TWI693068B - Use of nerolidol for manufacturing compositions for treating acute lung injury - Google Patents

Abstract

The invention relates to a use of nerolidol for manufacturing compositions for treating acute lung injury. It comprises administrating an effective dose of nerolidol to a subject in need for reducing hyaline membrane formation, neutrophil infiltration, lung edema and oxidative stresses so as to achieve efficacy of treating acute lung injury.

Description

Use of nerol to prepare a composition for treating acute lung injury

The present invention relates to the use of nerol to prepare a composition for treating acute lung injury, and improves the related symptoms of acute lung injury by nerol.

Due to the seriousness of air pollution, respiratory-related diseases, such as pneumonia, lung cancer, chronic lung obstruction, asthma, bronchitis, atypical severe acute respiratory syndrome, emphysema, etc., have become one of the health issues that are valued globally. Acute lung injury (Acute lung injury, ALI for short) is one of the common and serious lung inflammatory diseases with high mortality. Infectious pneumonia and sepsis are the main causes of acute lung injury. Although new medical techniques have been developed to control acute lung injury and its worsening acute respiratory distress syndrome (ARDS), acute lung injury The mortality rate still exceeds 30%. If you can effectively control infection and inflammation, you can reduce the high mortality caused by these diseases.

U.S. Patent Announcement No. US8591896B2 discloses a chemotactic receptor binding polypeptides (chemokine receptor binding polypeptides), which mainly provides polypeptides that specifically bind to the chemokine receptor CXCR2 to treat diseases involving CXCR2 dysfunction, including cysts Fibrosis, asthma, severe asthma, worsening asthma, allergic asthma, acute lung injury, acute respiratory distress syndrome, idiopathic pulmonary fibrosis, tracheal remodeling, obstructive bronchiolitis syndrome or bronchopulmonary dysplasia, etc. However, the preparation method of the polypeptide is complicated and costly, and its effect on treating acute lung injury is still limited.

Nerolidol (nerolidol), also known as peruviol, chemical name 3,7,11-trimethyl-1,6,10-dodecatrien-3-ol, exists in a variety of plants, such as orange blossom, ginger, jasmine, Lavender, tea tree, lemon grass, pepper, camphor tree etc. Past studies have found that nerol has anti-tumor, anti-ulcer or anti-parasitic effects. In addition, Chinese Patent Publication No. CN106692514A also discloses that a pharmaceutical composition containing nerol and other medicinal raw materials, such as iris root, thirteen years old flower, fanghua flower in Guangxi, and betulinic acid, can be used to treat osteoporosis.

Since there is currently no effective treatment for acute lung injury, the development of various active substances with acute lung injury is still the focus of research and development in related fields.

Now, in view of the fact that the above existing acute lung injury treatment methods are still insufficient in practical use, the inventors assisted by their rich professional knowledge and many years of practical experience, accordingly developed the present invention.

The main object of the present invention is to provide a use of Nerolidol in the preparation of a composition for treating acute lung injury, which comprises administering an effective dose of nerolidol to a desired individual to reduce Injuries caused by acute lung injury.

In one embodiment of the present invention, nerolidol reduces lung vitreous membrane formation, neutrophil infiltration, and pulmonary edema.

In one embodiment of the present invention, nerolidol further reduces lung lipid peroxidation and improves the activity of antioxidant enzymes, the antioxidant enzymes including superoxide dismutase (superoxide dismutase), catalase (catalase ) And glutathione peroxidase.

In an embodiment of the present invention, the effective dose of nerol can be, for example, 10-100 μmol/kg.

In this way, nerol can be used as a composition for treating acute lung injury, such as a pharmaceutical composition or a food composition.

The purpose of the present invention and its structural and functional advantages will be explained based on the structure shown in the following drawings and in conjunction with specific embodiments, so that the reviewing committee can have a more in-depth and specific understanding of the present invention.

The invention provides a use of Nerolidol for preparing a composition for treating acute lung injury (ALI), which comprises an effective dose (for example, 10-100 μmol/kg) of Nerolidol Alcohol is administered to a desired individual to treat acute lung injury; among them, Neroli tertiary alcohol reduces lung vitreous membrane formation, neutrophil infiltration, pulmonary edema and lung lipid peroxidation, and increases antioxidant enzyme activity , The superoxide dismutase (superoxide dismutase), catalase (catalase) and glutathione peroxidase (glutathione peroxidase).

In addition, through the following specific embodiments, the scope of the present invention can be further proved to be practical, but it is not intended to limit the scope of the present invention in any form.

preparation Example: Establishment of an experimental model of acute lung injury

In this case, BALB/c rats purchased from the National Laboratory Animal Center at 5 to 7 weeks of age were used for the experiment. The experimental rats were acclimated to the environment for one week before conducting various experiments. The groups of this preparation example include:

"Control group": Intratracheal injection (IT) was given to saline of experimental rats;

"LPS stimulation group": give 100 μg of lipopolysaccharide (LPS) to the experimental rats by IT, and give a total volume of 50 μL to induce ALI;

"Neroliterol group": First, intraperitoneal injection (IP) was given to experimental mice at 10, 30, and 100 μmol/kg nerolidol, and then 30 minutes later, IT was given 100 μg LPS; and

"Dexamethasone group": the rats were given 1 mg/kg dexamethasone (IP) by IP, and then 100 μg LPS was given by IT 30 minutes later.

The experimental rats in each group were sacrificed 6 hours after LPS was given, and bronchoalveolar lavage fluid (BAL) was obtained using bronchoalveolar lavage technique, and the blood cells were classified and counted to detect neutrophils. Activity, and measure BAL protein content. The lung tissues of the experimental mice were used for histopathological section analysis, pulmonary edema analysis, tissue lipid peroxidation analysis and antioxidant activity analysis.

Example 1: Analysis of tert-Nerol on pulmonary vitreous membrane formation

Neroli tertiary alcohol used in this experiment was purchased from Santa Cruz Biotechnology (commercial model SC-205767A), it was dissolved in dimethyl sulfoxide (DMSO), and was also diluted with DMSO to the required concentration when used .

The lung lobes of the experimental mice were taken for fixation, embedding, sectioning and other processing steps, and after dewaxing, stained with Hematoxylin & Eosin and observed to evaluate the lung tissue. When the permeability of alveolar microvessels increases, protein-like fluid will leak into the alveoli and condense into a hyaline membrane, causing noncardiogenic pulmonary edema. There is a phenomenon of hyperplasia of inflammatory cells, so the occurrence of vitreous plasma membrane can be regarded as an indicator of increased permeability of alveolar microvessels.

Please refer to the first figure. Compared with the control group, the lung tissue presented by the LPS stimulation group has more vitreous membrane formation, and more immune cell infiltration can be observed; while treating different doses of nerol The concentration-dependent (dose dependent) method reduced LPS-induced lung vitreous membrane formation and immune cell infiltration; in addition, the dexamethasone group also significantly improved lung vitreous membrane formation and immune cell infiltration.

Example 2: Analysis of the ratio of wet/dry ratio of nerolidol to lung tissue

After sacrificing the experimental rat, weigh the lung tissue, which is the wet weight of the lung tissue; place the lung tissue in an oven at 80 ℃ for 48 hours, and then weigh the weight, which is the dry weight of the lung tissue (dry), the wet/dry weight ratio of the lung tissue of the experimental rat can be obtained by dividing the wet weight of the lung tissue by the dry weight.

Please refer to the second figure. Compared with the control group, the lung tissue wet/dry weight ratio of the LPS stimulation group was significantly higher than that of the control group, indicating that the experimental rats in the LPS stimulation group had significant pulmonary edema; treatment 10, 30, 100 μmol /kg Neroli tertiary alcohol group can reduce the lung tissue wet / dry weight ratio, it can be seen from the figure, Neroli tertiary alcohol improves LPS-induced pulmonary edema in a concentration-dependent manner, and 100 μmol/kg Neroli tertiary alcohol group The improvement effect is similar to the dexamethasone group. The above results are expressed as mean ± SD values (n=5), "#" indicates comparison with the control group value, p <0.05, and "*" indicates comparison with the LPS stimulation group value, p <0.05.

Example 3: Analysis of the integrity of alveolar microvascular barrier by tertiary nerol

When the alveolar microvascular barrier is damaged, the polymorphonuclear leukocytes and proteins in the microvessels will flow into the alveoli, so the integrity of the alveolar vascular barrier can be analyzed by analyzing the content of polymorphonuclear leukocytes and proteins in the alveolar effluent (BAL) The evaluation index; the test process is briefly described as follows: after sacrificing the experimental rat, irrigate its lungs with 1 X PBS buffer, collect the washing solution and centrifuge, the supernatant is measured by Bradford assay to measure the protein content, and precipitated at the bottom The polymorphonuclear cells were fixed and stained with 10% Giemsa stain, and then the number or type of neutrophils were analyzed with a microscope.

Please refer to the third figure. Compared with the control group, the LPS-stimulated group had higher amounts of protein in the alveolar effluent, while the group treated with nerol reduced the protein content in the alveolar effluent. It can be seen from the figure that nerolidol improves LPS-induced damage to alveolar microvascular integrity in a concentration-dependent manner, and the improvement effect of the 100 μmol/kg nerolidol group is similar to the dexamethasone group. The above results are expressed as mean ± SD values (n=5), "#" indicates comparison with the control group value, p <0.05, and "*" indicates comparison with the LPS stimulation group value, p <0.05.

Please refer to the fourth figure again. Compared with the control group, the lungs of the experimental rats in the LPS-stimulated group had a lot of neutrophil infiltration, and the treatment of nerol will reduce the neutral spheres of the experimental rats. This figure shows that nerolithol reduces the infiltration of neutrophils into the alveolar cavity by LPS in a concentration-dependent manner, and the number of BAL neutrophils in the 100 μmol/kg nerolithol group and the dexamethasone group The number of BAL neutral balls is similar. The above results are expressed as mean ± SD values (n=5), "#" indicates comparison with the control group value, p <0.05, and "*" indicates comparison with the LPS stimulation group value, p <0.05.

Example 4: Analysis of nerol to lung lipid peroxidation

Detecting the amount of malondialdehyde (MDA) produced as the final product of lipid peroxidation can be used to assess the lung tissue damage status. The detection steps are briefly described as follows. After sacrificing the experimental rat, take the lung tissue and homogenize it, then add acetic acid, sodium dodecyl sulfate and thiobarbituric acid, mix well Heat to 95°C; after cooling the above mixture to room temperature, add n-butanol and pyridine to mix and centrifuge, and measure the absorbance of the mixture at a wavelength of 532 nm.

Please refer to the fifth figure. Compared with the control group, the lipid peroxidation of the LPS-stimulated group is significantly higher than that of the control group, and the treatment of nerolidol can reduce the degree of lipid peroxidation. In a dependent manner, the prevention of LPS-induced lung lipid peroxidation; and the effect of 100 μmol/kg nerol group on lipid peroxidation was similar to the dexamethasone group. The above results are expressed as mean ± SD values (n=5), "#" indicates comparison with the control group value, p <0.05, and "*" indicates comparison with the LPS stimulation group value, p <0.05.

Example 5: Analysis of tertiary nerol on lung antioxidant enzyme activity

In acute lung injury, increased lung oxidative pressure is likely to cause lung tissue damage. In this case, the organism will be protected by antioxidant enzymes (antioxidant enzyme). For example, superoxide dismutase (superoxide dismutase, SOD) metabolizes superoxide free radicals to hydrogen peroxide (H ₂ O ₂ ), and catalase (CAT) and glutathione peroxidase (glutathione peroxidase, GSH) then converts hydrogen peroxide into non-toxic water. In this example, the lung tissue of the experimental rat was homogenized in a dissolution solution, and after centrifugation, the supernatant was retained and the activity of antioxidant enzymes was measured.

As shown in Figures 6 to 8, compared to the control group, the lung SOD, CAT, and GSH activities of the LPS-stimulated group were significantly reduced, but the treatment of nerolidol increased the three antioxidant enzymes in a concentration-dependent manner Activity, from this figure, we can see that nerol can prevent LPS from reducing the activity of antioxidant enzymes; and the effect of 100 μmol/kg nerol group on improving the activity of three enzymes is similar to the dexamethasone group. The above results are expressed as mean ± SD values (n=5), "#" indicates comparison with the control group value, p <0.05, and "*" indicates comparison with the LPS stimulation group value, p <0.05.

It can be seen from the above implementation description that the present invention has the following advantages compared with the prior art:

1. The nerolidol of the present invention can effectively reduce the lung damage caused by acute lung injury, and can prevent the increase of cell microvessel permeability and maintain the integrity of lung tissue microvessels.

2. The nerolidol of the present invention has excellent antioxidant capacity, can reduce the degree of lipid peroxidation, and enhance the activity of antioxidant enzymes in a concentration-dependent manner.

In summary, the use of nerolidol of the present invention in the preparation of a composition for the treatment of acute lung injury can indeed achieve the expected use effect through the embodiments disclosed above, and the present invention has not been disclosed before the application , Cheng has fully complied with the provisions and requirements of the Patent Law. I filed an application for a patent for invention in accordance with the law, pleaded for the review, and granted the patent.

However, the illustrations and descriptions disclosed above are only preferred embodiments of the present invention, and are not intended to limit the scope of protection of the present invention; those who are familiar with this skill, according to the characteristic scope of the present invention, do other things Equivalent changes or modifications should be regarded as not departing from the design scope of the present invention.

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The first picture: the sliced image of nerol in the present invention reduces the formation of pulmonary vitreous membrane.

Figure 2: Analytical diagram of nerol in the present invention for inhibiting pulmonary edema.

Figure 3: Analytical chart of nerol in the present invention to reduce the protein content of alveolar effluent.

Figure 4: Analysis chart of nerol in the present invention to reduce the content of lung neutrophils.

Fifth figure: Analytical chart of nerol in the present invention reducing lung lipid peroxidation.

Figure 6: Analysis chart of nerol tertiary alcohol promoting superoxide dismutase activity of the present invention.

Figure 7: Analysis chart of the catalase activity promoted by nerol in the present invention.

Figure 8: Analysis chart of nerol in the present invention promoting glutathione peroxidase activity.

Claims (3)

A use of Nerolidol for preparing a composition for treating acute lung injury, which comprises administering an effective dose of nerolidol to a desired individual to treat acute lung injury. The use as described in item 1 of the patent application scope, wherein the nerolidol reduces lung vitreous membrane formation, neutrophil infiltration and pulmonary edema. The use as described in item 1 of the patent application range, wherein the effective dose is 10-100 μmol/kg.

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