COMPOSITION FOR TREATMENT AND PREVENTION OF INFLAMMATORY DISEASES COMPRISING COMPOSITE EXTRACT OF BEET, RED ONION AND PURPLE SWEET POTATO AS ACTIVE INGREDIENTS

Abstract

A method for treating or suppressing inflammatory disease includes administering a composition to a subject in need thereof. The composition includes a composite extract of beet, red onion, and purple sweet potato as an active ingredient. The composition has no cytotoxicity, effectively inhibits NO-mediated inflammatory reactions, inhibits the production of IL-6 protein, and inhibits the expression of iNOS COX-2 genes, which are representative inflammation-related genes, thus having an excellent anti-inflammatory effect, and so it can be usefully used for the treatment and prevention of inflammatory diseases in cosmetics, food, and quasi-drug fields. In addition, the composition of the present invention is not only very safe to human body but also excellent in stability.

Claims

1: A method for treating or suppressing inflammatory disease, the method comprising administering to a subject in need thereof a composition comprising a composite extract of beet, red onion, and purple sweet potato as an active ingredient.

2: The method of claim 1, wherein the inflammatory disease is nitrogen monoxide (NO)-mediated inflammatory disease.

3: A method for inhibiting production of IL-6 (interleukin-6) protein and expression of inducible NO synthase (iNOS) and COX-2 genes in a subject, the method comprising administering to a subject in need thereof.

4: The method of claim 1, wherein the beet, red onion, and purple sweet potato are mixed at a weight ratio of 1:0.5-1.5:0.5-1.5.

5. (canceled)

6: The method of claim 1, wherein the composition is a functional food composition.

7. (canceled)

8: The method of claim 1, wherein the composition is a pharmaceutical composition.

Description

DETAILED DESCRIPTION OF THE DRAWINGS

[0052] FIG. 1 is a graph showing the analysis of cell proliferation of human keratinocytes (HaCaT) according to the composite extract according to the treatment of beet, red onion, and purple sweet potato of the present invention.

[0053] FIG. 2 is a graph showing the measurement of the change of the NO production in the mouse macrophage cell line (RAW264.7) according to the treatment of the composite extract of beet, red onion, and purple sweet potato of the present invention.

[0054] FIG. 3 is a graph showing the measurement of the change of the IL-6 protein production in human keratinocytes (HaCaT) according to the treatment of the composite extract of beet, red onion, and purple sweet potato of the present invention.

[0055] FIG. 4 is a diagram showing the results of measuring the change of the expression of iNOS and COX-2 genes according to the treatment of the composite extract of beet, red onion, and purple sweet potato of the present invention.

DETAILED DESCRIPTION

[0056] Hereinafter, the present invention will be described in more detail through Examples. Since these Examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Preparation of Extract of Beet, Red Onion, and Purple Sweet Potato

[0057] After pulverizing 100 g of dried beet, 3 L of 50% ethanol was used as a solvent and the resulting mixture was subject to heated reflux extraction for 12 hours and cold immersion, and then filtered through a filter paper having a 1.2 μm permeation size to obtain a filtrate. The obtained filtrate was concentrated under reduced pressure and dried under reduced pressure to prepare dried beet powder (Preparation Example 1).

[0058] Dried powder of red onion (Preparation Example 2) and dried powder of purple sweet potato (Preparation Example 3) were prepared by the same method as the preparation of the beet extract.

[0059] In addition, the powder of the beet, red onion, and purple sweet potato was mixed at a weight ratio of 1:1:1 to prepare a composite extract thereof (Preparation Example 4).

Example 2. Cytotoxicity Experiment

[0060] The effect of the beet, red onion, and purple sweet potato extract obtained in Example 1 on the growth of human keratinocytes (HaCaT) was confirmed by the method described below.

[0061] First, human keratinocytes were inoculated at a concentration of 1×10.sup.5 in a 24-well cell culture dish in Dulbeccos modified Eagles medium (DMEM), which is an exclusive medium containing 10% fetal bovine serum (FBS, Cambrex), and then incubated at 37° C. under wet conditions with 5% CO.sub.2 for 24 hours. Thereafter, the medium was removed, and then the cells were treated with the single extracts of beet, red onion, and purple sweet potato of Example 1 diluted with serum-free DMEM medium and the composite extract thereof (weight ratio 1:1:1) at different concentrations (1, 10, and 100 μg/mL) and incubated for 24 hours. After 24 hours, the cells were treated with 1 mg/mL of MTT was treated and after 2 hours, formazan produced by the MTT treatment with the cells was dissolved in DMSO to measure light absorbance at 570 nm.

[0062] As a result, it was confirmed that the single extracts of beet, red onion, and purple sweet potato of Example 1 and the composite extract thereof did not significantly affect the cell viability of human keratinocytes; rather it was shown that the composite extraction of beet, red onion, and purple sweet potato promoted the survival of human keratinocytes at concentrations of 1, 10, and 100 μg/mL. The results described above confirmed that the single and composite extracts of beet, red onion, and purple sweet potato did not show toxicity to human body (see FIG. 1).

Example 3. Inhibition of NO Production

[0063] The change of NO production was measured to measure the anti-inflammatory effect of the single extracts of beet, red onion, and purple sweet potato and the composite extracts thereof. NO is a representative cytotoxic substance involved in causing inflammation.

[0064] After preparing RAW264.7 cells, which are mouse macrophages, LPS (1 μg/mL) was treated as a cell activity and inflammatory factor in all groups except the normal group, and after culturing for 24 hours, the cell culture solution was collected.

[0065] The NO donor and the Griess reagent were treated together, and the NO appearing over time was measured to quantify the NO inhibitory capacity.

[0066] In the experiment, to quantify the amount of NO observed at a specified time, the cells were treated with the single extracts of beet, red onion, and purple sweet potato and the composite extract thereof (mixed at a weight ratio of 1:1:1) at concentrations of 1, 10, and 100 ug/mL and incubated at 37° C. for 2 hours, and then the Griess reagent, a reagent for detecting NO, was mixed to measure the light absorbance at 540 nm

[0067] As a result, it was found that the production of NO induced by LPS was inhibited in a concentration-dependent manner in the groups to which the composite extract comprising the 3 ingredients of beet, red onion, and purple sweet potato was added, and the production of NO was inhibited up to 53% at concentration of 100 ug/mL, compared with the untreated group. On the contrary, in the groups treated with single extracts of beet, red onion, and purple sweet potato, the NO production inhibitory activity was negligible. Compared with the groups treated with the single extracts of beet, red onion, and purple sweet potato, the group treated with the 3-ingredient composite extract thereof showed 51%, 57%, and 50% improved NO inhibitory activity, respectively.

[0068] Through the experimental results described above, it was confirmed that the 3-ingredient composite extract of beet, red onion, and purple sweet potato of the present invention has a synergic effect of concentration-dependently inhibiting the production of NO, which mediates an inflammatory reaction (FIG. 2).

Example 4. Inhibition of IL-6 Protein Expression

[0069] In the inflammatory response mechanism caused by cell damage, inflammatory mediators such as IL-6 are involved, the inflammatory response is amplified due to the action of the inflammatory mediators, and when the expression of IL-6 is inhibited, the inflammatory response can be effectively blocked.

[0070] To verify the effect of the composite extract of beet, red onion, and purple sweet potato on the expression of IL-6 protein, an experiment was performed on human keratinocytes (HaCaT, Human immortalized keratinocyte). The HaCaT cells were inoculated in a 96-well plate at a concentration of 1.0×10.sup.5, and incubated at 37° C. under wet conditions with 5% CO.sub.2 for 24 hours. Thereafter, after washing the cells with PBS solution, 1 μg/ml of LPS was treated in each well either alone or with samples of various concentrations, and then incubated for 24 hours. In the normal control group, only the medium was treated.

[0071] After the incubation is finished, the supernatant was taken and the amount of the produced IL-6 protein was analyzed by using an enzyme-linked immunosorbent assay (ELISA) kit.

[0072] As a result, it was found that the production of IL-6 protein induced by LPS was inhibited in a concentration-dependent manner in the groups to which the 3-ingredient composite extract of beet, red onion, and purple sweet potato was added, and the production of IL-6 protein was inhibited up to 58% at concentration of 100 ug/mL, compared with the untreated group. On the contrary, in the groups treated with single extracts of beet, red onion, and purple sweet potato, the IL-6 protein production inhibitory activity was negligible. Compared with the groups treated with the single extracts of beet, red onion, and purple sweet potato, the group treated with the 3-ingredient composite extract showed 50%, 60%, and 55% improved NO IL-6 protein production inhibitory activity, respectively.

[0073] Through the results described above, it was confirmed that the composite extract of beet, red onion, and purple sweet potato exhibited a significantly improved IL-6 protein production inhibitory effect, compared to the groups treated with the single extracts thereof, and had a synergic effect was due to the combination of beet, red onion, and purple sweet potato.

Example 5. Effect of Controlling Inflammation-Inducing Factors

[0074] With the composite extract of beet, red onion, and purple sweet potato, which was found to have the best inhibitory effect on the production of NO and the production of IL-6 protein in Examples 3 and 4, the effect of regulating gene expression was verified with respect to iNOS and COX-2 genes, which are inflammation-causing factors.

[0075] First, human keratinocytes (HaCaT) were inoculated at a concentration of 1×10.sup.5 in a 24-well cell culture dish in Dulbeccos modified Eagles medium (DMEM), which is an exclusive medium containing 10% fetal bovine serum (FBS, Cambrex), and then incubated at 37° C. under wet conditions with 5% CO.sub.2 for 24 hours. Thereafter, the medium was removed, and then the cells were treated with samples prepared by diluting the composite extract of beet, red onion, and purple sweet potato (weight ratio 1:1:1) of Example 1 with serum-free DMEM medium at concentrations of 10 and 100 μg/mL and at the same time, with a new medium containing LPS (μg/mL), which is known as an endotoxin.

[0076] The experiment was performed by the RT-PCR method to confirm the mRNA expression levels of iNOS and COX-2. The used primers are described below: [0077] iNOS: Forward 5′-AAT GGC AAC ATC AGG TCG GCC ATC ACT-3′(SEQ ID NO:1), Reverse 5′-GCT GTG TGT CAC AGA AGT CTC GAA CTC-3′(SEQ ID NO:2) [0078] COX-2: Forward 5′-TGC TGG TGG AAA AAC CTC GT-3′ (SEQ ID NO:3), Reverse 5′-AAA ACC CAC TTC GCC TCC AA-3′(SEQ ID NO:4) [0079] GAPDH: Forward 5′-TGA AGG TCG GTG TGA ACG GAT TTT GGC-3′ (SEQ ID NO:5), Reverse 5′-TGG TTC ACA CCC ATC ACA AAC ATG G-3′(SEQ ID NO:6).

[0080] RT-PCR was performed by using the primers described above, and the results are summarized in FIG. 4.

[0081] As a result, with regard to the effect of the composite extract of beet, red onion, and purple sweet potato for inhibiting iNOS and COX-2 expression, compared with the negative control group, the iNOS expression inhibitory effect was improved by 57% and 61% at the concentrations of 10 and 100 μg/mL, respectively, and the COX-2 expression inhibitory effect was improved by 25% and 28%, respectively (see FIG. 4).

[0082] Through the results described above, it was confirmed that the composition for the treatment and prevention of inflammatory diseases, the composition comprising the composite extract of beet, red onion, and purple sweet potato of the present invention, has no cytotoxicity, and directly inhibits the production of NO and IL-6 protein, and inhibits the expression of iNOS and IL-6 genes, thus having an effect of treating and preventing inflammatory diseases.

[0083] Since the specific parts of the present invention have been described above in detail, it is clear that for those skilled in the art, this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereto.

[0084] Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

[0085] A sequence listing electronically submitted with the present application on Sep. 19, 2022 as an ASCII text file named 20220919_Q90522YG03_TU_SEQ.TXT, created on Sep. 19, 2022 and having a size of 1,206 bytes, is incorporated herein by reference in its entirety.