medicine extract for treating asthma and inflammation, characterized in that, It is prepared using *Smilax glabra* as raw material, specifically as total flavonoid extract or total polysaccharide extract of *Smilax glabra*.

2. The method for extracting the anti-asthmatic and anti-inflammatory traditional Chinese medicine extract as described in claim 1, characterized in that, Total flavonoids were extracted from *Smilax glabra* using alcohol extraction, and total polysaccharides were extracted from *Smilax glabra* using water extraction.

3. The method for extracting the anti-asthmatic and anti-inflammatory traditional Chinese medicine extract as described in claim 2, characterized in that, The specific steps for extracting total flavonoids from *Smilax glabra* using alcohol extraction are as follows: (1) After crushing the ox-duck-duck, mix the crushed material with 70% ethanol at a mass-volume ratio of 1:30~100 (w / v) and heat under reflux for 0.5-2 hours; (2) Take the product obtained in step (1), filter it, take the filtrate, place it in a refrigerated centrifuge and centrifuge at 3000 rpm for 10 min to separate it by centrifugation; (3) Take the supernatant obtained in step (2) and concentrate it under reduced pressure at 50~65℃ for 2~4 h to obtain the concentrate; (4) After the concentrate from step (3) is frozen at -80℃ for 12-24h, it is then placed in a vacuum freeze dryer at -50~-65℃ for 32-36h to obtain the total flavonoid extract of *Smilax glabra*.

4. The extraction method for the anti-asthmatic and anti-inflammatory traditional Chinese medicine extract as described in claim 3, characterized in that, In step (1), the mass-to-volume ratio of the pulverized material to 70% ethanol is 1:60; the mixture is heated under reflux for 1 hour.

5. The method for extracting the anti-asthmatic and anti-inflammatory traditional Chinese medicine extract as described in claim 2, characterized in that, The extraction steps for extracting total polysaccharides from *Achyranthes bidentata* using water extraction are as follows: (1) After crushing the powder, mix it with ultrapure water at a mass-volume ratio of 1:60~100 (w / v) and heat extract at 95-100℃ for 1-2 hours; (2) After filtering the product obtained in step (1), the filtrate is concentrated under reduced pressure at 50~70℃ for 4~6 h until it becomes viscous; (3) Add 80% ethanol to the concentrate obtained in step (2), place it in a refrigerator at 4°C and let it stand for 5 hours to precipitate alcohol. (4) After removing the upper liquid from the product obtained in step (3), place the remaining mixture in a centrifuge and centrifuge at 3000 rpm for 10 min. (5) After removing the supernatant from the product obtained in step (4), the flocculent precipitate is frozen at -80℃ for 12-24h, and then freeze-dried at -50~-65℃ in a freeze dryer for 32-36h to obtain the total polysaccharide extract of *Smilax glabra*.

6. The extraction method for the anti-asthmatic and anti-inflammatory traditional Chinese medicine extract as described in claim 5, characterized in that, In step (1), the mass-to-volume ratio of *Smilax glabra* powder to ultrapure water is 1:80; the hot extraction time in step (1) is 1.5 h.

7. The extraction method for the anti-asthmatic and anti-inflammatory traditional Chinese medicine extract as described in claim 5, characterized in that, In step (3), add 80% ethanol, which is 3 times the volume of the concentrate.

8. The application of an anti-asthmatic and anti-inflammatory traditional Chinese medicine extract in the preparation of anti-asthmatic and anti-inflammatory drugs.

9. The application of the anti-asthmatic and anti-inflammatory traditional Chinese medicine extract as described in claim 8 in the preparation of anti-asthmatic and anti-inflammatory drugs, characterized in that, The specific active ingredients are total flavonoid extract of *Smilax glabra* and / or total polysaccharide extract of *Smilax glabra*.

10. The application of the anti-asthmatic and anti-inflammatory traditional Chinese medicine extract as described in claim 8 in the preparation of anti-asthmatic and anti-inflammatory drugs, characterized in that, Its dosage form is any oral preparation.

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