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1. (WO2018226447) MÉTHODE D'INHIBITION D'UNE INFLAMMATION PROVOQUÉE PAR UNE INFECTION PAR DES BACTÉRIES À GRAM NÉGATIF
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METHOD FOR INHIBITING INFLAMMATION CAUSED BY GRAM-NEGATIVE BACTERIA INFECTION

BACKGROUND OF THE INVENTION

[0001 ] 1. Field of the Invention

[0002] The present invention relates to a method for inhibiting inflammation caused by Gram-negative bacteria infection and, more particularly, to a method for inhibiting inflammation caused by Gram-negative bacteria infection using an aqueous extract of Asplenium australasicum (J. Sm.) Hook..

[0003] 2. Description of the Related Art

[0004] Lipopolysaccharide (LPS), also known as endotoxin, is the major component of the outer membrane of Gram-negative bacteria, contributing to the structural integrity of the bacteria, and protecting the membrane from certain kinds of chemical attack. When an animal body is infected by the Gram-negative bacteria, macrophages of the animal body secrete the proinflammatory cytokines such as TNF-a, IL-Ιβ and IL-6. These cytokines increase vascular permeability, leading to signs of inflammation, such as fever, pain and swelling. The cytokines can also stimulate the activation of the macrophages to secrete more cytokines.

[0005] Generally, inflammation is a protective response for removing harmful pathogens and stimulating repair of tissues. However, prolonged inflammation leads to discomfort to the human body, or even destroy tissues and cause necrosis. Therefore, once an inflammatory reaction occurs, one should pay attention to the inflammatory reaction to prevent from deterioration.

[0006] Indomethacin, a conventional medicament for inhibiting infection of Gram-negative bacteria, is mainly used for inhibit signs of inflammation, such as fever, pain and swelling. However, indomethacin may cause peptic ulcers, leading to bleeding or perforated peptic ulcer. In light of this, it is necessary to develop a method for inhibiting inflammation caused by Gram-negative bacteria infection.

SUMMARY OF THE INVENTION

[0007] It is therefore an objective of the present invention to provide a method for inhibiting inflammation caused by Gram-negative bacteria infection by using an aqueous extract of Asplenium australasicum (J. Sm.) Hook, which is rich in active substances against inflammation.

[0008] One embodiment of the present invention discloses a method for inhibiting inflammation caused by Gram-negative bacteria infection in a subject in need thereof. The method includes administering an effective amount of an aqueous extract of Asplenium australasicum (J. Sm.) Hook, to the subject. The aqueous extract is obtained by extracting a sample of Asplenium australasicum (J. Sm.) Hook, by water under 0.35-0.5 Kg/cm2G and 115-125°C for 1.5-3 hours. Accordingly, with the active substances obtained from the sample of Asplenium australasicum (J. Sm.) Hook., the aqueous extract of Asplenium australasicum (J. Sm.) Hook, according to the present invention shows ability of inhibiting secretion of the proinflammatory cytokines (TNF-a, IL-Ι β, IL-6, etc.) induced by lipopolysaccharides, reducing the inflammatory response. Therefore, the aqueous extract of Asplenium australasicum (J. Sm.) Hook, can be applied to inhibiting inflammation caused by Gram-negative bacteria infection.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1 depicts a bar chart illustrating the relative survival rates of RAW264.7 cells of groups A0-A8 after being administrated by the aqueous extract of Asplenium australasicum (J. Sm.) Hook, according to the present invention in trial (A).

[0010] FIG. 2a depicts a bar chart illustrating the relative TNF-a RNA levels of RAW264.7 cells of groups B0-B4 after being administrated by the aqueous extract of Asplenium australasicum (J. Sm.) Hook, according to the present invention in trial (B).

[0011] FIG. 2b depicts a bar chart illustrating the relative IL-Ιβ RNA levels of RAW264.7 cells of groups B0-B4 after being administrated by the aqueous extract of Asplenium australasicum (J. Sm.) Hook, according to the present invention in trial (B).

[0012] FIG. 2c depicts a bar chart illustrating the relative IL-6 RNA levels of RAW264.7 cells of groups B0-B4 after being administrated by the aqueous extract of Asplenium australasicum (J. Sm.) Hook, according to the present invention in trial (B).

[0013] FIG. 3a depicts a bar chart illustrating the relative TNF-a protein levels secreted by RAW264.7 cells of groups C0-C4 after being administrated by the aqueous extract of Asplenium australasicum (J. Sm.) Hook, according to the present invention in trial (C).

[0014] FIG. 3b depicts a bar chart illustrating the relative IL-Ιβ protein levels secreted by RAW264.7 cells of groups C0-C4 after being administrated by the aqueous extract of Asplenium australasicum (J. Sm.) Hook, according to the present invention in trial (C).

[0015] FIG. 3c depicts a bar chart illustrating the relative IL-6 protein levels secreted by RAW264.7 cells of groups C0-C4 after being administrated by the aqueous extract of Asplenium australasicum (J. Sm.) Hook, according to the present invention in trial (C).

DETAILED DESCRIPTION OF THE INVENTION

[0016] Asplenium australasicum (J. Sm.) Hook, is a plant which belongs to the family of Aspleniaceae and the genus of Asplenium. The young emerging frond of Asplenium australasicum (J. Sm.) Hook, is one kind of edible leaves.

[0017] An aqueous extract of Asplenium australasicum (J. Sm.) Hook, according to the present invention shows ability of inhibiting secretion of the proinflammatory cytokines (TNF-a, IL-Ιβ, IL-6, etc.) induced by lipopolysaccharides, reducing the inflammatory response. Therefore, the aqueous extract of Asplenium australasicum (J. Sm.) Hook, can be applied to inhibiting inflammation caused by Gram-negative bacteria infection. The aqueous extract of Asplenium australasicum (J. Sm.) Hook, can be used individually, or in combination with pharmaceutical acceptable vehicles, excipients, salts or other nutrients, being in a composite. In addition, the aqueous extract of Asplenium australasicum (J. Sm.) Hook, can be further manufactured into any oral type that is easy to take, such as pastil, capsule, powder, pill, solution, or fermented products. Yet, the aqueous extract of Asplenium australasicum (J. Sm.) Hook, can be combined with other food products or drinks, being manufactured into a more convenient type for taking.

[0018] The aqueous extract of Asplenium australasicum (J. Sm.)

Hook, can be preferably obtained by a method including the following steps: providing a sample of Asplenium australasicum (J. Sm.) Hook.; extracting the sample of Asplenium australasicum (J. Sm.) Hook, by water as an extractant to obtain a rough extract of Asplenium australasicum (J. Sm.) Hook.; and concentrating the rough extract of Asplenium australasicum (J. Sm.) Hook, to obtain the aqueous extract of Asplenium australasicum (J. Sm.) Hook..

[0019] Specifically, the sample of Asplenium australasicum (J. Sm.) Hook, can be the 3 -year-old mature frond with spores on the underside thereof. The sample of Asplenium australasicum (J. Sm.) Hook, can be preferably dried in advance at 110-120 C to obtain a dried sample of Asplenium australasicum (J. Sm.) Hook. (100 grams of the sample of Asplenium australasicum (J. Sm.) Hook, are dried into 20 grams of the dried sample of Asplenium australasicum (J. Sm.) Hook.). Besides, the sample of Asplenium australasicum (J. Sm.) Hook, can also be milled to a size ranging from 1 to 2 mm in advance as well. With such performance, the contacting surface area of the sample of Asplenium australasicum (J. Sm.) Hook, with water is increased, and therefore, the efficiency of the extraction is also increased.

[0020] In an example, 4.8 kilograms of the sample of Asplenium

australasicum (J. Sm.) Hook, can be mixed with the extractant until the overall volume of the sample of Asplenium australasicum (J. Sm.) Hook, and the extractant is 80 liters. The sample of Asplenium australasicum (J. Sm.) Hook, is then extracted under 0.35-0.5 Kg/cm2G and 115-125°C for 1.5-3 hours. The extraction process can be repeated several times, assuring the active substances of the sample of Asplenium australasicum (J. Sm.) Hook, can be completely dissolved in the extractant, which can be appreciated by a person having ordinary skill in the art.

[0021] Furthermore, the rough extract of Asplenium australasicum (J. Sm.) Hook., can be further concentrated by concentration under reduced pressure and lyophilization to obtain the aqueous extract of Asplenium australasicum (J. Sm.) Hook.. The active substances of the aqueous extract of Asplenium australasicum (J. Sm.) Hook, are further concentrated. The preferable efficacy for inhibiting inflammation caused by Gram-negative bacteria infection will be achieved by only a limited amount of the aqueous extract of Asplenium australasicum (J. Sm.) Hook..

[0022] To validate the aqueous extract of Asplenium australasicum (J. Sm.) Hook, according to the present invention can be used to effectively inhibit secretion of the proinflammatory cytokines such as TNF-a, IL-Ιβ and IL-6, the following trial is carried out.

[0023 Trial (A).

[0024] Referring to TABLE 1, the aqueous extracts of Asplenium australasicum (J. Sm.) Hook, in different dosages are administrated to RAW264.7 cells of groups A1-A8 for 1 hour. LPS (1 μg/mL) is then administrated to RAW264.7 cells of groups A0-A8. 24 hours later, survival rates of RAW264.7 cells of groups A0-A8 are detected using CellTiter 96 AQueous One Solution Cell Proliferation Assay, and the survival rate of RAW264.7 cells of group AO is used as 100%.

TABLE 1


[0025] Referring to FIG. 1, the aqueous extract of Asplenium australasicum (J. Sm.) Hook, in a dosage lower than 50 shows no

cytotoxicity to RAW264.7 cells (groups A1-A4).

[0026] Trial (B).

[0027] Referring to TABLE 2, the aqueous extract of Asplenium australasicum (J. Sm.) Hook, in different dosages are administrated to RAW264.7 cells of groups B2-B4 for 1 hour. LPS (1 μg/mL) is then administrated to RAW264.7 cells of groups B 1-B4. 24 hours later, total RNAs are collected from RAW264.7 cells of groups B0-B4, and cDNAs of groups B0-B4 are obtained via reverse transcription reaction. Corresponding primers are used to detect relative RNA levels of proinflammatory cytokines such as TNF-a, IL-Ιβ and IL-6, as shown in FIGS. 2a, 2b and 2c, respectively.

TABLE 2


[0028] Referring to FIGS. 2a, 2b and 2c, the administration of the aqueous extract of Asplenium australasicum (J. Sm.) Hook, can effectively inhibit the RNA expression of the proinflammatory cytokines such as TNF-a, IL-Ιβ and IL-6. Moreover, the inhibition is dose-dependent.

[0029] Trial (C).

[0030] Referring to TABLE 3, the aqueous extract of Asplenium australasicum (J. Sm.) Hook, in different dosages are administrated to RAW264.7 cells of groups C2-C4 for 1 hour. LPS (1 μg/mL) is then administrated to RAW264.7 cells of groups C1-C4. 24 hours later, medium is collected from RAW264.7 cells of groups C0-C4. Relative protein levels of the proinflammatory cytokines such as TNF-a, IL-Ιβ and IL-6 are detected by ELISA, as shown in FIGS. 3a, 3b and 3c, respectively.

TABLE 3


[0031] Referring to FIGS. 3a, 3b and 3c, the administration of the aqueous extract of Asplenium australasicum (J. Sm.) Hook, can effectively inhibit the protein secretion of the proinflammatory cytokines such as TNF-a, IL-Ιβ and IL-6. Moreover, the inhibition is dose-dependent.

[0032] Accordingly, with the active substances obtained from the sample of Asplenium australasicum (J. Sm.) Hook., the aqueous extract of Asplenium australasicum (J. Sm.) Hook, according to the present invention shows ability of inhibiting secretion of the proinflammatory cytokines (TNF-a, IL-Ιβ, IL-6, etc.) induced by lipopolysaccharides, reducing the inflammatory response. Therefore, the aqueous extract of Asplenium australasicum (J. Sm.) Hook, can be applied to inhibiting inflammation caused by Gram-negative bacteria infection.

[0033] Although the invention has been described in detail with reference to its presently preferable embodiment, it will be understood by one of ordinary skill in the art that various modifications can be made without departing from the spirit and the scope of the invention, as set forth in the appended claims.