COMPOSITION COMPRISING A COMPLEX COMPRISING A CURCUMINOID COMPOUND, AND STEVIOL GLYCOSIDES OR A LICORICE EXTRACT OR A FRACTION THEREOF, AND USES THEREOF

Abstract

The present disclosure relates to a composition comprising a complex that includes a curcuminoid-based compound and a steviol glycoside, or a curcuminoid-based compound and a licorice extract or a fraction thereof; and a use of the composition for enhancement of immunity according to an enhancement of activity or a cell number of immune cells and/or a use of the composition for prevention, improvement, or treatment of COVID-19.

The complex including curcumin and a steviol glycoside, or a licorice extract or a fraction thereof according to the present disclosure, which are food materials that have been registered with the Ministry of Food and Drug Safety and have received the Generally Recognized as Safe (GRAS) rating from the U.S. FDA, and whose safety has already been confirmed, inhibits symptoms of COVID-19 by activating Th1 cells, CD8 T cells, and NK cells. Therefore, the complex can be provided as a food and pharmaceutical composition for preventing, improving, or treating COVID-19.

Claims

1. A method for preventing or treating COVID-19, the method comprising administering a pharmaceutical composition for preventing or treating COVID-19 comprising, as an active ingredient, a complex including a curcuminoid-based compound represented by the following Formula 1 or a pharmaceutically acceptable salt thereof; and a steviol glycoside or a pharmaceutically acceptable salt thereof, or a licorice extract or a fraction thereof to a subject: ##STR00003## wherein R.sup.1 and R.sup.2 are each independently hydrogen, a hydroxyl group, or a C.sub.1-10 alkoxy group, and n and m are 1≤n≤5 and 1≤m≤5.

2. The method of claim 1, wherein the curcuminoid-based compound represented by the following Formula 1 is a compound represented by any one of the following Formulas 2 to 4 or a mixture thereof: ##STR00004##

3. The method of claim 1, wherein the steviol glycoside is derived from Stevia rebaudiana Bertoni.

4. The method of claim 1, wherein the licorice is any one or more selected from the group consisting of Glycyrrhiza inflata BATALIN, Glycyrrhiza uralensis FISCHER, and Glycyrrhiza glabra LINNE.

5. The method of claim 1, wherein the composition is for oral administration.

6. A method for enhancing immunity, the method comprising administering a composition for enhancing immunity comprising, as an active ingredient, a complex including a curcuminoid-based compound represented by the following Formula 1 or a pharmaceutically acceptable salt thereof; and a steviol glycoside or a pharmaceutically acceptable salt thereof, or a licorice extract or a fraction thereof to a subject: ##STR00005## wherein R.sup.1 and R.sup.2 are each independently hydrogen, a hydroxyl group, or a C.sub.1-10 alkoxy group, and n and m are 1≤n≤5 and 1≤m≤5.

7. The method of claim 6, wherein the immunity enhancement is an enhancement of activity or a cell number of any one or more selected from the group consisting of Th1 cells, CD8+ T cells, and NK cells.

8. The method of claim 6, wherein the curcuminoid-based compound represented by Formula 1 above is a compound represented by any one of the following Formulas 2 to 4 or a mixture thereof: ##STR00006##

9. The method of claim 6, wherein the steviol glycoside is derived from Stevia rebaudiana Bertoni.

10. The method of claim 6, wherein the licorice is any one or more selected from the group consisting of Glycyrrhiza inflata BATALIN, Glycyrrhiza uralensis FISCHER, and Glycyrrhiza glabra LINNE.

11. The method of claim 6, wherein the composition is for oral administration.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0111] FIG. 1 shows an image illustrating a Tencumin S plus (TSP) complex containing water-solubilized curcumin and a steviol glycoside complex; water-solubilized curcumin; and a Tencumin G plus (TGP) complex containing water-solubilized curcumin and a licorice extract complex in this order, all before vacuum freeze-drying.

[0112] FIG. 2 shows a schematic diagram illustrating an experiment performed using an immunocompromised animal model.

[0113] FIG. 3 shows results confirming the ability of recovering CD4+ and CD8+ T cells by TSP administration in an immunocompromised animal model.

[0114] FIG. 4 shows results confirming the ability of recovering CD4+ and CD8+ T cells by TGP administration in an immunocompromised animal model.

[0115] FIG. 5 shows results confirming the ability of recovering Th1 and activated CD8+ T cells by TSP administration in an immunocompromised animal model.

[0116] FIG. 6 shows results confirming the ability of recovering Th1 and activated CD8+ T cells by TGP administration in an immunocompromised animal model.

[0117] FIG. 7 shows results confirming the ability of recovering multifunctional Th1 or multifunctional CD8+ T cells by TSP administration in an immunocompromised animal model.

[0118] FIG. 8 shows results confirming the ability of recovering multifunctional Th1 or multifunctional CD8+ T cells by TGP administration in an immunocompromised animal model.

[0119] FIG. 9 shows results confirming the ability of recovering activated NK cells by TSP administration in an immunocompromised animal model.

[0120] FIG. 10 shows results confirming the ability of recovering activated NK cells by TGP administration in an immunocompromised animal model.

[0121] FIG. 11 shows images illustrating lung tissue collected by autopsy for each test group after TSP administration.

[0122] FIG. 12 shows images illustrating lung tissue collected by autopsy for each test group after TGP administration.

[0123] FIG. 13 shows a graph illustrating the macroscopic cure rate of lung lesions according to TSP administration. Abbreviations: negative control (NC), virus control (VC), and positive control (PC).

[0124] FIG. 14 shows a graph illustrating the macroscopic cure rate of lung lesions according to TGP administration. Abbreviations: negative control (NC), virus control (VC), and positive control (PC).

DETAILED DESCRIPTION OF THE INVENTION

[0125] Hereinafter, the present disclosure will be described in detail through exemplary embodiments. However, these exemplary embodiments are for illustrative purposes of the present disclosure and are not intended to limit the scope of the present disclosure.

EXAMPLE 1: PREPARATION OF COMPLEX

[0126] In order to prepare a Tencumin S plus (hereinafter, “TSP”) complex containing water-solubilized curcumin and a steviol glycoside, 200 g of a steviol glycoside was dissolved in 1 L of water (20% concentration; [v/v]) and then 9 g of a turmeric pigment with a purity of about 95% or more produced in India (curcumin:demethoxycurcumin:bisdemethoxycurcumin=75:15:10 [w/w]) was added thereto. When the turmeric pigment was mixed with an aqueous solution of the steviol glycoside, about 30% to 35% of the turmeric pigment was dissolved, and the dissolved mixture was added into a microwave stirring extractor (Korean Patent No. 10-1436464), extracted with stirring at 12,000 W for 60 minutes, purified with a filter paper, and then vacuum freeze-dried to obtain TSP powder.

[0127] The TSP contained less than 200 g of the steviol glycoside and 3.0 g or more of the turmeric pigment per 203 g of the total weight of the powder.

[0128] In order to prepare a Tencumin G plus (hereinafter, “TGP”) complex containing water-solubilized curcumin and a licorice extract (Dodam Herb Chinese Medicien Products Co., Ltd., Korea; produced in Uzbekistan), 200 g of a steviol glycoside was dissolved in 1 L of water (20% concentration; [v/v]), and then 3 g of a turmeric pigment with a purity of about 95% or more produced in India (curcumin:demethoxycurcumin:bisdemethoxycurcumin=75:15:10 [w/w]) was added thereto. When the turmeric pigment was mixed with an aqueous solution of the licorice extract, about 30% to 35% of the turmeric pigment was dissolved, and the dissolved mixture was added into a microwave stirring extractor (Korean Patent No. 10-1436464), extracted with stirring at 12,000 W for 60 minutes, purified with a filter paper, and then vacuum freeze-dried to obtain TGP powder.

[0129] The TSP contained less than 200 g or less of the licorice extract and more than 1.0 g or more of the turmeric pigment per 201 g of the total weight of the powder.

[0130] The TSP, curcumin, and TGP before vacuum freeze-drying are as shown in FIG. 1.

EXAMPLE 2: EVALUATION OF ABILITY OF RECOVERING IMMUNE CELLS BY COMPLEXES IN IMMUNOCOMPROMISED ANIMAL MODEL

2-1. Confirmation of Ability of Recovering CD4+ and CD8+ T Cells

[0131] TSP (50 mg/kg and 100 mg/kg) or TGP (10 mg/kg and 50 mg/kg) prepared in Example 1 was orally administered to mice once a day for a total of 5 times. Three days thereafter, cyclophosphamide (CTX), which is an immunosuppressant that rapidly reduces the number of T cells in the spleen, was intraperitoneally injected once a day for a total of 5 times (FIG. 2). Three days after the CTX injection, the splenocytes of the mice were separated and treated with a T cell activator (including PMA/Ino; phorbol 12-myristate 13-acetate (PMA), ionomycin, brefeldin A, monensin, and a protein transport inhibitor) for 4 hours.

[0132] In order to measure the number of T cells, the splenocytes were treated with live/dead cell staining reagent, anti-CD3, anti-CD4, and anti-CD8 antibodies, left at room temperature for 20 minutes to be stained, washed several times with phosphate-buffered saline (PBS), and then analyzed by flow cytometry.

[0133] As a result, it was confirmed that when TSP (100 mg/kg) was administered 5 times before the CTX treatment, the number of CD4+ and CD8+ T cells decreased by CTX was recovered (FIG. 3).

[0134] Additionally, it was confirmed that when TGP (10 mg/kg or 50 mg/kg) was administered 5 times before the CTX treatment, the number of CD4+ and CD8+ T cells decreased by CTX was recovered (FIG. 4).

2-2. Confirmation of Ability of Recovering Th1 and Activated CD8+ T Cells

[0135] In order to measure the number of Th1 or activated CD8+ T cells in the same animal model as in Example 2-1 according to the administration of the complex (TSP or TGP) of Example 1, the splenocytes were treated with PMA/Ino for 4 hours, treated with live/dead cell staining reagent, anti-CD3, anti-CD4, anti-CD8, and anti-CD25 antibodies, left at room temperature for 20 minutes to be stained, and washed several times with PBS. While fixing the cell surfaces using the Fix & Perm Cell permeabilization kit, holes were made on the cell surfaces simultaneously, and the cells were left at room temperature for 20 minutes to be stained using anti-IFN-gamma, anti-IL-5, anti-IL-17A, and anti-Foxp3 antibodies, and then analyzed by flow cytometry.

[0136] As a result, it was confirmed that when CTX was intraperitoneally injected, all of the number of Th1 cells (IFN-gamma+CD3+CD4+ cells), Th2 cells (IL-5+CD3+CD4+ cells), Th17 cells (IL-17A+CD3+CD4+ cells), regulatory T cells (Foxp3+CD25+CD3+CD4+ cells), and activated CD8+ T cells (IFN-gamma+CD3+CD8+ cells) was reduced, whereas when TSP (100 mg/kg) was administered 5 times before the CTX treatment, the ratio of Th1 cells and activated CD8+ T cells decreased by CTX was recovered (FIG. 5).

[0137] Additionally, it was confirmed that when TGP (10 mg/kg or 50 mg/kg) was administered 5 times before the CTX treatment, the number of Th1 cells and activated CD8+ T cells decreased by CTX was recovered (FIG. 6).

2-3. Confirmation of Ability of Recovering Multifunctional Th1 and Multifunctional CD8+ T Cells

[0138] CD4+ or CD8+ T cells simultaneously induce IFN-gamma, TNF-alpha, and IL-2, which are Th1 cytokines, and are thus also referred to as multifunctional T cells (multifunctional Th1 or multifunctional CD8+ T cells, respectively).

[0139] In order to measure the number of multifunctional T cells according to the administration of the complex (TSP or TGP) of Example 1 in the same animal model as in Example 2-1, the splenocytes were treated with PMA/Ino for 4 hours, treated with live/dead cell staining reagent, anti-CD3, anti-CD4, and anti-CD8 antibodies, left at room temperature for 20 minutes to be stained, and washed several times with PBS. While fixing the cell surfaces using the Fix & Perm Cell permeabilization kit, holes were made on the cell surfaces simultaneously, and the cells were left at room temperature for 20 minutes to be stained using anti-IFN-gamma, anti-TN F-alpha, and anti-IL-2 antibodies, and then analyzed by flow cytometry.

[0140] As a result, it was confirmed that when CTX was intraperitoneally injected, the numbers of both multifunctional Th1 cells (CD3+CD4+ cells which simultaneously induce IFN-gamma, TN F-alpha, and IL-2) and multifunctional CD8+ T cells (CD3+CD8+ cells which simultaneously induce IFN-gamma, TN F-alpha, and IL-2) were reduced, whereas when TSP (100 mg/kg) was administered 5 times before the CTX treatment, the ratio of multifunctional Th1 or CD8+ T cells decreased by CTX was recovered (FIG. 7).

[0141] Additionally, it was confirmed that when TGP (50 mg/kg) was administered 5 times before the CTX treatment, the number of multifunctional Th1 cells or CD8+ T cells decreased by CTX was recovered (FIG. 8).

2-4. Confirmation of Ability of Recovering NK Cells

[0142] In order to measure the number of activated NK cells according to the administration of the complex (TSP or TGP) of Example 1 in the same animal model as in Example 2-1, the splenocytes were treated with PMA/Ino for 4 hours, treated with live/dead cell staining reagent, lineage antibodies (anti-CD19, anti-CD14, and anti-CD3e), and anti-NK1.1 antibodies, and the cells were left at room temperature for 20 minutes to be stained, and were washed several times with PBS. While fixing the cell surfaces using the Fix & Perm Cell permeabilization kit, holes were made on the cell surfaces simultaneously, and the cells were left at room temperature for 20 minutes to be stained using anti-IFN-gamma, anti-CD107a, and anti-Granzyme B antibodies, and then analyzed by flow cytometry.

[0143] As a result, it was confirmed that when TSP (50 mg/kg or 100 mg/kg) was administered 5 times before the CTX treatment, the number of activated NK cells (IFN-gamma+CD107a+Granzyme B+ NK cells) reduced by CTX was recovered (FIG. 9).

[0144] Additionally, it was confirmed that when TGP (10 mg/kg or 50 mg/kg) was administered 5 times before the CTX treatment, the number of NK cells decreased by CTX was recovered (FIG. 10).

[0145] According to the results above, it was confirmed that both TSP and TGP have an effect on recovering multifunctional Th1 cells, multifunctional CD8+ T cells, and activated NK cells, all of which are reduced by immunosuppressive agents.

EXAMPLE 3: EVALUATION OF DRUG EFFICACY IN COVID-19 ANIMAL MODEL

[0146] The therapeutic effect of the complex (TSP or TGP) of Example 1 on COVID-19 was confirmed using a Syrian hamster animal model infected with type 2 severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and exhibiting COVID-19 symptoms.

[0147] The animal model was prepared by administering COVID-19 according to the method shown in Table 1 below, and the negative control group (NC), the positive control virus group (VC), and the test groups administered with TSP were divided as shown in Table 2 below.

[0148] Specifically, respiratory anesthesia was performed for 10 minutes using isoflurane. After infecting the hamsters by instilling 100 μL of the prepared SARS-CoV-2 virus into the nasal cavity of the hamsters according to Table 2 below, the drug was orally administered (P.O.) at 10 mL/kg using an oral sonde for each test group.

TABLE-US-00001 TABLE 1 Hamsters Female, 5 n/Group Age 11 weeks old Virus Titer 2.0 × 10.sup.5 PFU/mL Inoculation Route intranasal (100 μL) Drug Administration per oral (P.O., 10 mL/kg)

TABLE-US-00002 TABLE 2 Test Group Group Dose Vehicle/Volume Route/Interval No. (n = 5) (mg/head) (mL/kg) (hours) Remarks 1 Negative 0 water/10 P.O./(24 — Control hours/a total (NC) of 4 times) 2 Virus 0 water/10 P.O./(24 h/a — Control total of 4 (VC) times) 3 TSP 218.75 water/10 P.O./simultaneous curcumin (water administration content solubilization) after infection (25 mg/kg/day) (24 hours/a total of 4 times) 4 TSP 39 water/10 P.O./simultaneous curcumin (water administration content solubilization) after infection (8.9 mg/kg/day) (24 hours/a total of 8 times) 5 TSP:Curcumin = 9:1 39 water/10 P.O./simultaneous curcumin (water administration content solubilization) after infection (8.9 mg/kg/day) (24 hours/a total of 8 times) 6 TGP 39 water/10 P.O./simultaneous curcumin (water administration content solubilization) after infection (3.0 mg/kg/day) (24 hours/a total of 8 times) 7 TGP:Curcumin = 9:1 39 water/10 P.O./simultaneous curcumin (water administration content solubilization) after infection (3.0 mg/kg/day) (24 hours/a total of 8 times)

[0149] On the 4th day after infection (PID4), macroscopic lung lesions were evaluated for lung tissues collected through autopsy from each test group (FIG. 11 and FIG. 12).

[0150] Specifically, each lung tissues were divided into (i) to (v) according to the lung lobe as shown in Table 3 below, and the improvement rate of the lung lesions was visually evaluated according to each weight, and the cure rate of the lung lesions was calculated by substituting the resultant into the following formula.

[0151] Macroscopic cure rate of lung lesion=pneumonia incidence rate in TSP group/pneumonia incidence rate in VC group×100

[0152] As a result, compared to the VC group infected with SARS-CoV-2 (a severe infection model, lung lesions (62%), an improvement rate of lung lesions (1%)), in TSP groups (TSP 78 and TSP-218.75, respectively), where water-soluble TSP (78 mg/head/day, curcumin content (8.9 mg/kg/day); Test Group 4 in Table 2) or water-soluble TSP (218.75 mg/head/day, curcumin content (25 mg/kg/day); Test Group 3 in Table 2) was administered for 4 consecutive days, the rate of improvement of lung lesions was shown to be 14.9% and 31.2%, respectively, without abnormal findings, and lung lesions were significantly improved. However, when a complex (TSP-C 78; TSP:water-insoluble curcumin=9:1 [w/w]) (78 mg/head/day, curcumin content (8.9 mg/kg/day); Test Group 5 in Table 2) was administered under the same conditions, and an improvement rate of 6.1% of lung lesions was confirmed (FIG. 13).

[0153] Additionally, in the TGP group (TGP 78), where water-soluble TSP (78 mg/head/day, curcumin content (3.0 mg/kg/day); Test Group 6 in Table 2) was administered for 4 consecutive days under the same conditions, the rate of improvement in lung lesions was 20.2% without abnormal findings, and thus a significant improvement was confirmed. However, when a complex additionally including water-insoluble curcumin (TGP-C 78; TGP:water-insoluble curcumin=9:1 [w/w]) (78 mg/head/day, curcumin content (3.0 mg/kg/day); Test Group 7 in Table 2) was administered under the same conditions, the efficacy was also significantly lowered, and an improvement rate of 15.3% of lung lesions was confirmed (FIG. 14).

[0154] In particular, TSP showed excellent effects of improving bilateral pneumonia, pulmonary edema, and pulmonary hemorrhage caused by SARS-CoV-2 infection.

[0155] According to the results above, it was confirmed that when water-insoluble curcumin was additionally added to the complex (TSP or TGP) of Example 1 and orally administered, the efficacy of improving lung lesions was significantly lowered.

[0156] From the results of Examples above, it was confirmed that the TSP (which contains water-solubilized curcumin and a steviol glycoside) or the TGP (which contains water-solubilized curcumin and a licorice extract) of the present disclosure enhances the activity and cell number of multifunctional Th1 cells, multifunctional CD8+ T cells, and activated NK cells and significantly improves lung lesions caused by SARS-CoV-2 infection.

[0157] In particular, curcumin and a steviol glycoside constituting the TSP, and a licorice extract constituting the TGP of the present disclosure are food materials that have been registered with the Ministry of Food and Drug Safety (Korea) and have received the Generally Recognized as Safe (GRAS) rating from the U.S. FDA, and their safety has already been confirmed. Therefore, these ingredients can be provided as a food and pharmaceutical composition for preventing, improving, or treating COVID-19.

[0158] From the foregoing, a skilled person in the art to which the present disclosure pertains will be able to understand that the present disclosure may be embodied in other specific forms without modifying the technical concepts or essential characteristics of the present disclosure. In this regard, the exemplary embodiments disclosed herein are only for illustrative purposes and should not be construed as limiting the scope of the present disclosure. On the contrary, the present disclosure is intended to cover not only the exemplary embodiments but also various alternatives, modifications, equivalents, and other embodiments that may be included within the spirit and scope of the present disclosure as defined by the appended claims.