TWI698246B - Use of elsholtzia ciliata hylander extracts for treating lung tissue damage and inhibiting pulmonary fibrosis - Google Patents

Abstract

The present invention provides use of an Elsholtzia ciliata Hylander extract for manufacture of a pharmaceutical composition for treating lung tissue damage and inhibiting pulmonary fibrosis. The Elsholtzia ciliata Hylander extract is preferably prepared by extraction of Elsholtzia ciliata Hylander using water as solvents. Said extract improves the wound healing ability of lung epithelial cells, inhibits secretion of proinflammatory cytokines and enhances E-cadherin expression in lung epithelial cells, and reduces glycated protein formation.

Description

Purplish lemongrass extract is used to treat lung tissue damage and inhibit pulmonary fibrosis

The present invention relates to the health care use of a plant extract, in particular to the use of a Citronella extract for preparing a pharmaceutical composition for treating lung tissue damage and inhibiting lung fibrosis.

The lungs are respiratory organs and filter organs. The lungs usually secrete mucus to form a protective layer to prevent pathogens (such as viruses, bacteria, fungi) or suspended particles in the inhaled air from adsorbing to the lungs. However, the lung's ability to remove foreign bodies is limited, and some foreign bodies may penetrate this protective layer and infect or damage the lungs, thereby causing lung inflammation. In addition, lung contusion, sepsis, hemorrhagic shock, etc. can cause severe lung inflammation. A variety of immune cells involved in inflammation (such as macrophages, neutrophils) and stimulated secretion of pro-inflammatory cytokines (such as interleukin, tumor necrosis factor-α (TNF-α)) may cause lung damage , Including alveolar injury, increased microvascular permeability, microthrombosis, and even pulmonary edema and acute respiratory distress in severe cases. The aforementioned factors that cause lung tissue damage may cause idiopathic pulmonary fibrosis along with inflammation. Symptoms include dry cough and progressive dyspnea, which may eventually lead to respiratory failure and death.

In order to maintain the normal function of the lungs, the exposure to air pollutants and pathogens should be minimized to avoid lung tissue damage and pulmonary fibrosis. Possible methods include avoiding smoking, wearing masks, and using air purification devices. In addition, if lung tissue damage has already formed, drugs that can promote the healing of the damage or inhibit inflammation should be taken. However, currently known drugs that can effectively inhibit the development of idiopathic pulmonary fibrosis are limited, and studies have shown that anti-inflammatory drugs may not necessarily inhibit the deterioration of pulmonary fibrosis. Therefore, it is necessary to develop a novel composition that can effectively promote the healing of lung tissue damage, resist pulmonary inflammation, and inhibit pulmonary fibrosis.

For this reason , one object of the present invention is to provide an extract of Elsholtzia ciliata Hylander for the preparation of a pharmaceutical composition for treating lung tissue damage and inhibiting pulmonary fibrosis, wherein the extract of Citronella is a It is obtained by solvent extraction of Citronella.

In an embodiment of the present invention, the weight ratio of the solvent to the lemongrass ranges from 20:1 to 2:1, and the extraction is performed at 50°C to 100°C.

In an embodiment of the present invention, the solvent is water, and the concentration of the Citronella citronella extract is 0.25 to 1 mg/mL.

In an embodiment of the present invention, the Citronella extract promotes the wound healing power of a lung epithelial cell.

In an embodiment of the present invention, the Citronella extract inhibits a lung epithelial cell to secrete a pro-inflammatory cytokine, such as interleukin-8 (IL-8).

In an embodiment of the present invention, the Citronella purpurea extract promotes a lung epithelial cell to express an E-cadherin (E-cadherin).

In an embodiment of the present invention, the Citronella purpurea extract inhibits protein glycation.

The extract of Citronella citronella of the present invention can improve the wound repair ability of lung tissue, at the same time inhibit the secretion of proinflammatory cytokine by lung tissue and promote its expression of E-cadherin, and can inhibit the production of glycated protein, and ultimately reduce lung tissue damage and anti-inflammatory , And the effect of inhibiting pulmonary fibrosis, thus helping to maintain lung function. Therefore, the present invention provides the use of the extract of Citronella citronella for the preparation of a pharmaceutical composition for treating lung tissue damage and inhibiting pulmonary fibrosis. The pharmaceutical composition can be powder, granule, solution, colloid or paste, which can be administered to a body by oral administration or inhalation through the respiratory tract.

The following will further illustrate the implementation of the present invention in conjunction with the drawings. The following examples are used to illustrate the features and applications of the present invention, rather than to limit the scope of the present invention. Anyone familiar with the art will not depart from Within the spirit and scope of the present invention, some changes and modifications can be made. Therefore, the protection scope of the present invention shall be subject to those defined by the appended patent scope.

Figure 1A shows photomicrographs of a single layer of human lung epithelial cells treated with or without Citronella extracts for 0 hours and 16 hours.

Figure 1B shows the percentage of wound healing of a single layer of human lung epithelial cells treated with or without Citronella extract for 16 hours.

Figure 2 shows the IL-8 secretion of human lung epithelial cells stimulated by lipopolysaccharide (LPS) after treatment with or without Citronella extracts; the control group cells were not treated with lipopolysaccharide and Citronella extracts.

Figure 3 shows the fluorescence micrographs of human lung epithelial cells treated with lipopolysaccharides with or without Citronella extracts; the control group cells were not treated with lipopolysaccharides and Citronella extracts.

Figure 4 shows the inhibitory effect of Citronella extract on collagen glycation.

The present invention provides a use of a Citronella extract for preparing a pharmaceutical composition for treating lung tissue damage and inhibiting pulmonary fibrosis. The Citronella purpurea extract of the present invention is obtained by extracting a Citronella purpurea with a solvent, wherein the solvent is preferably water, and the weight ratio of the solvent to the Citronella purpurea ranges from 20:1 to 2:1, and The extraction is performed at 50°C to 100°C. The following examples show that the Citronella extract significantly improves the wound repair ability of lung epithelial cells, and significantly inhibits the secretion of pro-inflammatory cytokine by lung epithelial cells under inflammatory stimulation and promotes the performance of E-cadherin for anti-pulmonary fibrosis. In addition, lemongrass extract can still inhibit the production of collagen glycation end products, so it helps prevent lung fibrosis.

definition

The numerical values used herein are approximate values, and all experimental data are expressed in the range of 20%, preferably in the range of 10%, and most preferably in the range of 5%.

"Treatment of lung tissue injury" as used herein refers to reducing the degree of damage to the entire lung tissue (including alveoli). Its judgment indicators include the reduction of lung inflammation and the increase in the speed of lung wound repair. Those skilled in the art should be able to assess lung tissue damage according to known techniques in the art, such as the biochemical and cell culture techniques described herein.

Materials and Methods Cell culture

The following examples use the human normal lung epithelial cell line BEAS-2b (ATCC CRL-9609) purchased from the American Type Culture Collection (ATCC). The cell line was cultured in bronchial epithelial cell basal medium (Lonza ^TM Bronchial epithelial cell basal medium; Thermo Fisher Scientific) under the conditions of 37° C. and 5% carbon dioxide, referred to as BEBM medium.

IL-8 quantitative analysis

The enzyme-linked immunosorbent assay (ELISA) kit (Human CXCL8/IL-8 ELISA kit; R&D systems) was used to determine the protein content of IL-8. According to the manufacturer’s instructions, block the 96-well plate with the capture antibody immobilized with phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA) at 37°C. After 3 hours, rinse with cleaning solution. Add a fixed amount of the aforementioned 1% BSA solution and 100 μL/well of the test sample or standard to the 96-well plate and react at 37°C for 2 hours. Thereafter, 100 μL/well of detection antibody was added to the washed 96-well plate and reacted at 37°C for 2 hours. Finally, 100μL/well of Streptavidin-Horseradish peroxidase (HRP) solution was added to the washed 96-well plate and reacted at room temperature for 20 minutes, and then 100μL/well of substrate solution was added After washing the 96-well plate and performing a color reaction at room temperature for 20 minutes, the reaction is stopped. Detect the absorbance value of the 96-well plate at 450 nm using an ELISA plate reader (BioTek).

Immunofluorescence staining of anti-pulmonary fibrosis index protein

In order to assess the occurrence of pulmonary fibrosis, immunofluorescence staining was used to determine the expression level of E-cadherin (E-cadherin, also known as CD324) in lung epithelial cells as an indicator of anti-fibrosis. The steps are briefly described as follows. According to the manufacturer's instructions, the cells to be tested were washed with PBS solution (Thermo Fischer Scientific), and then fixed with PBS solution containing 4% formaldehyde at room temperature for 15 minutes. After washing with PBS solution, the fixed cells are treated with 0.5% Triton X-100 (Triton X-100) in PBS solution at room temperature for 3 to 5 minutes, and then washed with PBS solution for immunostaining. The cells were blocked in a PBS solution containing 1% BSA for 1 hour at room temperature, and then combined with the mouse anti-human E-cadherin antibody (anti-human CD324 antibody; Biolegend) diluted in the 1% BSA solution. React at room temperature for 2 hours. After washing with PBS solution, the cells and the red fluorescent-labeled goat anti-mouse immunoglobulin G secondary antibody (Alexa Flour ^TM 594 goat anti-mouse IgG; Thermo Fischer Scientific) diluted in 1% BSA solution were kept at room temperature React for 30 minutes, then wash with PBS solution. Finally, the cells were stained with a DNA fluorescent dye (Hoechst 33342; Thermo Fischer Scientific) at room temperature for 3 to 5 minutes. The cells were washed to observe the red fluorescence distribution with a fluorescence microscope.

Statistical Analysis

The statistically significant differences are determined by using the student T test in Excel software.

Example 1 Preparation of Lemongrass Extract

Elsholtzia ciliata Hylander ( Elsholtzia ciliata Hylander ), also known as Elsholtzia and Honeygrass , is an annual herb, mainly distributed in Asia. The plant is known to be used to treat acute vomiting, diarrhea and heatstroke, and has a diuretic effect.

In order to obtain the extract of Citronella purpurea, firstly, the whole plant of Citronella purpurea is washed and dried, and then coarsely crushed with a grinder. After that, the crude citronella was extracted with water as a solvent. The weight ratio of the solvent and the crude citronella crumb is in the range of 20:1 to 2:1. The extraction temperature is between 50°C and 100°C, preferably between 80°C and 90°C. The extracts of Citronella purpurea in the following Examples 2-5 are all extracted with water, and the extraction time is 0.5 to 3 hours.

After the lemongrass extract obtained by the above extraction step is cooled to room temperature, it can be filtered with a 400 mesh filter to remove residual solids. The filtered lemongrass extract can be further concentrated under reduced pressure at 45°C to 70°C to obtain a concentrated product. In order to obtain a solid Citronella citronella extract, the concentrated Citronella citronella extract can be dried to remove the solvent by drying methods such as freeze drying, spray drying, etc., thereby obtaining a dried product of the Citronella citronella extract.

Example 2 Citronella extract promotes the wound healing power of lung epithelial cells

In order to study whether the extract of Citronella citronella affects the wound healing ability of lung tissue, this example uses a microscope to observe the changes in wound healing of the cell monolayer of the human lung epithelial cell line BEAS-2b after treatment with Citronella extract. In short, cells were seeded in a 24-well plate containing BEMB medium at 1.5×10 ⁵ cells/well and cultured at 37°C overnight to form a cell monolayer. Thereafter, a wound was formed in the cell monolayer, the culture medium was removed and the cells were washed with PBS solution, and then 1 mL of BEMB medium containing 0.25 mg/mL lemongrass extract was used to treat the cells, or only BEMB medium was used to treat the cells to As a control group. The two groups of cell monolayers (triple repeat test) were observed and photographed with a microscope (ZEISS) after wound formation (0 hour) and after 16 hours of culture at 37°C.

Figure 1A shows the micrographs of the two groups of cell monolayers at 0 hours and 16 hours (magnification 50X); Figure 1B is the wound healing percentage calculated based on Figure 1A, and its value is the wound area of each group at 16 hours The percentage of the total cell area relative to the total cell area of the wound area at 0 hours. According to Figures 1A-1B, the treatment of Citronella extract increased the area of wound repaired by lung epithelial cells by about 30%. This result shows that Citronella extract promotes the wound healing power of lung epithelial cells, and therefore has the effect of treating lung tissue damage.

Example 3 Citronella extract inhibits the secretion of pro-inflammatory cytokine by lung epithelial cells

In order to investigate whether the extract of Citronella purpurea has anti-inflammatory effects on lung tissues, this example uses lipopolysaccharide (LPS; Sigma) to stimulate the inflammatory response of the human lung epithelial cell line BEAS-2b, and uses ELISA to measure the passage of the cells through Citronella purpurea Changes in IL-8 secretion after extract treatment. In short, BEAS-2b cells were seeded on a 24-well plate with 5×10 ⁴ cells/well, and each well contained 1% penicillin/streptomycin and 10% fetal bovine serum. , FBS) 500μL DMEM medium (Dulbecco's modified Eagle's medium; Thermo Fisher Scientific), hereinafter referred to as DMEM cell culture medium. After culturing the cells at 37°C for 24 hours, the medium was removed and the cells in each well were treated in the following manner: (a) 500 μL DMEM cell culture medium (control group); (b) 500 μL DMEM cell culture medium containing 1 mg/ mL lipopolysaccharide (LPS group); or (c) apply 500 μL DMEM medium containing 1 mg/mL lipopolysaccharide and 0.25 mg/mL citronella extract (LPS+ citronella extract group). After the cells of each group (triple repeat test) were cultured at 37°C for 2 hours, the DMEM cell culture medium was updated and cultured for another 24 hours, that is, 120 μL of supernatant was collected for IL-8 quantitative analysis.

Figure 2 shows the amount of IL-8 secreted by lung epithelial cells in the aforementioned groups; ^*** means p<0.001 compared to the LPS group. According to Figure 2, compared with the control group, the treatment of lipopolysaccharide caused a large amount of IL-8 secretion, but the simultaneous administration of 0.25 mg/mL citronella extract significantly reduced the IL-8 secretion of lung epithelial cells by about 60%, even It was slightly lower than the IL-8 secretion of the control group. This result indicates that Citronella extract can inhibit the inflammation of lung tissue (such as lung epithelial cells), thus reducing the possibility of further damage to individual lungs due to inflammation (especially chronic inflammation).

Example 4 Citronella extract inhibits lung fibrosis

Previous studies have pointed out that the expression of E-cadherin, which is related to the maintenance of normal cell types, is reduced in lung fibrotic cells, so E-cadherin is regarded as an anti-pulmonary fibrosis indicator protein. In order to test whether the extract of Citronella purpurea prevents the occurrence of pulmonary fibrosis, this example uses lipopolysaccharide to stimulate the inflammatory response and fibrosis pattern of the human lung epithelial cell line BEAS-2b, and the immunofluorescence staining method is used to determine the cell growth rate. E-cadherin expression after treatment with Citronella extract. In short, BEAS-2b cells were seeded on a 24-well plate containing BEBM medium at 2×10 ⁴ cells/well. After culturing the cells overnight at 37°C, the nutrient medium was removed, and the cells in each well were treated in the following manner: (a) 500μL DMEM cell culture medium (control group); (b) 500μL DMEM cell culture medium containing 1 mg /mL lipopolysaccharide (LPS group); or (c) 500 μL of DMEM cell culture medium containing 1 mg/mL lipopolysaccharide and 0.25 mg/mL citronella extract (LPS + citronella extract group). After each group of cells (triple repeat test) were cultured at 37°C for 2 hours, the DMEM cell medium was renewed and cultured for another 24 hours, that is, washed with PBS solution and used for immunofluorescence staining.

Figure 3 shows the fluorescence micrographs of the aforementioned lung epithelial cells (magnification 100X); red fluorescence indicates E-cadherin, and blue fluorescence indicates cell nuclei. According to the photo, the cells of the control group showed a lot of red fluorescence, but the treatment of lipopolysaccharide almost disappeared the red fluorescence, indicating that the inflammatory stimulus inhibits the expression of E-cadherin in lung epithelial cells and makes lung epithelial cells change into a fibrotic form . However, the simultaneous administration of 0.25 mg/mL Lemongrass extract restored E-cadherin performance. This result shows that Citronella extract can directly inhibit the development of lung epithelial cells to pulmonary fibrosis, and therefore has the potential to inhibit lung dysfunction caused by pulmonary fibrosis.

Example 5 Lemongrass extract inhibits protein glycation

The glycation reaction of protein in the body can lead to loss of protein function, which in turn leads to tissue or organ (such as lung) degradation. In addition, studies have suggested that the end-products of protein glycation are related to the development of pulmonary fibrosis. In order to investigate whether the extract of Citronella purpurea can inhibit protein saccharification, this example uses an anti-glycation analysis to determine the inhibitory effect of 1 mg/mL Citronella purpurea extract on the saccharification reaction of porcine collagen. In short, a 60 mg/mL collagen solution (containing 0.06% sodium azide) and a 1.5 M fructose solution were prepared using 200 mM phosphate buffer solution (pH 7.4). In order to carry out the collagen saccharification reaction, the mixture of 0.2mL collagen solution and 0.2mL fructose solution and 0.2mL of Citronella extract sample or deionized water (control group) were evenly mixed, reacted at 50℃ for 24 hours, and then added Aminoguanidine (AG, purchased from Sigma) was used to stop the glycation reaction. Use a spectrofluorometer (FLx 800, BioTek) to measure the fluorescence intensity (excitation wavelength 360nm, emission wavelength 460nm) of the aforementioned reaction solution (0.1mL) at 0 hours and 24 hours, and calculate the final protein glycation product according to the following formula Generation rate: [(sample fluorescence intensity for _{24 hours} -sample fluorescence intensity for _{0 hours} )/(control group fluorescence intensity for _{24 hours} -control group fluorescence intensity for _{0 hours} )]×100%.

Figure 4 shows the inhibitory effect of Citronella extract on collagen glycation. According to this figure, 0.25mg/mL Lemongrass extract reduces the production of collagen glycation end products by about 33%. This result indicates that the extract of Citronella fragrans has the effect of inhibiting pulmonary fibrosis by reducing the end-products of protein glycation, and can delay organ degeneration by inhibiting protein glycation in the body.

In summary, Citronella extract can improve the wound repair ability of lung tissues, at the same time inhibit the secretion of pro-inflammatory cytokine and promote the expression of E-cadherin in lung tissues, and inhibit the production of glycated protein, ultimately reducing lung tissue damage , Anti-inflammatory, and inhibit pulmonary fibrosis, so it helps maintain lung function. Therefore, the present invention provides the use of the extract of Citronella citronella for the preparation of a pharmaceutical composition for treating lung tissue damage and inhibiting pulmonary fibrosis. The pharmaceutical composition can be powder, granule, solution, colloid or paste, which can be administered to a body by oral administration or inhalation through the respiratory tract.

Claims (9)

A citronella extract is used to prepare a pharmaceutical composition for treating lung tissue damage and inhibiting pulmonary fibrosis, wherein the citronella extract is obtained by extracting a citronella with a solvent, wherein the solvent is water. The use as described in item 1 of the scope of patent application, wherein the weight ratio of the water to the purple lemongrass ranges from 20:1 to 2:1. For the use described in item 1 of the scope of patent application, the extraction is carried out at 50°C to 100°C. The use described in item 1 of the scope of patent application, wherein the concentration of the Citronella extract is at least 0.25 to 1 mg/mL. The use as described in item 1 of the scope of the patent application, wherein the Citronella extract promotes the wound healing power of a lung epithelial cell. The use described in item 1 of the scope of the patent application, wherein the Citronella fragrans extract inhibits a lung epithelial cell from secreting a pro-inflammatory cytokine. The use described in item 1 of the scope of the patent application, wherein the citronella extract promotes the expression of an E-cadherin in a lung epithelial cell. The use as described in item 1 of the scope of patent application, wherein the Citronella extract inhibits protein glycation. The use described in item 1 of the scope of the patent application, wherein the pharmaceutical composition has a powder, granule, solution, gel, or paste dosage form.

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