Use of perilla leaf extract in preparation of drugs for treating asthma-chronic obstructive pulmonary disease overlap (ACO)

Abstract

The application belongs to the technical field of medicine, and discloses application of a perilla leaf extract in preparation of a medicine for treating and / or preventing asthma-chronic obstructive pulmonary disease (ACO). The perilla leaf extract has a significant curative effect in treating and / or preventing ACO disease, can significantly improve aggregation of inflammatory cells in the lung of an ACO mouse and generation levels of inflammatory factors, and improve lung function and pathological changes of lung tissue inflammation; in addition, the perilla leaf extract can significantly inhibit generation of neutrophil inflammatory factors TNF-alpha, IL-6 and IL-1beta, and inhibit generation of an eosinophil inflammatory factor TNF-alpha.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical or food technology, and in particular relates to a type of perilla leaf extract, its preparation method and its application in the preparation of drugs for the prevention and / or treatment of asthma-chronic obstructive pulmonary disease overlap (ACO). Background Technology

[0002] Asthma-COPD overlap (ACO) is a complex respiratory disease characterized by various combinations of pathological features from both asthma and chronic obstructive pulmonary disease (COPD). Epidemiological studies of ACO show that the prevalence of ACO in the general population ranges from 0.9% to 11.0%, among asthma patients from 11.1% to 61.0%, and among COPD patients from 4.6% to 66.0%. Unlike asthma or COPD alone, the causes of ACO are more complex, resulting from the combined influence of numerous risk factors, including genetics and environment. ACO patients experience a heavier symptom burden, a poorer quality of life, more frequent and severe respiratory exacerbations requiring hospitalization, and require more medical services, leading to a significant economic and social burden.

[0003] ACO (Acute Colitis Occurs) is characterized by the simultaneous accumulation and infiltration of large numbers of neutrophils and eosinophils in the respiratory tract, accompanied by increased pro-inflammatory cytokines, leading to aggravated lung tissue damage. Inflammatory cells release large amounts of tumor necrosis factor-α (TNF-α), which plays a crucial role in the inflammatory flare-up of ACO. TNF-α can induce the release of IL-6, IL-8, oxygen free radicals, and matrix metalloproteinases, ultimately leading to an inflammatory cytokine cascade, oxidative stress, airway hyperresponsiveness, airway remodeling, and alveolar wall destruction. Furthermore, ACO is associated with elevated levels of inflammatory vesicles such as NLRP3 and sputum IL-1β. Activated neutrophils secrete elastase and histone G, effectively promoting the release of IL-1β and IL-33, key factors in triggering Th2-type inflammation such as eosinophilic inflammation. Therefore, reducing TNF-α release is crucial for alleviating inflammation and reducing tissue damage. Drugs used to treat chronic obstructive pulmonary disease (COPD) and asthma, such as infliximab and golimumab, have been identified as biological inhibitors of TNF-α. However, these drugs are associated with increased infections and potential drug resistance, limiting their use and long-term management in ACO. The combined use of long-acting beta-agonists (LABAs) and long-acting muscarinic antagonists (LAMAs) with inhaled corticosteroids (ICS) in ACO patients still lacks evidence-based medical support. Moreover, such multidrug combination therapy increases the risk of adverse reactions such as infections and exacerbates the burden on the liver and kidneys. Therefore, the development of innovative drugs and treatments for ACO is essential.

[0004] The multi-component, multi-target, and holistic approach of traditional Chinese medicine gives it unique efficacy in treating complex diseases. Perilla (Perilla frutescens (L.)) is a plant of the Lamiaceae family, with a fragrant aroma and slightly pungent taste. The Pharmacopoeia of the People's Republic of China (2020 edition) records its functions of relieving exterior syndromes, dispersing cold, regulating qi, and harmonizing the stomach; it can be used for colds due to wind-cold, cough, and nausea. Modern pharmacological studies have found that it possesses anti-inflammatory, antioxidant, antibacterial, and anti-allergic activities, and has significant efficacy in improving lung inflammation. Currently, there are no reports on perilla leaf extract and its treatment of acute lung cancer (ACO). This invention prepared perilla leaf extract and studied its anti-ACO inflammatory activity. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the technical problem solved by the present invention is to provide the application of perilla leaf extract in the preparation of drugs for the prevention and / or treatment of ACO.

[0006] To solve the technical problem of this invention, the present invention provides the following technical solution:

[0007] This invention provides an application of perilla leaf extract in the preparation of drugs for the prevention and / or treatment of acute colic (ACO), characterized in that the preparation method of the perilla leaf extract comprises the following steps:

[0008] 1) Using dried perilla leaves as raw material, weigh 1.00 kg, add 70% ethanol aqueous solution, heat and reflux for extraction, recover the ethanol from the filtrate under reduced pressure to obtain an aqueous solution, concentrate the aqueous solution under reduced pressure to a density of 1.090–1.100 (60℃), and the mass is 319.81.

[0009] g of extract is ready for use;

[0010] 2) Extract the extract obtained in step 1) twice with ethyl acetate, 500 ml each time, and concentrate the remaining aqueous solution to obtain the extract (density 1.090-1.100);

[0011] 3) The extract obtained in step 2) was precipitated with 90% ethanol aqueous solution, filtered, and the precipitate was dried and pulverized to obtain 189.07g of perilla leaf extract, with a yield of 18.9%.

[0012] In step 1) of the above preparation method,

[0013] The extraction solvent is an aqueous solution of C1-C5 lower alcohols, preferably an aqueous solution of C1-C3 lower alcohols, and more preferably an aqueous solution of ethanol;

[0014] The concentration of the lower alcohol in the aqueous solution of C1 to C5 lower alcohols is 0-95% (volume / volume), preferably 50-80% (volume / volume), and more preferably 65%-75% (volume / volume).

[0015] The extraction method is maceration, decoction or reflux, preferably decoction or reflux, with the reflux temperature set at 80-85°C for 2 hours;

[0016] The filtrate obtained after heating and reflux extraction needs to be sieved to a mesh size of 400.

[0017] Concentration can be performed at atmospheric pressure or under reduced pressure, with reduced pressure concentration being preferred.

[0018] In step 2) of the above preparation method,

[0019] The low-polarity organic solvent used for extraction is cyclohexane, petroleum ether, dichloromethane, chloroform, ethyl acetate, n-butanol, isoamyl alcohol, or a mixture thereof, preferably ethyl acetate or n-butanol, and more preferably n-butanol.

[0020] The extraction is performed 1-5 times, preferably 2-3 times.

[0021] Concentration can be performed at atmospheric pressure or under reduced pressure, with reduced pressure concentration being preferred.

[0022] In step 3) of the above preparation method,

[0023] The aqueous solution of C1-C5 lower alcohols used for alcohol precipitation is preferably an aqueous solution of C2-C3 lower alcohols, and more preferably an aqueous solution of ethanol;

[0024] The concentration of C1 to C5 lower alcohols is 50-90% (volume / volume), preferably 60-85% (volume / volume), and more preferably 75%-85% (volume / volume);

[0025] The alcohol precipitation time is 8-72 hours, preferably 12-24 hours;

[0026] The drying methods include atmospheric pressure drying, vacuum drying, freeze drying, and spray drying, with vacuum drying and freeze drying being preferred.

[0027] Asthma-COPD overlap (ACO) differs from the single disease entity of asthma or COPD, referring to various combinations of pathological features of both asthma and COPD. The main inflammatory feature of ACO is the simultaneous and massive infiltration of neutrophils and eosinophils. This invention induces an ACO animal model by intraperitoneal injection of a suspension of ovalbumin OVA and aluminum hydroxide gel, and by intratracheal instillation of ovalbumin OVA and lipopolysaccharide LPS. Oral administration of Perilla leaf extract at doses of 25 mg / kg, 50 mg / kg, and 100 mg / kg improved lung function and airway hyperresponsiveness in ACO mice, significantly inhibited the production of TNF-α, IL-1β, and IL-6 in the bronchoalveolar lavage fluid (BALF) supernatant, and suppressed the recruitment of neutrophils and eosinophils in the lungs.

[0028] A second aspect of the present invention is to provide the use of a pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of asthma-chronic obstructive pulmonary disease overlap (ACO), wherein the pharmaceutical composition comprises the perilla leaf extract described in the first aspect and a pharmaceutically acceptable carrier or excipient. The method for preparing the perilla leaf extract is as described in the first aspect of the present invention.

[0029] When used for this purpose, if desired, the active ingredient may be combined with one or more solid or liquid pharmaceutical excipients and / or adjuvants to form an appropriate administration or dosage form suitable for human use.

[0030] The pharmaceutical composition of the present invention can be administered in unit dose form, and the route of administration can be enteric or non-enteric, such as oral, intramuscular, subcutaneous, nasal, oral mucosa, skin, peritoneum or rectum, etc.

[0031] The drug composition of the present invention can be administered via injection. Injection includes intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, and acupoint injection. The dosage form can be a liquid or a solid dosage form. For example, liquid dosage forms can be true solutions, colloids, microparticles, emulsions, or suspensions. Other dosage forms include tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, suppositories, and lyophilized powder injections.

[0032] The compositions of the present invention can be formulated into ordinary formulations, sustained-release formulations, controlled-release formulations, targeted formulations, and various microparticle delivery systems.

[0033] To formulate unit-dose dosage forms into tablets, a wide variety of carriers known in the art can be used. Examples of carriers include, for instance, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; humectants and binders such as water, glycerol, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, etc.; and disintegrants. Examples of active ingredients include dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitol fatty acid esters, sodium dodecyl sulfate, methylcellulose, and ethylcellulose; disintegration inhibitors, such as sucrose, tristearate, cocoa butter, and hydrogenated oils; absorption enhancers, such as quaternary ammonium salts and sodium dodecyl sulfate; and lubricants, such as talc, silica, corn starch, stearates, boric acid, liquid paraffin, and polyethylene glycol. Tablets can also be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets.

[0034] To formulate the drug delivery unit into a pellet, a wide variety of carriers known in the art can be used. Examples of carriers include, for instance, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, gelucire, kaolin, talc, etc.; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste, etc.; and disintegrants such as agar powder, dried starch, alginate, sodium dodecyl sulfate, methylcellulose, ethylcellulose, etc.

[0035] To formulate the drug delivery unit into a suppository, a wide variety of carriers known in the art can be used. Examples of carriers include, for instance, polyethylene glycol, lecithin, cocoa butter, higher alcohols, enzymes of higher alcohols, gelatin, semi-synthetic glycerolases, etc.

[0036] To encapsulate the drug delivery unit, the active ingredient is mixed with the various carriers described above, and the resulting mixture is placed in a hard gelatin capsule or a soft capsule. Alternatively, the active ingredient can be formulated as a microcapsule, suspended in an aqueous medium to form a suspension, or filled into a hard capsule or formulated as an injectable preparation.

[0037] For example, the compositions of the present invention can be formulated into injectable formulations, such as solutions, suspension solutions, emulsions, and lyophilized powders for injection. These formulations can be aqueous or non-aqueous and may contain one or more pharmacodynamically acceptable carriers, diluents, binders, lubricants, preservatives, surfactants, or dispersants. Diluents may be selected from water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, polyoxyethylene sorbitan fatty acidase, etc. Furthermore, to prepare isotonic injections, appropriate amounts of sodium chloride, glucose, or glycerol can be added to the injectable formulations. In addition, conventional solubilizers, buffers, pH adjusters, etc., may also be added. These excipients are commonly used in the art.

[0038] In addition, colorants, preservatives, flavorings, tasters, sweeteners or other materials may be added to pharmaceutical preparations if necessary.

[0039] The dosage of the pharmaceutical composition of this invention depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight, personality, and individual response of the patient or animal, the route of administration, and the frequency of administration. Therefore, the therapeutic dosage of this invention can vary widely. Generally speaking, the dosage of the pharmaceutical components used in this invention is well known to those skilled in the art. The actual effective amount of the drug contained in the final formulation of the pharmaceutical composition of this invention can be appropriately adjusted to achieve the required therapeutic dose and fulfill the purpose of treating acute coronary syndrome (ACO) of this invention. Typically, for patients weighing approximately 75 kg, the daily dose of the extract is 0.001 mg / kg body weight to 500 mg / kg body weight, preferably 3 mg / kg body weight to 100 mg / kg body weight. The above dosage can be administered as a single dose or divided into several doses, such as two, three, or four doses, depending on the clinical experience of the attending physician and the administration regimen.

[0040] Compared with the prior art, the present invention has the following advantages:

[0041] (1) The present invention found that the perilla leaf extract described in the present invention has a significant inhibitory effect on the release of IL-1β from neutrophils, while the positive control drug dexamethasone did not show a significant improvement effect.

[0042] (2) Neutrophils and eosinophils are the main characteristic inflammatory cells recruited and infiltrating in acute lung cancer (ACO), and are closely related to excessive inflammatory response and tissue damage. This invention found that bronchoalveolar lavage fluid (BALF) from mice treated with the Perilla frutescens leaf extract of this invention showed a significant decrease in the number of neutrophils and eosinophils compared to the ACO model group, and this decrease was dose-dependent. Therefore, the anti-ACO effect of Perilla frutescens leaf extract may be related to its simultaneous inhibition of neutrophil and eosinophil inflammation. Thus, the Perilla frutescens leaf extract of this invention, as an active component against ACO, has a good anti-neutrophil and eosinophil inflammation effect, and its efficacy in improving lung inflammation, lung function, and lung tissue pathological indicators is significant, meeting the requirements for pharmaceutical formulation development.

[0043] (3) As a versatile herb, perilla leaves are not demanding in terms of soil requirements, are easy to cultivate, and have good application prospects. The perilla leaf extract preparation process described in this invention is simple and easy to standardize. In addition to the medicinal and edible values ​​discovered in existing research, the discovery of this invention not only expands the new efficacy of perilla plant in terms of medicinal value, but also enriches the source of natural active components for combating ACO diseases. Attached Figure Description

[0044] Figure 1 This invention investigates the effect of perilla leaf extract on the lung function indicators FEF25 / 50 / 75 in ACO mice. # p < 0.05, ## p < 0.01, ### p < 0.001; compared with the model control group, * p < 0.05, ** p < 0.01, *** p < 0.001.

[0045] Figure 2 This invention relates to the effect of perilla leaf extract on specific airway resistance (sRAW) in ACO mice. # p < 0.05, ## p < 0.01, ### p < 0.001; compared with the model control group, * p < 0.05, ** p < 0.01, *** p < 0.001.

[0046] Figure 3 This invention relates to the effect of perilla leaf extract on pathological damage in lung tissue of ACO mice. # p < 0.05, ## p < 0.01, ### p < 0.001; compared with the model control group, * p < 0.05,** p < 0.01, *** p < 0.001. Detailed Implementation

[0047] The following embodiments further illustrate the present invention, but the present invention is not limited to these embodiments.

[0048] Example 1: Preparation of Perilla Leaf Extract

[0049] 1) Using dried perilla leaves as raw material, weigh 1.00 kg, add 12.00 L of 70% ethanol solution (12 times the amount), soak at room temperature for 2 hours, heat and reflux at 80-85℃ for 2 hours, cool naturally to about 60℃, filter through a 400-mesh sieve, and obtain 10 L of filtrate for later use.

[0050] 2) The filter residue was treated again using the same method as in 1), yielding 11 L of filtrate;

[0051] 3) Combine the filtrates, heat under reduced pressure (-0.075 MPa) at 50℃ and reflux to recover ethanol, yielding an aqueous solution;

[0052] 4) The aqueous solution was concentrated under reduced pressure (-0.097 MPa) at 60℃ to obtain an extract with a density of 1.090~1.100 (60℃) and a mass of 319.81g. The extract was then extracted twice with 500mL of ethyl acetate.

[0053] 5) The remaining water was concentrated to obtain an extract (density 1.090-1.100), anhydrous ethanol was added to dissolve it to a final ethanol concentration of 90%, and precipitation was carried out at room temperature for 24 hours. The precipitate was filtered under reduced pressure, and the filter residue was freeze-dried and pulverized to obtain 189.07g of perilla leaf extract, with a yield of 18.9%.

[0054] Example 2: Dosage Form Preparation

[0055] Capsule preparation: 10g of perilla leaf extract is added to 14.75g of dextrin and 0.25g of magnesium stearate. After thorough mixing, 100 capsules are prepared according to the capsule preparation method and filled into 250mg capsules. Each capsule contains 100mg of perilla leaf extract, and the total amount is not less than 50%.

[0056] Preparation of tablets: Take 10g of perilla leaf extract, add 10g of starch, 8g of dextrin, and 1.5g of sucrose, stir and mix thoroughly, make into granules, dry at below 60℃, add appropriate amount of magnesium stearate and talc, mix well, compress into 100 tablets, and coat with sugar or film to obtain the tablets.

[0057] Preparation of granules: Take 10g of perilla leaf extract, add appropriate amount of sucrose and dextrin, make granules according to the granule preparation method, dry, and make 100g.

[0058] Preparation of oral liquid: Take perilla leaves, extract twice with 70% ethanol under reflux for 2 hours, filter, combine the filtrates, recover ethanol under reduced pressure, and concentrate into an extract; extract twice with ethyl acetate; dissolve the water portion in an appropriate amount of anhydrous ethanol, precipitate at room temperature for 24 hours, filter under reduced pressure, dissolve the residue in an appropriate amount of water, adjust the pH to 7.0 with 40% sodium hydroxide, stir well, refrigerate at 4-8℃ for 48 hours, filter, add 300g of sucrose to the filtrate, stir well to dissolve, add an appropriate amount of flavoring and adjust the pH to 7.0, add water to make 1000mL, stir well, let stand for 12 hours, filter, fill, sterilize, and the product is obtained.

[0059] Pharmacological experiments

[0060] Experimental Example 1: Pharmacodynamic effects of Perilla Leaf Extract on ACO in Mice

[0061] 1. Experimental materials

[0062] method:

[0063] ACO Model Preparation and Grouping: Male SPF-grade BALB / c mice (18-20g) were randomly divided into a normal control group, a model control group, a positive control group (dexamethasone, 0.5mg / kg, administered by gavage), and a drug treatment group (Perilla leaf extract 25, 50, and 100mg / kg, administered by gavage), with 12 mice in each group. Except for the blank control group, mice in each group were sensitized on days 1, 7, and 14 by intraperitoneal injection of 200μL of a mixture (100μL aluminum hydroxide gel + 100μL 0.3mg / mL OVA solution); on day 15, 50μL of 0.6mg / mL LPS was administered via intravenous drip. Mice were exposed to cigarette smoke (200ppm) from day 16 to day 40. On day 26, mice were intratracheally infused with 50 μL of 1.2 mg / mL OVA; on days 28 and 40, mice were intratracheally infused with 50 μL of a mixture (25 μL of 1.2 mg / mL LPS solution + 25 μL of 1.2 mg / mL OVA solution); on day 42, the specific airway resistance (sRaw) values ​​of mice were measured using a small animal pulmonary function analyzer (NAM) (Buxco / DSI) at concentration gradients of 0, 6.25, 12.5, and 25 mg / mL; on day 43, mice were anesthetized with 0.6% pentobarbital, intubated after exposing the trachea through a cervical incision, and lung function was measured using a small animal respiratory pulmonary function measurement instrument (PFT) (Buxco / DSI). Blood, bronchoalveolar lavage fluid, and lung tissue samples were subsequently collected from all animals for further analysis.

[0064] Mouse administration: Starting on day 29, mice were administered perilla leaf extract at doses of 25, 50, and 100 mg / kg by gavage, while control and model mice were given the same volume of physiological saline, once daily for 14 consecutive days.

[0065] Indicator Measurement:

[0066] Mice in each group underwent non-invasive respiratory airway resistance testing using a FinePointe NAM instrument to detect airway hyperresponsiveness. Mice in each group were anesthetized with sodium phenobarbital and then intubated to detect lung function (FinePointe PFT). Lung tissue from mice was collected for histopathological examination and evaluation. Bronchoalveolar lavage fluid (BALF) was collected from mice in each group after perfusion with 0.6 mL of pre-cooled physiological saline three times and centrifuged at 4°C. The supernatant was used to determine the levels of relevant inflammatory factors (ELISA method). Cells were resuspended in PBS buffer and white blood cell differential count was performed using a fully automated modular animal blood analyzer (XN-1000V[B1], Sysmex).

[0067] Experimental results:

[0068] (1) Effect of Perilla Leaf Extract on the Number of Inflammatory Cells in BALF of ACO Mice

[0069] Airway inflammation is one of the main pathological features of ACO patients. Inflammation manifests as the simultaneous accumulation of neutrophils and eosinophils in lung tissue. ACO patients show a significant increase in the production of inflammatory mediators in the airways, forming a complex cellular and molecular network that promotes airway inflammation and remodeling. This experiment aimed to investigate the effects of perilla leaf extract on the total number and differential count of inflammatory cells in the bronchoalveolar lavage fluid (BALF) of ACO mice.

[0070] Results: The results are shown in Table 1. Compared with the blank control group, ACO mice had higher total white blood cell count (WBC), neutrophil count (NEU), and eosinophil count (EOS) in their BALF. This invention found that gavage administration of Perilla leaf extract at doses of 25, 50, and 100 mg / kg can significantly reduce the number of neutrophils and eosinophils in the BALF of ACO mice. In addition, it also has a significant inhibitory effect on the total white blood cell count, which is dose-dependent.

[0071] Table 1. Effects of Perilla Leaf Extract on Total Inflammatory Cell Count and Differential Count in BALF of ACO Mice (Mean±Std)

[0072]

[0073] Note: Compared with the blank control group, # p < 0.05, ## p < 0.01, ### p < 0.001; compared with the model control group, * p < 0.05, ** p < 0.01, *** p < 0.001

[0074] (2) Inhibitory effect of perilla leaf extract on the production of inflammatory factors in bronchoalveolar lavage fluid (BALF) of ACO mice

[0075] One characteristic of acute lung cancer (ACO) is chronic airway inflammation, in which various inflammatory cytokines participate in the pathophysiological mechanisms of airway inflammation, damaging lung tissue and bronchi. TNF-α, a potent pro-inflammatory cytokine, can activate various inflammatory cells, leading to their accumulation in lung tissue. It also affects airway wall integrity by promoting apoptosis and necrosis of airway epithelial cells, resulting in airway remodeling and narrowing. IL-6 not only participates in local inflammatory responses in the lungs but also promotes the proliferation of airway epithelial cells and smooth muscle cells, and enhances the activity of airway mucus-secreting cells, leading to increased airway secretions and impairing airway patency. IL-1β, an important pro-inflammatory cytokine, can activate various inflammatory cells and endothelial cells, further amplifying the inflammatory response. This experiment aims to investigate the effects of perilla leaf extract on the production of inflammatory factors in the bronchoalveolar fluid (BALF) of ACO mice.

[0076] Results: As shown in Table 2, compared with the model control group, the perilla leaf extract administered by gavage at doses of 25, 50, and 100 mg / kg significantly inhibited the production of inflammatory factors IL-6, IL-1β, and TNF-α in the BALF of ACO mice.

[0077] Table 2. Effects of Perilla Leaf Extract on the Release of Inflammatory Factors in BALF of ACO Mice (Mean±Std)

[0078]

[0079] Note: Compared with the blank control group, # p < 0.05, ## p < 0.01, ### p < 0.001; compared with the model control group, * p < 0.05, ** p < 0.01, *** p < 0.001

[0080] (3) Effects of Perilla Leaf Extract on Lung Function in ACO Mice

[0081] For ACO, its FEV 50The / FVC ratio may not be as normal or only slightly decreased as in asthma, nor as low as in pure COPD; this is a hallmark distinguishing ACO from any of these conditions. MMEF is an indicator of small airway function; in ACO patients, small airway function is impaired, resulting in a lower MMEF value. Peak expiratory flow (PEF) often reflects airway obstruction in ACO patients. Residual volume (RV) in ACO patients is often significantly increased compared to normal individuals. Forced expiratory flow (FEF), which reflects small airway function, is significantly reduced, especially in the mid-to-late stages. This experiment aimed to investigate the effects of perilla leaf extract on various lung function indicators in ACO mice.

[0082] Results: The results are shown in Table 3 and... Figure 1 The results showed that, compared with the model control group, perilla leaf extract administered by gavage at doses of 25, 50, and 100 mg / kg had an effect on the pulmonary function test FEV1. 50 / FVC, Mid-expiratory Flow Rate (MMEF), Peak Expiratory Flow Rate (PEF), Residual Volume (RV), Forced Expiratory Flow Rate (FEF) 25 / 50 / 75 All indicators showed significant improvement, and this improvement was dose-dependent.

[0083] Table 3. Effects of Perilla Leaf Extract on MMEF, PEF, and FEV in ACO Mice 50 The effect of / FVC (Mean±Std)

[0084]

[0085]

[0086] Note: Compared with the blank group, # p < 0.05, ## p < 0.01, ### p < 0.001; compared with the model group, * p < 0.05, ** p < 0.01, *** p < 0.001

[0087] (4) The effect of perilla leaf extract on improving airway hyperresponsiveness in ACO mice

[0088] Airway hyperresponsiveness (AHR) refers to an abnormal, excessive contraction response of the airways after inhalation of small amounts of irritants or allergens, leading to airway narrowing and a significant increase in airway resistance. Specific airway resistance (sRAW) values ​​monitored by NAM (Natural Aspirin) are an important indicator of airway hyperresponsiveness. Methacholine (Mch) is an airway constrictor and a primary tool for clinical airway hyperresponsiveness testing. In asthmatic mice, nebulized methacholine resulted in hyperresponsive airway resistance, which increased with increasing methacholine dosage. This experiment aimed to investigate the effect of Perilla frutescens leaf extract on airway hyperresponsiveness in ACO (Acute Airway Hyperresponsiveness) mice.

[0089] Results: The results are as follows Figure 2 As shown, compared with the normal control group, the airway resistance sRAW of ACO model mice was significantly increased. Gavage administration of Perilla leaf extract at doses of 25, 50 and 100 mg / kg significantly inhibited the sRAW value of ACO mice and improved their airway hyperresponsiveness.

[0090] (5) Effects of Perilla Leaf Extract on Inflammatory Pathological Changes in Lung Tissue of ACO Mice

[0091] Microscopic observation and analysis of lung tissue, along with pathological examination, can assess the severity and progression of lung diseases. The purpose of this experiment was to investigate the effects of perilla leaf extract on inflammatory lesions in the lung tissue of ACO mice.

[0092] Results: The results are as follows Figure 3 As shown, pathological observation of the lungs of ACO mice revealed swollen and thickened alveolar walls, dilated alveolar cavities, and extensive inflammatory cell infiltration around the small and incisors. Administration of perilla leaf extract significantly improved the inflammatory pathological changes in the lungs, with a significantly lower lung tissue inflammatory pathological score compared to the model group.

[0093] Experimental Example 2: Pharmacodynamic effects of Perilla Leaf Extract on neutrophil inflammation

[0094] Neutrophil TNF-α is one of the most widely used cytokines in patients with acute lung cancer (ACO), and it can amplify the inflammatory response. Neutrophil activation can recruit monocytes and macrophages to the site of lung lesions, exacerbate endothelial dysfunction, and promote foam cell formation. This study aimed to investigate the effects of perilla leaf extract on the release of neutrophil inflammatory factors TNF-α, IL-6, and IL-1β.

[0095] method:

[0096] Isolation, purification, and inflammatory factor assay of mouse bone marrow-derived neutrophils: Long bones of the hind limbs of mice were collected. After dissecting muscles and connective tissue, bone marrow was collected into clean 1.5 mL centrifuge tubes. Red blood cells were extracted from the bone marrow using erythrocyte lysis buffer; after filtering through a 70 μm filter, the cells were resuspended in PBS buffer containing 1% FBS and 1% penicillin / streptomycin. Gradient separation was performed using HISTOPAQUE (SIGMA) at 1.075 g / mL and 1.092 g / mL. The white, misty cell layer between the two density layers was collected, and cell viability was assessed using trypan blue. Cells were purified using a MACS separator via magnetic beads, and CD193 was measured. (+) CD11b after reverse selection of cells (+) Primary bone marrow-derived neutrophils were obtained through positive selection. After pre-protection with different doses of Perilla leaf extract (0.2, 0.5, 1, 2.5, 5, 10 μg / mL) for 1.5 hours, all cells except the control group were induced with 0.2 μg / mL LPS for 7 hours. The culture supernatant was collected, and the levels of extracellular inflammatory factors TNF-α, IL-6, and IL-β in neutrophils were measured using an ELISA kit (BioLegend).

[0097] Experimental results:

[0098] The results are shown in Table 4. Compared with the model control group, the production levels of neutrophil inflammatory factors TNF-α, IL-6 and IL-1β were significantly reduced after administration of Perilla leaf extract in a dose-dependent manner.

[0099] Table 4. Effects of Perilla Leaf Extract on the Release of Neutrophil Inflammatory Factors TNF-α, IL-6, and IL-1β (Mean ± Std)

[0100]

[0101] Note: Compared with the blank control group, # p < 0.05, ## p < 0.01, ### p < 0.001; compared with the model control group, * p < 0.05, ** p < 0.01, *** p < 0.001

[0102] Experimental Example 3: Pharmacodynamic effects of Perilla Leaf Extract on Eosinophilic Inflammation

[0103] TNF-α released by eosinophils is a key pro-inflammatory cytokine that promotes airway inflammation. TNF-α, along with other inflammatory factors, may contribute to airway hyperresponsiveness and epithelial cell proliferation. This study aimed to investigate the effect of perilla leaf extract on the release of the eosinophilic inflammatory factor TNF-α.

[0104] method:

[0105] In vitro isolation and culture of mouse bone marrow-derived eosinophils and determination of inflammatory factors: Long bones of the hind limbs of mice were collected. After separating the muscle and connective tissue, the bone marrow was collected into clean 1.5 mL centrifuge tubes. Red blood cells were extracted from the bone marrow using erythrocyte lysis buffer; after filtering through a 70 μm filter and centrifuging, the cells were resuspended in DMEM medium containing 10% FBS and 1% penicillin / streptomycin. Mature bone marrow-derived eosinophils were cultured in 100 ng / mL stem cell factors SCF and FLT3 and incubated at 37°C in a 5% CO2 environment for 4 days. Then, 10 ng / mL IL-5 was added to induce differentiation and maturation for 18 days. After pre-protection with different doses of perilla leaf (0.2, 0.5, 1, 2.5, 5, 10 μg / mL) for 2 hours, except for the control group, all other groups were stimulated with LPS (1 μg / mL) and IL-5 (100 ng / mL) for 12 hours. Collect cell culture supernatant and use ELISA to detect the level of TNF-α cytokine production. Follow the instructions in the kit manual.

[0106] Experimental results:

[0107] The results are shown in Table 5. Compared with the model control group, different doses (0.2, 0.5, 1, 2.5, 5, 10 μg / mL) of Perilla leaf extract significantly reduced the production level of eosinophil inflammatory factor TNF-α in a dose-dependent manner.

[0108] Table 5. Effects of Perilla Leaf Extract on TNF-α Release from Eosinophils (Mean ± Std)

[0109]

[0110] Note: Compared with the blank control group, ### p < 0.001; compared with the model control group, *** p < 0.001.

Claims

1. The use of a perilla leaf extract in the preparation of a drug for the prevention and / or treatment of asthma-chronic obstructive pulmonary disease overlap (ACO), characterized in that, The preparation method of the perilla leaf extract is as follows: 1) Using perilla leaves as raw material, 70% ethanol aqueous solution was heated and refluxed for extraction. The ethanol was recovered from the filtrate under reduced pressure to obtain an aqueous solution. The aqueous solution was concentrated under reduced pressure at 60℃ to a density of 1.090~1.100 (60℃) to obtain an extract for later use. 2) Extract the extract obtained in step 1) twice with ethyl acetate, and concentrate the remaining aqueous solution to obtain the extract; 3) The extract obtained in step 2) was precipitated with 90% ethanol, filtered, and the precipitate was dried and pulverized to obtain perilla leaf extract.

2. The application according to claim 1, characterized in that: The perilla leaf extract can simultaneously inhibit inflammation of ACO neutrophils and eosinophils, including inhibiting the production of TNF-α, IL-6, and IL-1β in neutrophils and TNF-α in eosinophils.

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