MODIFIED TAURINE AND PREPARATION METHOD THEREFOR
20190152905 ยท 2019-05-23
Inventors
Cpc classification
C07C303/42
CHEMISTRY; METALLURGY
A61K31/7004
HUMAN NECESSITIES
C07C309/14
CHEMISTRY; METALLURGY
A61K31/185
HUMAN NECESSITIES
International classification
C07C309/14
CHEMISTRY; METALLURGY
A61K31/185
HUMAN NECESSITIES
Abstract
The present invention relates to modified taurine and a method for preparing the same and, more particularly, to modified taurine different from conventional taurine in a distance between an intramolecular carbon (C) atom and a sulfur (S) atom adjacent thereto, and a preparation method therefor. Superior in terms of prophylactic and therapeutic effect on metabolic diseases, the modified taurine of the present invention is expected to find a wide spectrum of applications in the medical field.
Claims
1. A modified taurine, which has an interatomic distance between carbon (C) and sulfur (S) of 1.7730 to 1.7779(), an average interatomic distance between sulfur (S) and three oxygen atoms (O) of 1.452 to 1.462(), and a maximum interatomic distance between sulfur (S) and three oxygen atoms (O) of 1.458 to 1.468().
2. The modified taurine of claim 1, wherein the modified taurine has 891/847, 1182/1256 and 1427/1458 absorption band intensity ratio at the position 847, 891, 1182, 1256, 1427 and 1458 cm.sup.1 in Raman spectrum are less than 1.
3. The modified taurine of claim 1, having an onset point of melting at 330 to 340 C.
4. The modified taurine of claim 1, having a solubility of 75 to 79 g/L.
5. The modified taurine of claim 1, having a maximum density of 1.74 to 1.76g/cm.sup.3.
6. The modified taurine of claim 1, having an absorption wavelength of FT-IR at the position 1650 to 2800 cm.sup.1 is different from unmodified taurine.
7. The modified taurine of claim 1, produced by dissolving taurine with heating in a first polar solvent, adding a second polar solvent thereto to form a semi-solid material, and removing the solvents to obtain the modified taurine in the semi-solid material, wherein a difference in polarity between the first polar solvent and the second polar solvent is 5 or less.
8. The modified taurine of claim 7, wherein the first polar solvent is water and is used in an amount of 1 to 20 times of amount of water in an aqueous solution saturated with the taurine.
9. The modified taurine of claim 1, wherein the modified taurine has monoclinic crystal system and P2(1)/c of space group.
10. A method of treating obesity, diabetes, or thrombotic diseases, comprising administering to a patient in need thereof a pharmaceutical composition comprising the modified taurine according to claim 1, and one or more selected from the group consisting of sugar, polyphenol, and amino acid.
11. The method of claim 10, wherein the modified taurine has an onset point of melting at 330 to 340 C.
12. The method of claim 10, wherein the modified taurine has a solubility of 75 to 79 g/L.
13. The method of claim 10, wherein the modified taurine has a maximum density of 1.74 to 1.76g/cm.sup.3.
14. The method of claim 10, wherein the modified taurine has monoclinic crystal system and P2(1)/c of space group.
15. The method of claim 10, wherein the sugar is at least one selected from the group consisting of xylose, arabinose, ribose, glucose, mannose, and fructose.
16. The method of claim 10, wherein the polyphenol is at least one selected from the group catechin and epigallocatechin gallate.
17. The method of claim 10, wherein the amino acid is betaine.
18. The method of claim 10, wherein the sugar is included in an amount of 0.1 to 2.0 parts by weight relative to the modified taurine.
19. The method of claim 10, wherein the amino acid is included in an amount of 0.1 to 0.5 parts by weight relative to the modified taurine.
20. A composition comprising the modified taurine according to claim 1, and one or more selected from the group consisting of sugar, polyphenol, and amino acid.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE EMBODIMENTS
[0054] Hereinafter, the present invention will be described in further detail. It will be obvious to those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
[0055] Design of Compositions
[0056] Prior to experiments, the components and component ratios of examples and comparative examples to be used in each experiment were designed. The results of the design are shown in Table 1 below. Specific methods for preparation of each component and composition are described in Examples 1 and 2 and Comparative Examples 1 and 2.
TABLE-US-00001 TABLE 1 Example 1-2 TauAlc DT15 Example 2-1 TauAlc 8.6* + Ara 2.5 DT16 TauAlc 8.6 + Xyl 3.5 DT20 Example 2-2-1 TauAlc 8.6 + Ara 1.04 TauAlc 8.6 + Ara 5.2 TauAlc 8.6 + Ara 7.8 TauAlc 8.6 + Xyl 1.04 TauAlc 8.6 + Xyl 5.2 TauAlc 8.6 + Xyl 7.8 TauAlc 4.3 + Rib 2.6 TauAlc 4.3 + Rib 6.5 Example 2-2-2 TauAlc 8.6 + Ara 1.04 TauAlc 8.6 + Ara 5.2 TauAlc 8.6 + Ara 7.8 TauAlc 8.6 + Xyl 1.04 TauAlc 8.6 + Xyl 5.2 TauAlc 8.6 + Xyl 7.8 Example 2-3 TauAlc 4.3 + Glu 3.1 TauAlc 4.3 + Glu 7.75 TauAlc 4.3 + Mann 3.1 TauAlc 4.3 + Mann 7.75 TauAlc 4.3 + Fruc 7.75 Example 2-4-1 TauAlc 8.6 + Cat 3 + Bet 4 DT7 Example 2-4-2 TauAlc 8.6 + EGCG 1.5 + Bet 4 DT10 Example 2-5 TauAlc 8.6 + EGCG 1.5 + Bet DT4 4 + Xyl 3.5 Comparative Tau** DT19 Example 1 Comparative Ara 1.04 Example 2-1 Ara 5.2 Ara 7.8 Xyl 1.04 Xyl 5.2 Xyl 7.8 Rib 5.2 Rib 10.4 Comparative Glu 6.2 Example 2-2 Glu 12.4 Mann 3.1 Mann 6.2 Mann 12.4 Fruc 6.2 Fruc 12.4 Comparative Tau 8.6 + Ara 1.04 Example 2-3-1 Tau 8.6 + Ara 5.2 Tau 8.6 + Ara 7.8 Tau 8.6 + Xyl 1.04 Tau 8.6 + Xyl 5.2 Tau 8.6 + Xyl 7.8 Tau 4.3 + Rib 2.6 Tau 4.3 + Rib 6.5 Comparative Tau 8.6 + Ara 2.5 DT18 Example 2-3-2 Tau 8.6 + Xyl 3.5 DT21 Comparative Tau 4.3 + Glu 3.1 Example 2-4 Tau 4.3 + Glu 7.75 Tau 4.3 + Mann 3.1 Tau 4.3 + Mann 7.75 Tau 4.3 + Fruc 7.75 Comparative Tau 8.6 + Cat 3 + Bet 4 DT11 Example 2-5-1 Comparative Tau 8.6 + EGCG 1.5 + Bet 4 DT14 Example 2-5-2 Comparative Tau 8.6 + EGCG 1.5 + Bet DT6 Example 2-6 4 + Xyl 3.5 *TauAlc 8.6 means 8.6 g of modified taurine. **Tau is conventional taurine.
[0057] In the above table, Tau: taurine, TauAlc: modified taurine, Ara: arabinose, Xyl: xylose, Rib: ribose, Glu: glucose, Mann: mannose, Fruc: fructose, Cat: catechin, Bet: betaine, EGCG: epigallocatechin gallate.
Example 1: Preparation of Modified Taurine
[0058] 1-1: Preparation of Composition Containing Modified Taurine
[0059] Taurine (8.6 g) was added to 30 ml of purified water and dissolved by heating in a microwave oven for about 60 seconds, and then immediately, the solution was stirred with a rod while 60 ml of alcohol (ethanol (ethanol was used hereinafter), methanol, propanol or butanol) or acetone was added thereto, thereby preparing compositions (
[0060] 1-2: Preparation of Modified Taurine Crystal
[0061] (1) Taurine (1.72 g) was added to 6 ml of purified water, or (2) taurine (4.3 g) was added to 15 ml of purified water, or (3) taurine (8.6 g) was added to 28 ml of purified water. The added taurine was dissolved by heating in a microwave oven for 20-60 seconds, and then immediately, the taurine solution was stirred with a rod while 12 ml of ethanol (for (1) above), 30 ml of ethanol (for (2) above) or 60 ml of ethanol (for (3) above) was added at room temperature, thereby preparing mixtures comprising a formed semi-solid material.
[0062] Next, 40 ml of purified water or 32 ml of purified water (for (3) above), heated to about 100 C., was added to each of the prepared mixtures, and then heated in a microwave oven for about 3-5 minutes until the alcohol was removed, thereby preparing compositions.
[0063] A solid taurine crystal was prepared according to the case of (3) above, and then dried in a hot-air dryer. (See Table 1 above for the specific component ratio of each composition).
Example 2: Preparation of Pharmaceutical Compositions for Prevention or Treatment of Metabolic Disease
[0064] 2-1: Preparation 1 of Modified Taurine+Pentose Composition
[0065] A predetermined amount (2.5 g or 3.5 g) of pentose (xylose or arabinose) was added to 32 ml of purified water and completely dissolved by heating in a microwave oven for 30 seconds. Meanwhile, taurine (8.6 g) was added to 28 ml of purified water and dissolved by heating in a microwave oven for 60 seconds, and then immediately, the taurine solution was stirred with a rod while 60 ml of ethanol was added, thereby preparing a mixture comprising a formed white semi-solid material. Next, each of the aqueous pentose solutions prepared as described above was added to the mixture, and then heated in a microwave oven for about 5 minutes until the alcohol was completely removed, thereby preparing compositions. (See Table 1 above for the specific component ratio of each composition).
[0066] 2-2-1: Preparation 2 of Modified Taurine+Pentose Composition
[0067] A predetermined amount (1.04 g, 2.6 g, 5.2 g, 6.5 g or 7.8 g) of pentose (xylose, arabinose or ribose) was added to 40 ml of purified water and completely dissolved by heating in a microwave oven for about 30-50 seconds. Meanwhile, (1) taurine (4.3 g) was added to 15 ml of purified water, or (2) taurine (8.6 g) was added to 30 ml of purified water, and the added taurine was dissolved by heating in a microwave oven for 40-60 seconds, and then immediately, the taurine solution was stirred with a rod while 30 ml (for (1) above) or 60 ml (for (2) above) of ethanol was added thereto, thereby preparing mixtures comprising a formed white semi-solid material. Next, the aqueous pentose solution prepared as described above was added to the mixture, and then heated in a microwave oven for about 3-5 minutes until the alcohol was completely removed. Purified water was added to the mixture to a total volume of 100 ml before use in experiments. (See Table 1 above for the specific component ratio of each composition).
[0068] 2-2-2: Preparation 3 of Modified Taurine+Pentose Composition
[0069] 40 ml of purified water was boiled by heating in a microwave oven for about 30 seconds. Then, taurine (8.6 g) was added to 30 ml of purified water and dissolved by heating in a microwave oven for about 60 seconds, and then immediately, the taurine solution was stirred with a rod while 60 ml of ethanol was added thereto, thereby preparing a mixture comprising a formed white semi-solid material. Next, 40 ml of the boiled purified water prepared as described above was added to the mixture, and then heated in a microwave oven at 100 C. for about 5 minutes until the alcohol was completely removed, thereby preparing a composition.
[0070] Furthermore, a predetermined amount (1.04 g, 5.2 g or 7.8 g) of pentose (xylose or arabinose) was added to 40 ml of purified water and completely dissolved by heating in a microwave oven for about 30 seconds. To the solution, the composition prepared as described above was added, and the mixture was heated in a microwave oven for about 30 seconds, after which purified water was added to the mixture to a total volume of 100 ml before use in experiments. (See Table 1 above for the specific component ratio of each composition).
[0071] 2-3: Preparation of Modified Taurine+Hexose Composition
[0072] A predetermined amount (3.1 g or 7.75 g) of hexose (mannose, glucose or fructose) was added to 40 ml of purified water and completely dissolved by heating in a microwave oven for about 50 seconds. Meanwhile, taurine (4.3 g) was added to 15 ml of purified water and dissolved by heating in a microwave oven for about 40 seconds, and then immediately, the taurine solution was stirred with a rod while 30 ml of ethanol was added thereto, thereby preparing a mixture comprising a formed white semi-solid material. Next, the aqueous hexose solution prepared as described above was added to the mixture, and then heated in a microwave oven for about 3 minutes and 30 seconds until the alcohol was completely removed, after which purified water was added to the mixture to a total volume of 100 ml before use in experiments. (See Table 1 above for the specific component ratio of each composition).
[0073] 2-4-1: Preparation 1 of Modified Taurine+Polyphenol+Amino Acid Composition
[0074] Taurine (8.6 g) was added to 30 ml of purified water and completely dissolved by heating in a microwave oven for about 1 minute and 20 second, and then immediately, the taurine solution was stirred with a rod while 60 ml of ethanol was added thereto, thereby preparing a mixture comprising a formed white semi-solid material. Next, 40 ml of purified water was heated to about 100 C. in a microwave oven for 1 minute, and catechin (3 g) and betaine (4 g) were added thereto and dissolved, after which the solution was added to the mixture, and then heated in a microwave oven for 5 minutes to remove the ethanol. (See Table 1 above for the specific component ratio of each composition).
[0075] 2-4-2: Preparation 2 of Modified Taurine+Polyphenol+Amino Acid Composition
[0076] Taurine (8.6 g) was added to 30 ml of purified water and completely dissolved by heating in a microwave oven for about 1 minute and 20 second, and then immediately, the taurine solution was stirred with a rod while 60 ml of ethanol was added thereto, thereby preparing a mixture comprising a formed white semi-solid material. Meanwhile, 40 ml of purified water was heated to about 100 C. in a microwave oven for 1 minute, and EGCG (1.5 g) and betaine (4 g) were added thereto and dissolved, after which the solution was added to the mixture, and then heated in a microwave oven for 5 minutes to remove the ethanol. (See Table 1 above for the specific component ratio of each composition).
[0077] 2-5: Preparation of Modified Taurine+Polyphenol+Amino Acid Composition+Pentose Composition
[0078] Taurine (8.6 g) was added to 30 ml of purified water and completely dissolved by heating in a microwave oven for about 1 minute and 20 second, and then immediately, the taurine solution was stirred with a rod while 60 ml of ethanol was added thereto, thereby preparing a mixture comprising a formed white semi-solid material. Meanwhile, 40 ml of purified water was heated to about 100 C. in a microwave oven for 1 minute, and EGCG (1.5 g), betaine (4 g) and xylose (3.5 g) were added thereto and dissolved, after which the solution was added to the mixture, and then heated in a microwave oven for 5 minutes to remove the ethanol. (See Table 1 above for the specific component ratio of each composition).
Comparative Example 1: Preparation of Aqueous Taurine Solution
[0079] 1-1: Preparation 1 of Aqueous Taurine Solution
[0080] 60 ml of taurine was heated in a microwave oven for about 1 minute, and taurine (8.6 g) was added and dissolved, and then heated in a microwave oven for 3 minutes.
[0081] 1-2: Preparation 2 of Aqueous Taurine Solution
[0082] 40 ml of purified water was boiled by heating in a microwave oven for about 30 seconds. Meanwhile, taurine (1.72 g, 4.3 g or 8.6 g) was added to 30 ml of purified water and dissolved by heating to 100 C. in a microwave oven for 1 minute. The taurine solution was added to 40 ml of the boiled purified water prepared as described above, and was heated to boiling in a microwave oven for about 2 minutes, after which purified water was added to the mixture to a total volume of 100 ml before use in experiments. (See Table 1 above for the specific component ratio of each composition).
Comparative Example 2: Preparation of Pharmaceutical Compositions for Prevention or Treatment of Metabolic Disease
[0083] 2-1: Preparation of Pentose Composition
[0084] A predetermined amount (1.04 g, 5.2 g, 7.8 g or 10.4 g) of pentose (xylose, arabinose or ribose) was added to 40 ml of purified water and completely dissolved by heating in a microwave oven for 30 seconds, after which 30 ml of fresh purified water boiled in a microwave oven for about 30 seconds was added thereto. The pentose solution was heated to boiling in a microwave oven for about 3-4 minutes, after which purified water was added thereto to a total volume of 100 ml before use in experiments. (See Table 1 above for the specific component ratio of each composition).
[0085] 2-2: Preparation of Hexose Composition
[0086] A predetermined amount (3.1 g, 6.2 g or 12.4 g) of hexose (mannose, glucose or fructose) was added to 40 ml of purified water and completely dissolved by heating in a microwave oven for about 50 seconds, after which 30 ml of fresh purified water boiled in a microwave oven for about 30 seconds was added thereto. The hexose solution was heated to boiling in a microwave oven for about 2-3 minutes, after which purified water was added thereto to a total volume of 100 ml before use in experiments. (See Table 1 above for the specific component ratio of each composition).
[0087] 2-3-1: Preparation 1 of Taurine+Pentose Composition
[0088] A predetermined amount (1.04 g, 2.6 g, 5.2 g, 6.5 g or 7.8 g) of pentose (xylose, arabinose or ribose) was added to 40 ml of purified water and completely dissolved by heating in a microwave oven for about 390-50 seconds. Meanwhile, (1) taurine (4.3 g) was added to 15 ml of purified water, or (2) taurine (8.6 g) was added to 30 ml of purified water, and the added taurine was dissolved by heating in a microwave oven for 40-60 seconds, after which the pentose solution prepared as described above was added thereto. The mixture was heated to boiling in a microwave oven for about 2-4 minutes, after which purified water was added thereto to a total volume of 100 ml before use in experiments. (See Table 1 above for the specific component ratio of each composition).
[0089] 2-3-2: Preparation 2 of Taurine+Pentose Composition
[0090] 60 ml of purified water was heated to about 100 C. in a microwave oven for 1 minute, and taurine (8.6 g) and each of arabinose (2.5 g) and xylose (3.5 g) was added thereto and completely dissolved, followed by heating in a microwave oven for 3 minutes. (See Table 1 above for the specific component ratio of each composition).
[0091] 2-4: Preparation of Taurine+Hexose Composition
[0092] A predetermined amount (3.1 g or 7.75 g) of hexose (mannose, glucose or fructose) was added to 40 ml of purified water and completely dissolved by heating in a microwave oven for about 50 seconds. Meanwhile, taurine (4.3 g) was added to 15 ml of purified water and dissolved by heating in a microwave oven for 40 seconds, and then the hexose solution prepared as described above was added thereto. The mixture was heated to boiling in a microwave oven for about 2 minutes, after which purified water was added thereto to a total volume of 100 ml before use in experiments. (See Table 1 above for the specific component ratio of each composition).
[0093] 2-5-1: Preparation 1 of Taurine+Polyphenol+Amino Acid Composition
[0094] Taurine (8.6 g) was added to 30 ml of purified water and completely dissolved by heating in a microwave oven for 1 minute and 20 seconds to thereby prepare an aqueous taurine solution. Meanwhile, 40 ml of purified water was heated to about 100 C. in a microwave oven for 1 minute, and catechin (3 g) and betaine (4 g) were added thereto and dissolved, after which the solution was added to the aqueous taurine solution and heated in a microwave oven for about 4 minutes. (See Table 1 above for the specific component ratio of each composition).
[0095] 2-5-2: Preparation 2 of Taurine+Polyphenol+Amino Acid Composition
[0096] Taurine (8.6 g) was added to 30 ml of purified water and completely dissolved by heating in a microwave oven for 1 minute and 20 seconds to thereby prepare an aqueous taurine solution. Meanwhile, 40 ml of purified water was heated to about 100 C. in a microwave oven for 1 minute, and EGCG (1.5 g) and betaine (4 g) were added thereto and dissolved, after which the solution was added to the aqueous taurine solution and heated in a microwave oven for about 4 minutes. (See Table 1 above for the specific component ratio of each composition).
[0097] 2-6: Preparation of Taurine+Polyphenol+Amino Acid+Pentose
[0098] Taurine (8.6 g) was added to 30 ml of purified water and completely dissolved by heating in a microwave oven for 1 minute and 20 seconds to thereby prepare an aqueous taurine solution. Meanwhile, 40 ml of purified water was heated to about 100 C. in a microwave oven for 1 minute, and EGCG (1.5 g), betaine (4 g) and xylose (3.5 g) were added thereto and dissolved, after which the solution was added to the aqueous taurine solution and heated in a microwave oven for about 4 minutes. (See Table 1 above for the specific component ratio of each composition).
Example 3: Analysis of Properties of Modified Taurine
Experimental Example 1: Single Crystal XRD Analysis
Experimental Example 1-1: Single Crystal Measurement at 100K
[0099] Single crystal X-ray analysis was performed by using XRD equipment (Bruker SMART APEX II X-ray Diffractometer) with Mo tube, graphite-monochromator and CCD area-detector on 50 KV, 40 mA, and 100K conditions, and the structural analysis was performed with Bruker SHELXTL software. The above-mentioned specific performance conditions are shown in Table 2 below.
TABLE-US-00002 TABLE 2 Identification code 201412241t_0m Empirical formula C2 H7 N O3 S Formula weight 125.15 Temperature 100(1) K Wavelength 0.71073 Crystal system Monoclinic Space group P2(1)/c Unit cell dimensions a = 5.2607(2) = 90 b = 11.6303(4) = 94.015(2) c = 7.7903(3) = 90 Volume 475.47(3) .sup.3 Z 4 Density (calculated) 1.748 Mg/m.sup.3 Absorption coefficient 0.569 mm.sup.1 F(000) 264 Crystal size 0.30 0.06 0.04 mm.sup.3 Theta range for data collection 3.15 to 28.36. Index ranges 6 <= h <= 6, 0 <= k <= 15, 0 <= l <= 10 Reflections collected 1164 Independent reflections 1164 [R(int) = 0.0000] Completeness to theta = 28.36 98.2% Absorption correction Multi-scan Max. and min. transmission 0.9776 and 0.8478 Refinement method Full-matrix least-squares on F.sup.2 Data/restraints/parameters 1164/0/73 Goodness-of-fit on F.sup.2 1.102 Final R indices [I > 2sigma(I)] R1 = 0.0324, wR2 = 0.0779 R indices (all data) R1 = 0.0387, wR2 = 0.0797 Largest diff. peak and hole 0.433 and 0.509 e..sup.3
[0100] From the single crystal X-ray analysis result of the modified taurine, the X-atom coordinates (10.sup.4) and the equivalent isotropic displacement parameter (.sup.210.sup.3) for the modified taurine are shown in Table 3, and the bond length () and angle () in the modified taurine are shown in Table 4, and anisotropic displacement parameters (10.sup.3) for modified taurine are shown in Table 5, the hydrogen coordinates (10.sup.4) and the isotropic displacement parameter (.sup.210.sup.3) of the modified taurine are listed in Table 6, and the twist angle () of the modified taurine is shown in Table 7, and the hydrogen bonds ( and ) of the modified taurine are shown in Table 8.
TABLE-US-00003 TABLE 3 x y z U(eq) S(1) 2983(1) 8499(1) 1500(1) 9(1) O(1) 5677(2) 8384(1) 2094(2) 14(1) O(2) 2700(2) 9122(1) 137(2) 12(1) O(3) 1588(2) 7424(1) 1465(2) 14(1) N(1) 2330(3) 11304(1) 1673(2) 11(1) C(1) 1616(4) 9399(1) 3033(2) 11(1) C(2) 2909(4) 10567(1) 3210(2) 12(1) U(eq) is defined as one third of the trace of the orthogonalized U.sup.ij tensor.
TABLE-US-00004 TABLE 4 S(1)O(3) 1.4495(12) S(1)O(2) 1.4653(12) S(1)O(1) 1.4659(13) S(1)C(1) 1.7775(18) N(1)C(2) 1.487(2) N(1)H(1A) 0.85(2) N(1)H(1B) 0.84(3) N(1)H(1C) 0.84(2) C(1)C(2) 1.520(2) C(1)H(1D) 0.9900 C(1)H(1E) 0.9900 C(2)H(2B) 0.9900 C(2)H(2C) 0.9900 O(3)S(1)O(2) 112.98(8) O(3)S(1)O(1) 113.83(7) O(2)S(1)O(1) 110.89(8) O(3)S(1)C(1) 107.02(8) O(2)S(1)C(1) 105.80(8) O(1)S(1)C(1) 105.62(8) C(2)N(1)H(1A) 106.3(15) C(2)N(1)H(1B) 112.0(16) H(1A)N(1)H(1B) 111(2) C(2)N(1)H(1C) 112.4(15) H(1A)N(1)H(1C) 108(2) H(1B)N(1)H(1C) 108(2) C(2)C(1)S(1) 112.80(13) C(2)C(1)H(1D) 109.0 S(1)C(1)H(1D) 109.0 C(2)C(1)H(1E) 109.0 S(1)C(1)H(1E) 109.0 H(1D)C(1)H(1E) 107.8 N(1)C(2)C(1) 112.14(14) N(1)C(2)H(2B) 109.2 C(1)C(2)H(2B) 109.2 N(1)C(2)H(2C) 109.2 C(1)C(2)H(2C) 109.2 H(2B)C(2)H(2C) 107.9 Symmetry transformations used to generate equivalent atoms:
TABLE-US-00005 TABLE 5 U.sup.11 U.sup.22 U.sup.33 U.sup.23 U.sup.13 U.sup.12 S(1) 8(1) 7(1) 13(1) 0(1) 2(1) 0(1) O(1) 10(1) 11(1) 22(1) 2(1) 0(1) 1(1) O(2) 12(1) 11(1) 13(1) 1(1) 2(1) 0(1) O(3) 15(1) 9(1) 20(1) 2(1) 5(1) 5(1) N(1) 10(1) 8(1) 13(1) 1(1) 2(1) 1(1) C(1) 11(1) 10(1) 12(1) 0(1) 2(1) 1(1) C(2) 11(1) 11(1) 12(1) 0(1) 1(1) 1(1) The anisotropic displacement factor exponent takes the form: 2.sup.2[h.sup.2 a*.sup.2U.sup.11 + . . . + 2 h k a* b* U.sup.12]
TABLE-US-00006 TABLE 6 x y z U(eq) H(1A) 2930(40) 11960(20) 1930(30) 16 H(1B) 760(50) 11347(18) 1410(30) 16 H(1C) 3040(40) 11067(19) 800(30) 16 H(1D) 213 9512 2686 13 H(1E) 1739 9010 4166 13 H(2B) 2336 10962 4241 14 H(2C) 4775 10455 3381 14
TABLE-US-00007 TABLE 7 O(3)S(1)C(1)C(2) 179.82(12) O(2)S(1)C(1)C(2) 59.46(14) O(1)S(1)C(1)C(2) 58.19(14) S(1)C(1)C(2)N(1) 71.01(18) Symmetry transformations used to generate equivalent atoms:
TABLE-US-00008 TABLE 8 DH . . . A d(DH) d(H . . . A) d(D . . . A) <(DHA) N(1)H(1A) . . . O(1)#1 0.85(2) 1.94(2) 2.7808(19) 170(2) N(1)H(1A) . . . S(1)#1 0.85(2) 2.99(2) 3.7591(16) 152.0(19) N(1)H(1B) . . . O(2)#2 0.84(3) 2.08(2) 2.870(2) 156(2) N(1)H(1B) . . . O(3)#3 0.84(3) 2.47(2) 2.910(2) 113.3(18) N(1)H(1B) . . . S(1)#2 0.84(3) 2.90(2) 3.6064(17) 143(2) N(1)H(1C) . . . O(2)#4 0.84(2) 2.34(2) 2.992(2) 134.0(18) N(1)H(1C) . . . O(1)#4 0.84(2) 2.48(2) 3.205(2) 144.1(19) N(1)H(1C) . . . S(1)#4 0.84(2) 2.89(2) 3.6186(18) 145.4(18) Symmetry transformations used to generate equivalent atoms: #1 x + 1, y + 1/2, z + 1/2 #2 x, y + 2, z #3 x, y + 1/2, z + 1/2 #4 x + 1, y + 2, z
Experimental Example 1-2: Single Crystal X-ray Analysis at 296K
[0101] Single crystal X-ray analysis was performed by using XRD equipment (Bruker SMART APEX II X-ray Diffractometer) with Mo tube, graphite-monochromator and CCD area-detector on 50 KV, 40 mA, 296K conditions, and structural analysis was performed with Bruker SHELXTL software. The above-mentioned specific performance conditions are shown in Table 9 below.
TABLE-US-00009 TABLE 9 Identification code och08-1 Empirical formula C2 H7 N O3 S Formula weight 125.15 Temperature 296(2) K Wavelength 0.71073 Crystal system Monoclinic Space group P2.sub.1/c Unit cell dimensions a = 5.2771(2) = 90. b = 11.6376(4) = 94.113(2). c = 7.9190(3) = 90. Volume 485.08(3) .sup.3 Z 4 Density (calculated) 1.714 Mg/m.sup.3 Absorption coefficient 0.558 mm.sup.1 F(000) 264 Crystal size 0.30 0.20 0.10 mm.sup.3 Theta range for data collection 3.117 to 26.406. Index ranges 6 <= h <= 6, 14 <= k <= 14, 9 <= l <= 9 Reflections collected 6712 Independent reflections 995 [R(int) = 0.0305] Completeness to theta = 25.242 99.9% Refinement method Full-matrix least-squares on F.sup.2 Data/restraints/parameters 995/0/66 Goodness-of-fit on F.sup.2 1.106 Final R indices [I > 2sigma(I)] R1 = 0.0271, wR2 = 0.0690 R indices (all data) R1 = 0.0279, wR2 = 0.0702 Extinction coefficient 0.74(3) Largest diff. peak and hole 0.340 and 0.410 e..sup.3
[0102] From the single crystal X-ray analysis result of the modified taurine, the X-atom coordinates (10.sup.4) and the equivalent isotropic displacement parameter (.sup.210.sup.3) for the modified taurine are shown in Table 10, and the bond length () and angle () in the modified taurine atom are shown in Table 11, and anisotropic displacement parameters (10.sup.3) for modified taurine are shown in Table 12, the hydrogen coordinates (10.sup.4) and the isotropic displacement parameter (.sup.210.sup.3) of the modified taurine are listed in Table 13, and the twist angle () of the modified taurine is shown in Table 14, and the hydrogen bonds ( and ) of the modified taurine are shown in Table 15.
TABLE-US-00010 TABLE 10 x Y z U(eq) S(1) 2967(1) 8487(1) 1492(1) 22(1) O(1) 2682(2) 9108(1) 114(1) 29(1) O(3) 5640(2) 8371(1) 2072(2) 36(1) O(2) 1581(3) 7415(1) 1460(2) 36(1) C(2) 2894(3) 10548(1) 3186(2) 27(1) C(1) 1606(3) 9384(1) 2997(2) 25(1) N(1) 2361(3) 11292(1) 1684(2) 25(1) U(eq) is defined as one third of the trace of the orthogonalized U.sup.ij tensor.
TABLE-US-00011 TABLE 11 S(1)O(2) 1.4452(12) S(1)O(3) 1.4582(12) S(1)O(1) 1.4609(12) S(1)C(1) 1.7750(16) C(2)N(1) 1.482(2) C(2)C(1) 1.518(2) C(2)H(2A) 0.9700 C(2)H(2B) 0.9700 C(1)H(1A) 0.9700 C(1)H(1B) 0.9700 N(1)H(1C) 0.8900 N(1)H(1D) 0.8900 N(1)H(1E) 0.8900 O(2)S(1)O(3) 113.75(8) O(2)S(1)O(1) 113.06(8) O(3)S(1)O(1) 110.89(7) O(2)S(1)C(1) 106.89(8) O(3)S(1)C(1) 105.75(8) O(1)S(1)C(1) 105.80(7) N(1)C(2)C(1) 112.59(12) N(1)C(2)H(2A) 109.1 C(1)C(2)H(2A) 109.1 N(1)C(2)H(2B) 109.1 C(1)C(2)H(2B) 109.1 H(2A)C(2)H(2B) 107.8 C(2)C(1)S(1) 113.03(11) C(2)C(1)H(1A) 109.0 S(1)C(1)H(1A) 109.0 C(2)C(1)H(1B) 109.0 S(1)C(1)H(1B) 109.0 H(1A)C(1)H(1B) 107.8 C(2)N(1)H(1C) 109.5 C(2)N(1)H(1D) 109.5 H(1C)N(1)H(1D) 109.5 C(2)N(1)H(1E) 109.5 H(1C)N(1)H(1E) 109.5 H(1D)N(1)H(1E) 109.5 Symmetry transformations used to generate equivalent atoms:
TABLE-US-00012 TABLE 12 U.sup.11 U.sup.22 U.sup.33 U.sup.23 U.sup.13 U.sup.12 S(1) 20(1) 17(1) 29(1) 1(1) 3(1) 1(1) O(1) 32(1) 29(1) 26(1) 2(1) 5(1) 0(1) O(3) 22(1) 28(1) 55(1) 4(1) 2(1) 6(1) O(2) 41(1) 24(1) 45(1) 4(1) 11(1) 13(1) C(2) 30(1) 25(1) 26(1) 4(1) 3(1) 2(1) C(1) 26(1) 26(1) 25(1) 2(1) 5(1) 1(1) N(1) 24(1) 21(1) 29(1) 2(1) 4(1) 2(1) The anisotropic displacement factor exponent takes the form: 2.sup.2[h.sup.2a*.sup.2U.sup.11 + . . . + 2 h k a* b* U.sup.12]
TABLE-US-00013 TABLE 13 x y z U(eq) H(2A) 2317 10932 4176 32 H(2B) 4715 10437 3369 32 H(1A) 179 9498 2657 30 H(1B) 1717 9000 4087 30 H(1C) 3162 11016 820 37 H(1D) 2904 12002 1921 37 H(1E) 696 11305 1410 37
TABLE-US-00014 TABLE 14 N(1)C(2)C(1)S(1) 70.52(16) O(2)S(1)C(1)C(2) 179.40(11) O(3)S(1)C(1)C(2) 57.86(13) O(1)S(1)C(1)C(2) 59.84(13) Symmetry transformations used to generate equivalent atoms:
TABLE-US-00015 TABLE 15 DH . . . A d(DH) d(H . . . A) d(D . . . A) <(DHA) N(1)H(1C) . . . O(1) 0.89 2.35 2.9239(18) 122.5 N(1)H(1C) . . . O(1)#1 0.89 2.31 3.0121(18) 136.2 N(1)H(1C) . . . O(3)#1 0.89 2.52 3.250(2) 139.5 N(1)H(1D) . . . O(3)#2 0.89 1.92 2.7913(18) 166.5 N(1)H(1E) . . . O(1)#3 0.89 2.05 2.8931(18) 157.9 N(1)H(1E) . . . O(2)#4 0.89 2.50 2.9382(19) 111.0 Symmetry transformations used to generate equivalent atoms: #1 x + 1, y + 2, z #2 x + 1, y + 1/2, z + 1/2 #3 x, y + 2, z #4 x, y + 1/2, z + 1/2
Experimental Example 1-3: Comparison of Modified Taurine and Taurine in the Single Crystal X-ray Analysis
[0103] From the results of the single crystal X-ray analysis for the modified taurine in the results of Tables 2 to 15, the atomic characteristics of the modified taurine are shown in Table 16 in comparison with those of taurine. The data of taurine to be compared with the modified taurine are available from published articles (Y. Okaya, Acta Cryst. 1966. (21) 726-35; David E. Hibbs et al., Chem. Eur. J. 2003, 9, No. 5. 1075-84; and J. A. Beukes et al., Phys. Chem. Chem. Phys., 2007, 9, 4709-20)
TABLE-US-00016 TABLE 16 distance average average (maximum) average distance between distance distance distance between NH(3H) SC between CN between CH(4H) between SO(3O) atmos in ionic molecule atoms () atoms () density (Mg/m.sup.3) atoms () atom () atom () Temperature 1 modified 1.7775 1.487 1.748 0.99 1.4602 0.843 100 K taurine (1.4659) 2 modified 1.7750 1.482 1.714 0.97 1.4548 0.89 296 K taurine (1.4609) 3 Taurine 1.7858 1.4910 1.734 1.10 1.4659 1.045 100 k (1.4720) 4 Taurine 1.780 1.484 1.70 0.9525 1.458 0.847 293 k (1.465) 5 Taurine 1.7815 1.4862 1.738 1.10 1.4648 1.045 120 k (1.4696) 6 Taurine 1.7818 1.4809 1.709 1.10 1.4581 1.045 296 k (1.4639)
[0104] The atomic distances in Table 16 are summarized and shown in Table 17
TABLE-US-00017 TABLE 17 SC distance SO average distance SO maximum distance (Difference due to (Difference due to (Difference due to temperature change) temperature change) temperature change) {circle around (1)} Taurine 1.780~1.7858 1.458~1.4659 1.4639~1.4720 (0.0058) (0.0079) (0.0081) {circle around (2)} Modified 1.7750~1.7775 1.4548~1.4602 1.4609~1.4659 Taurine (0.0025) (0.0054) (0.005) Distance (0.0025/0.0058) 100 = (0.0054)/(0.0079) 100 = (0.005)/(0.0081) 100 = difference ratio 43.1% (56.9% decrease) 68.4% (31.6% decrease) 61.7% (38.3% decrease) with temperature change ({circle around (2)}/{circle around (1)}) 100%
[0105] As a result of the experiment, the bond length changes of the atoms in the XRD results of the temperature changes of 100 K and 296 K (room temperature) were as follows.
[0106] {circle around (1)} The bond length of SC and SO tended to be longer as the temperature decreased from 296K to 100K. This can be interpreted as a phenomenon caused by the decrease in the mobility of molecules as the temperature decreases and the hydrogen bonds with the direction of bonding become stronger with other adjacent molecules.
[0107] {circle around (2)} Changes in SC distance, SO mean distance, and SO maximum length indicating binding strength when temperature was decreased from 296K to 100K were 56.9%, 31.6% and 38.3% shorter than that of normal taurine, respectively. At the same temperature, the maximum distance between the SO atoms of the modified taurine was almost the same as the average distance between the SO atoms of the normal taurine and the distance between the SC atoms of the modified taurine was 0.007-0.008 shorter than that of the normal taurine. In general, the distance between the SC atoms was less than 1.78 for modified taurine and 1.78 or longer for normal taurine regardless of temperature. Thus, the difference in bond lengths means that the interatomic electron density distribution of the modified taurine molecule is different from that of normal taurine, which means that the CH3CH2 group in the taurine molecule is affected by the SO3 group.
[0108] {circle around (3)} In addition, the modified taurine showed less hydrogen bonding property than taurine. In Table 17, the maximum distance of the SO bond indicates the degree of hydrogen bonding with NH of the adjacent molecule. The longer the length, the stronger the hydrogen bond. At the same temperature, the SO maximum distance of normal taurine was found to be 0.0030.006 longer than that of modified taurine, the hydrogen bond of general taurine is stronger. This indicates that the distribution of the electron density in the molecule is more concentrated in the specific bond in the case of the modified taurine and does not show a large change in the external influence (hydrogen bonding).
Experimental Example 2: Raman Spectrum Analysis
[0109] In order to examine the structural difference between taurine and the modified taurine, Raman spectrum analysis was carried out by the Korea Polymer Testing & Research Institute Ltd., and the results of the analysis are shown in
[0110] The modified taurine crystals used in the analysis were the solid-state crystals prepared according to Example 1-2 (3).
[0111] For reference, an analysis instrument and analysis conditions are as follows.
[0112] (1) Analysis instrument: Nanofinder FLEX G (Lambda Ray)
[0113] (2) Source: 532 nm
[0114] (3) Range: 200-3600 cm1
[0115] (4) Exposure time, accumulation: 3 sec/20 time
[0116] (5) Spatial resolution: about 0.5 m
[0117] (6) Peak resolution: 1 cm1.
[0118] As shown in
Experimental Example 3: FT-IR (Fourier Transform Infrared Spectroscopy) Analysis
[0119] In order to examine the structural difference between taurine and the modified taurine, FT-IR spectroscopy analysis was carried out by the Korea Polymer Testing & Research Institute Ltd., and the results of the analysis are shown in
[0120] For reference, an analysis instrument and analysis conditions are as follows.
[0121] (1) Analysis instrument: JASCO FT-IR 4100
[0122] (2) Measurement mode: ATR mode
[0123] (3) Range: 600-4000 cm1
[0124] (4) Scan number: 32
[0125] (5) Peak resolution: 4 cm1.
[0126] As shown in
[0127] With reference to the data reported in Journal of Raman Spectroscopy, Vol. 27, 507-512(1996) and G. Socrates, Infrared and Raman Characteristic Group Frequencies, John Wiley & Sons, 2001, pp. 220, this difference appears to be a characteristic that appears when the SO3H of taurine is hydrated into SO3-H3O+ with an external water molecule (H2O). Furthermore, the vibration mode of NH3 shows IR absorption at wavelengths of 1173, 1508, 1614, 3044 and 3211 cm1, and the vibration mode of SO3 shows IR absorption at wavelengths of 1037, 1204 and 1303 cm1, and the IR absorption at 1527 and 3523 cm1 by hydrogen bonds does not appear. Thus, it can be seen that taurine and the modified taurine have the same ionized structure (H3N+CH2CH2SO3-), but there is a slight difference between the two with respect to the intensity of binding of SO3 to NH3 adjacent thereto in the crystals. This indicates that there is a difference in the intensity of binding between the molecules in the crystals of the ionized taurine and the ionized modified taurine.
Experimental Example 4: Scanning Electron Microscope (SEM) Analysis
[0128] To observe the surface and morphology of each of taurine and the modified taurine, SEM analysis was performed, and the results of the analysis are shown in
[0129] For reference, an analysis instrument and analysis conditions are as follows.
[0130] (1) Analysis instrument: HITACHI (S-2700), Japan
[0131] (2) Electron gun: Tungsten filament type
[0132] (3) Resolution: 4.0 nm
[0133] (4) Accelerating voltage: 15.0 kV.
[0134] As can be seen in
Experimental Example 5: Thermogravimetric Analysis (TGA)
[0135] To examine the difference between taurine and the modified taurine, thermogravimetric analysis was performed, and the results of the analysis are shown in
[0136] For reference, an analysis instrument and analysis conditions are as follows.
[0137] (1) Analysis instrument: TGA 7 (Perkin-Elmer)
[0138] (2) Atmosphere: N2 gas
[0139] (3) Heating rate: 20 C./min
[0140] (4) Range: 50 to 600 C.
[0141] As shown in
Experimental Example 6: Melting Point Analysis
[0142] To examine the difference between taurine and the modified taurine, malting point analysis was carried out, and the results of the analysis are shown in
[0143] For reference, an analysis instrument and analysis conditions are as follows.
[0144] (1) Analysis instrument: MPA100 (SRS; Stanford Research System)
[0145] (2) Start temperature: 200 C.
[0146] (3) Heating rate: 10 C./min.
[0147] As shown in
Experimental Example 7: Water Solubility Analysis
[0148] To examine the difference between taurine and the modified taurine, water solubility analysis (OECD Test Guideline 105) was carried by the Korea Polymer Testing & Research Institute Ltd., and the results of the analysis are shown in
[0149] As shown in
[0150] The convergence phenomenon of the electron distribution within a specific bond, as evidenced by the single crystal measurement of modified taurine, affects the polarization of the electron density around a specific bond, which shows a difference in the Raman spectrum. IR spectra showed that the absorbance peaks of NH.sup.3+ of conventional unmodified taurine were 1172.05 cm.sup.1, 3043.6 cm.sup.1 and absorption peaks of SO.sub.3.sup. was 1204.45 cm.sup.1, respectively. In the modified taurine, absorbance peaks of NH.sup.3+ were 1173.77 cm.sup.1 and 3044.57 cm.sup.1 and absorbance peak of SO.sub.3.sup. was 1205.5 cm.sup.1, and blue shifted of 12 cm.sup.1 compared to unmodified taurine, which indicates that the bond strength has been increased due to the shortened bond length, and that the electron density in the modified taurine molecule has been increased through the denaturation process.
[0151] That is, the modified taurine has a stronger electron delocalization in the bond of SO.sub.3.sup. than common taurine, the ionic character of NH.sup.3+ is stronger so that the ionic bonding force with the adjacent molecule is stronger as amphoteric molecule than the hydrogen bond. For the reason, it was found that the melting point and the water solubility were also high. Therefore, it was found that the modified taurine has a stronger ion-binding property than that of conventional taurine by changing the electron density distribution of the taurine molecule through the modification process.
Example 4: Examination of Therapeutic Effect Against Metabolic Disease
[0152] Evaluation of Anticoagulant Activity
[0153] Antithrombotic (anticoagulant) activity was evaluated according to a previously reported method. Each of thromborel S, actin and thrombin, which are reagents for Sysmex CA-1500 for analysis of PT, aPTT and TT, was mounted in Sysmex CA-1500 (Siemens Healthcare, Germany) which is an automated blood coagulation test device. According to the automated procedure of the test device, clotting time (sec) (prothrombin time (PT), activated partial thromboplastin time (aPTT) and thrombin time (TT)) was measured. A sufficient amount of a sample for each of the three analyses was about 400 uL of an 80:20 mixture of plasma and a test substance.
[0154] From healthy Korean adult men, a total of 23-25 ml of blood was sampled using a vacutainer (3.2% sodium citrate) for blood coagulation testing, and then immediately, centrifuged at 4 C. and 2500 rpm for 10 minutes to isolate plasma. The isolated plasma was used in an experiment in a fresh state within 5-6 hours.
[0155] In this experiment, for the relative comparison of the measured clotting time (sec) (PT, aPTT and TT) between test groups, clotting time prolongation (%) relative to triple distilled water (purified water) that is a normal control test sample was statistically analyzed. For statistical analysis, the in vivo anticoagulant activities of test materials were comparatively analyzed using SPSS IBM version 21.0 by one-way ANOVA at p<0.05, and significance comparison between groups was performed by Duncan's test.
[0156] As a control material, 37.5 mg of aspirin was completely dissolved in 1 ml of ethanol, and 4 ml of purified water was added thereto to an aspirin concentration of 7.5 mg/ml. The aspirin solution was immediately used at room temperature in a light-shielded state or was cold-stored.
[0157] 20 l of each of the samples prepared in Examples 1 and 2 was added to 80 l of plasma, and the plasma was measured for prothrombin time, activated partial thromboplastin time and thrombin time. The results of the measurement are shown in
TABLE-US-00018 TABLE 18 PT aPTT TT Test material (g/dL) N Mean S.D. Mean S.D. Mean S.D. control Purified water 3 0.00 0.00 0.00 0.00 0.00 0.00 control Asp 7.5 mg/ml 3 9.90 0.24 7.31 0.79 13.59 1.79 Comparative Ara 1.04 3 0.56 0.98 2.51 0.92 2.09 0.53 Example 2-1 Ara 5.2 3 5.93 1.47 3.72 1.76 13.40 1.01 Ara 7.8 3 9.60 1.29 6.39 1.90 22.92 1.22 Xyl 1.04 3 0.85 0.85 1.23 1.35 2.68 0.02 Xyl 5.2 3 4.52 1.29 5.70 0.91 14.59 1.49 Xyl 7.8 3 7.63 1.47 6.47 2.12 24.41 1.22 Comparative Tau 1.72 3 0.00 0.85 2.41 0.81 3.58 0.92 Example Tau 4.3 3 0.56 0.98 4.35 1.17 9.83 1.63 1-2 Tau 8.6 3 4.80 1.29 4.01 0.33 21.13 0.55 Comparative Tau 8.6 + Ara 1.04 3 4.80 0.49 4.01 0.41 22.03 0.69 Example Tau 8.6 + Ara 5.2 3 9.04 1.29 1.06 0.67 33.64 2.10 2-3-1 Tau 8.6 + Ara 7.8 3 12.71 1.47 0.83 1.35 42.57 1.38 Tau 8.6 + Xyl 1.04 3 4.80 0.98 3.73 0.85 24.11 0.22 Tau 8.6 + Xyl 5.2 3 8.76 0.49 0.47 1.97 36.32 1.33 Tau 8.6 + Xyl 7.8 3 12.99 1.29 1.73 1.95 45.54 0.77 Example 1-2 TauAlc 1.72 3 2.27 1.78 1.19 1.26 4.06 0.46 TauAlc 4.3 3 2.26 1.76 2.57 0.42 9.39 1.11 TauAlc 8.6 3 4.82 1.32 2.73 1.69 20.64 1.25 Example TauAlc 8.6 + Ara 1.04 3 7.66 2.99 0.18 1.56 21.25 0.59 2-2-1 TauAlc 8.6 + Ara 5.2 3 12.46 0.43 2.40 0.54 32.21 1.68 TauAlc 8.6 + Ara 7.8 3 16.71 0.54 4.03 1.08 40.35 3.18 TauAlc 8.6 + Xyl 1.04 3 6.80 0.03 0.67 1.93 25.64 1.75 TauAlc 8.6 + Xyl 5.2 3 11.05 0.05 1.11 0.30 35.33 1.43 TauAlc 8.6 + Xyl 7.8 3 14.73 0.56 4.89 0.50 43.77 2.47 Example TauAlc 8.6 + Ara 1.04 3 4.25 0.87 1.52 3.58 21.58 1.26 2-2-2 TauAlc 8.6 + Ara 5.2 3 11.62 0.52 1.04 1.70 37.19 0.52 TauAlc 8.6 + Ara 7.8 3 13.60 0.07 1.46 1.06 41.92 3.15 TauAlc 8.6 + Xyl 1.04 3 5.95 0.88 0.85 0.64 27.86 3.52 TauAlc 8.6 + Xyl 5.2 3 10.20 0.85 1.46 1.00 36.27 1.23 TauAlc 8.6 + Xyl 7.8 3 13.88 0.56 3.18 1.80 45.01 1.88
[0158] As shown in Table 18 above, when the modified taurine (TauAlc) was used alone or together with sugar, it showed an increase in PT prolongation compared to taurine (Tau). In the case in which the modified taurine (TauAlc) was used together with sugar, the composition containing 5.2-7.8 g of pentose (xylose (Xyl) or arabinose (Ara)) showed PT prolongation and TT prolongation, which were equal to or greater than those shown by aspirin (Asp) 7.5 mg/dL.
TABLE-US-00019 TABLE 19 PT aPTT *TT Test material (g/dL) N Mean S.D. Mean S.D. Mean S.D. control Purified water 3 0.00 0.00 0.00 0.00 0.00 0.00 control Asp 7.5 mg/ml 3 17.51 2.72 16.28 2.04 20.96 5.10 Comparative Rib 5.2 3 8.47 2.24 9.17 1.42 23.15 8.20 Example 2-1 Rib 10.4 3 14.97 2.59 12.46 3.16 52.50 7.63 Comparative Gluc 6.2 3 1.69 0.85 2.22 0.73 16.24 3.09 Example 2-2 Gluc 12.4 3 2.26 1.29 2.89 0.78 27.82 7.79 Comparative Tau 4.3 3 1.69 0.85 0.29 1.17 11.18 5.29 Example 1-2 Comparative Tau 4.3 + Rib 2.6 3 4.80 0.49 1.65 1.03 20.57 6.88 Example Tau 4.3 + Rib 6.5 3 10.73 0.49 4.25 0.98 34.61 6.87 2-3-1 Comparative Tau 4.3 + Gluc 3.1 3 0.00 0.85 1.25 1.61 19.23 3.82 Example 2-4 Tau 4.3 + Gluc 7.75 3 1.98 1.29 1.54 1.09 30.30 4.29 Example 1-2 TauAlc 4.3 3 0.85 0.85 2.03 2.03 12.49 7.15 Example TauAlc 4.3 + Rib 2.6 3 5.37 0.49 2.61 2.79 21.84 6.34 2-2-1 TauAlc 4.3 + Rib 6.5 3 11.02 0.85 3.96 0.78 35.88 6.33 Example TauAlc 4.3 + Gluc 3 0.85 0.85 1.06 1.18 24.16 4.40 2-3 3.1 TauAlc 4.3 + Gluc 3 3.11 2.13 0.56 2.30 41.80 15.69 7.75 *Values excluding one sample showing an extreme value.
[0159] As shown in Table 19, when the modified taurine (TauAlc) was used alone or together with sugar, it showed an increase in TT prolongation compared to taurine (Tau). In the case in which the modified taurine (TauAlc) was used together with sugar, the composition containing 2.6-7.75 g of sugar (ribose (Rib) or glucose (Gluc)) showed TT prolongation equal to or greater than that shown by aspirin (Asp) 7.5 mg/dL.
TABLE-US-00020 TABLE 20 PT aPTT TT Test material (g/dL) N Mean S.D. Mean S.D. Mean S.D. control Purified water 3 0.00 0.00 0.00 0.00 0.00 0.00 control Asp 7.5 mg/ml 3 26.02 1.41 29.82 1.78 23.95 2.18 Comparative Mann 3.1 3 2.71 0.94 0.65 3.67 11.67 1.56 Example 2-2 Mann 6.2 3 5.96 3.29 1.24 4.22 22.74 4.59 Mann 12.4 3 13.55 2.05 7.98 3.07 47.87 4.27 Fruc 6.2 3 0.54 0.94 0.20 3.04 13.19 1.43 Fruc 12.4 3 0.54 0.94 0.58 4.29 30.07 1.65 Comparative Tau 4.3 3 3.79 2.35 5.50 3.82 9.51 2.10 Example 1-2 Comparative Tau 4.3 + Mann 3.1 3 1.08 2.35 3.14 3.83 17.48 0.78 Example 2-4 Tau 4.3 + Mann 7.75 3 6.23 1.69 0.02 3.64 31.60 2.08 Tau 4.3 + Fruc 7.75 3 0.00 0.81 3.49 2.86 30.39 2.81 Example 1-2 TauAlc 4.3 3 3.79 2.35 5.45 2.18 10.12 0.78 Example TauAlc 4.3 + Mann 3 0.54 1.88 2.65 3.33 18.74 3.05 2-3 3.1 TauAlc 4.3 + Mann 2* 5.69 0.00 1.03 0.37 34.59 2.63 7.75 TauAlc 4.3 + Fruc 3 1.36 0.47 3.50 2.58 29.78 2.97 7.75 *Values excluding one sample showing an extreme value.
[0160] As shown in Table 20 above, when the modified taurine (TauAlc) was used alone or together with sugar, it showed an increase in TT prolongation compared to taurine (Tau). In the case in which the modified taurine (TauAlc) was used together with sugar, the composition containing 7.75 g of sugar (mannose (Mann) or fructose (Fruc)) showed TT prolongation equal to or greater than that shown by aspirin (Asp) 7.5 mg/dL.
[0161] Experiment on Anti-Diabetic and Anti-Obesity Effects
[0162] Experimental Method
[0163] Using 8-week-old male mice (C57BL/6) (purchased from CoreTech), anti-diabetic candidate materials were evaluated using a GTT (Glucose Tolerance Test) that is a typical method for diagnosis of diabetes.
[0164] Mice were housed at a temperature of 222 C. and a relative humidity of 55-60% with 12-hr light/12-hr dark cycles. Five animals were allotted to each group, and mice from the same mother were grouped into one group, because male mice tend to fight together.
[0165] As feed, high-fat diet (60% of calories from fat; Research Diet Inc., New Brunswick, N.J.) and water were fed ad libitum for 10 weeks. Herein, the water fed contained the samples prepared in Examples 1-2 (3), 2-1, 2-4-1, 2-4-2 and 2-5. As a positive control, metformin (M-072, Sigma) (250 mg/kg) was used, and as a control, the samples prepared in Comparative Examples 1-1, 2-3-2, 2-5-1, 2-5-2 and 2-6 were used.
[0166] For reference, each of the samples prepared in Examples 2-4-1, 2-4-2 and 2-5 was set to the amount to be taken by an adult man (60 kg) for 3 days, and the amount was converted into a mouse dose (12-fold/kg, see the US NIH guidance), thereby preparing animal feed. Namely, the amount to be taken by an adult man (60 kg) for 3 days is 180-day dose/kg, which corresponds to 15-day dose/kg for mice. Thus, the amount corresponds to 750-day dose/mouse (20 g), because the average weight of mice is about 20 g. Thus, one mouse was allowed to take 1/750 of the prepared sample each day.
[0167] In addition, each of the samples prepared in Examples 1-2 (3) and 2-1 was set to the amount to be taken by an adult man (60 kg) for 2 days, and the amount was converted into a mouse dose (12-fold/kg, see the US NIH guidance), thereby preparing animal feed. Namely, the amount to be taken by an adult man (60 kg) for 2 days is 120-day dose/kg, which corresponds to 10-day dose/kg for mice. Thus, the amount corresponds to 500-day dose/mouse (20 g), because the average weight of mice is about 20 g. Thus, one mouse was allowed to take 1/500 of the prepared sample each day.
[0168] The diet intake and the body weight gain were measured weekly for 8 weeks. The body weight and the diet intake were measured immediately before first drug administration, and then measured at one-week intervals.
[0169] At 8 weeks of high-fat diet feeding, a glucose tolerance test (GTT) was performed. For 8 hours for the test, mice were fasted. Then, blood was sampled from the tail vein, and initial blood glucose levels were measured with a blood glucose meter (AUTO-CHEK, Diatech Korea). Then, glucose was administered intraperitoneally to the mice at a concentration of 1 g/kg, and after 30 min, 60 min, 90 min and 120 min, blood was sampled from the mice, followed by measurement of blood glucose levels (each group consisting of five animals).
[0170] In blood biochemistry, insulin (AKRIN-011T, Shibayagi, Japan), glucose (AM202, Asan Pharmaceutical Co., Ltd., Korea), triglyceride (AM157, Asan Pharmaceutical Co., Ltd.), total cholesterol (AM202, Asan Pharmaceutical Co., Ltd.), AST and ALT (Asan Pharmaceutical Co., Ltd.) were analyzed using enzymatic assay kits.
[0171] At 10 weeks of high-fat diet feeding, the mice were sacrificed by cervical dislocation to obtain serum. For histological examination, the liver, white adipose tissue (WAT), brown adipose tissue (BAT) and kidney were fixed with formalin (50-00-0, Junsei, Japan), and the remaining organs were stored at 70 C. The blood sampled from the heart was coagulated to obtain serum which was then stored at 70 C.
[0172] For histological examination, the major organs and adipose were fixed in 4% neutral buffered formalin and embedded in paraffin blocks, and the paraffin blocks were sectioned at 5 m and stained with hymatoxylin (MHS-16, Sigma-Aldrich, USA) and eosin (HT110116, Sigma-Aldrich). The prepared tissue samples were mounted in glycerin gel mounting media (SP15-100, Fisher Scientific, USA), covered with cover glass, and observed with a microscope (IX71, OLYMPUS, USA). The tissue was imaged with the camera equipped in the microscope.
[0173] Meanwhile, experimental analysis results were expressed as meanS.E.M., and the significance between test groups was statistically processed using Student T-TEST, and then the significance was verified at *P<0.05.
[0174] Measurement Results
[0175] (1) Results of Measurement of Body Weight Gain
[0176] Body weight, body weight gain and body weight T-TEST results are shown in
TABLE-US-00021 TABLE 21 Weight (g) Before admis. 1 week 2 weeks 3 weeks 4 weeks 5 weeks 6 weeks 7 weeks 8 weeks Control Reference Mean 17.80 19.36 21.48 22.44 23.56 24.32 24.78 25.74 26.18 diet Deviation 0.17 0.35 0.54 0.56 0.54 0.57 0.66 0.78 0.81 (RD) Control High fat Mean 20.68 23.58 24.87 26.50 28.16 30.12 31.85 33.04 35.96 diet Deviation 0.29 0.54 0.53 0.57 0.62 0.68 0.52 0.47 0.42 (HFD) Example DT15 Mean 17.65 20.30 22.05 23.93 25.18 26.03 27.90 28.75 29.23 1-2 (3) (TauAlc) Deviation 0.16 0.17 0.27 0.26 0.27 0.22 0.41 0.38 0.37 Comparative DT19 Mean 19.26 21.46 23.62 24.60 25.78 27.38 27.20 28.06 28.78 Example (Tau) Deviation 0.28 0.35 0.59 0.66 0.63 0.77 0.84 0.97 1.10 1-1 Control Metformin Mean 19.30 21.00 22.78 23.84 25.18 25.72 26.34 27.52 28.20 (MET) Deviation 0.62 0.72 0.99 0.97 1.16 1.04 1.07 1.24 1.37
TABLE-US-00022 TABLE 22 Body weight gain (g) 1 week 2 weeks 3 weeks 4 weeks 5 weeks 6 weeks 7 weeks 8 weeks Control Reference Mean 1.56 3.68 4.64 5.76 6.52 6.98 7.94 8.38 diet Deviation 0.24 0.45 0.48 0.44 0.51 0.59 0.68 0.73 (RD) Control High fat diet Mean 2.90 4.19 5.82 7.48 9.44 11.17 12.36 15.28 (HFD) Deviation 0.52 0.50 0.51 0.49 0.52 0.36 0.35 0.29 Example DT15 Mean 2.65 4.40 6.28 7.53 8.38 10.25 11.10 11.58 1-2 (TauAlc) Deviation 0.25 0.28 0.34 0.40 0.36 0.52 0.46 0.40 (3) Comparative DT19 Mean 2.20 4.36 5.34 6.52 8.12 7.94 8.80 9.52 Example (Tau) Deviation 0.10 0.73 0.81 0.74 0.91 0.91 1.06 1.12 1-1 Control Metformin Mean 1.70 3.48 4.54 5.88 6.42 7.04 8.22 8.90 (MET) Deviation 0.30 1.10 0.99 1.19 1.11 1.14 1.29 1.44
TABLE-US-00023 TABLE 23 group 1 week 2 weeks 3 weeks 4 weeks 5 weeks 6 weeks 7 weeks 8 weeks HFD vs DT15 0.7736 0.8052 0.6050 0.9579 0.2443 0.1925 0.0656 0.0000 HFD vs DT19 0.3663 0.8499 0.6119 0.2908 0.1971 0.0016 0.0013 0.0000 DT15 vs DT19 0.1145 0.9645 0.3648 0.3044 0.8198 0.0801 0.1116 0.1623
[0177] From Tables 21 to 23 above, it can be seen that the DT15 group administered with the modified taurine (TauAlc) and the DT19 group administered with taurine (Tau) showed a significant reduction in body weight gain compared to the group administered with high-fat diet (HFD) alone, at 8 weeks after high-fat diet (HFD) feeding.
TABLE-US-00024 TABLE 24 Body weight (g) Before admis. 1 week 2 weeks 3 weeks 4 weeks 5 weeks 6 weeks 7 weeks 8 weeks control Reference Mean 17.80 19.36 21.48 22.44 23.56 24.32 24.78 25.74 26.18 diet Deviation 0.17 0.35 0.54 0.56 0.54 0.57 0.66 0.78 0.81 (RD) control High fat Mean 20.68 23.58 24.87 26.50 28.16 30.12 31.85 33.04 35.96 diet Deviation 0.29 0.54 0.53 0.57 0.62 0.68 0.52 0.47 0.42 (HFD) Example DT16 Mean 19.88 22.16 23.94 25.02 26.44 27.34 27.92 28.50 30.62 2-1 (TauAlc Deviation 0.32 0.32 0.43 0.53 0.75 0.71 0.79 0.74 0.84 8.6 + Ara 2.5) Comparative DT 18 Mean 20.20 22.29 23.47 25.17 26.70 27.67 29.03 30.77 32.03 Example (Tau Deviation 0.50 0.55 0.58 0.97 1.30 1.58 1.96 2.11 2.72 2-3-2 8.6 + Ara 2.5) control Metformin Mean 19.30 21.00 22.78 23.84 25.18 25.72 26.34 27.52 28.20 (MET) Deviation 0.62 0.72 0.99 0.97 1.16 1.04 1.07 1.24 1.37
TABLE-US-00025 TABLE 25 Body weight gain (g) 1 week 2 weeks 3 weeks 4 weeks 5 weeks 6 weeks 7 weeks 8 weeks control Reference Mean 1.56 3.68 4.64 5.76 6.52 6.98 7.94 8.38 diet Deviation 0.24 0.45 0.48 0.44 0.51 0.59 0.68 0.73 (RD) control High fat diet Mean 2.90 4.19 5.82 7.48 9.44 11.17 12.36 15.28 (HFD) Deviation 0.52 0.50 0.51 0.49 0.52 0.36 0.35 0.29 Example DT16 Mean 2.28 4.06 5.14 6.56 7.46 8.04 8.62 10.74 2-1 (TauAlc Deviation 0.29 0.71 0.75 1.00 1.00 1.05 1.02 1.15 8.6 + Ara 2.5) Comparative DT 18 Mean 2.09 3.27 4.97 6.50 7.47 8.83 10.57 11.83 Example (Tau Deviation 0.16 0.91 1.42 1.77 2.02 2.40 2.54 3.15 2-3-2 8.6 + Ara 2.5) control Metformin Mean 1.70 3.48 4.54 5.88 6.42 7.04 8.22 8.90 (MET) Deviation 0.30 1.10 0.99 1.19 1.11 1.14 1.29 1.44
TABLE-US-00026 TABLE 26 group 1 week 2 weeks 3 weeks 4 weeks 5 weeks 6 weeks 7 weeks 8 weeks HFD vs DT16 0.4351 0.8839 0.4636 0.3673 0.0722 0.0036 0.0008 0.0002 HFD vs DT18 0.4222 0.3970 0.4895 0.4556 0.1827 0.1121 0.2258 0.0582 DT16 vs DT18 0.6482 0.5201 0.9100 0.9753 0.9974 0.7355 0.4307 0.7065
[0178] From Tables 24 to 26 above, it can be seen that the DT16 group administered with the modified taurine (TauAlc) and arabinose (Ara) showed a significant reduction in body weight gain compared to the group administered with high-fat diet (HFD) alone, at 8 weeks after high-fat diet (HFD) feeding, whereas the DT18 group administered with taurine (Tau) and arabinose (Ara) showed no significant reduction in body weight gain.
[0179] Although there was no statistically significant difference in the inhibition of body weight gain between the DT16 group and the DT18 group, it was considered in view of the difference from HFD that the body weight control effect of DT16 was greater than that of DT18.
TABLE-US-00027 TABLE 27 Body weight (g) Before admis. 1 week 2 weeks 3 weeks 4 weeks 5 weeks 6 weeks 7 weeks 8 weeks control Reference Mean 17.80 19.36 21.48 22.44 23.56 24.32 24.78 25.74 26.18 diet Deviation 0.17 0.35 0.54 0.56 0.54 0.57 0.66 0.78 0.81 (RD) control High fat Mean 20.68 23.58 24.87 26.50 28.16 30.12 31.85 33.04 35.96 diet Deviation 0.29 0.54 0.53 0.57 0.62 0.68 0.52 0.47 0.42 (HFD) Example DT20 Mean 19.56 22.76 24.02 25.38 27.38 28.40 28.86 30.28 32.28 2-1 (TauAlc Deviation 0.49 0.67 0.48 0.76 0.77 0.88 1.05 0.94 0.96 8.6 + Xyl 3.5) Comparative DT 21 Mean 19.64 21.64 24.22 26.16 27.28 27.22 29.04 30.02 31.74 Example (Tau Deviation 0.15 0.15 0.10 0.32 0.27 0.27 0.16 0.34 0.81 2-3-2 8.6 + Xyl 3.5) control Metformin Mean 19.30 21.00 22.78 23.84 25.18 25.72 26.34 27.52 28.20 (MET) Deviation 0.62 0.72 0.99 0.97 1.16 1.04 1.07 1.24 1.37
TABLE-US-00028 TABLE 28 Body weight gain (g) 1 week 2 weeks 3 weeks 4 weeks 5 weeks 6 weeks 7 weeks 8 weeks control Reference Mean 1.56 3.68 4.64 5.76 6.52 6.98 7.94 8.38 diet Deviation 0.24 0.45 0.48 0.44 0.51 0.59 0.68 0.73 (RD) control High fat diet Mean 2.90 4.19 5.82 7.48 9.44 11.17 12.36 15.28 (HFD) Deviation 0.52 0.50 0.51 0.49 0.52 0.36 0.35 0.29 Example DT20 Mean 3.20 4.46 5.82 7.82 8.84 9.30 10.72 12.72 2-1 (TauAlc Deviation 0.36 0.72 0.56 1.11 0.85 1.01 1.02 1.13 8.6 + Xyl 3.5) Comparative DT 21 Mean 2.00 4.58 6.52 7.64 7.58 9.40 10.38 12.10 Example (Tau 8.6 + Xyl Deviation 0.14 0.14 0.41 0.41 0.32 0.27 0.41 0.95 2-3-2 3.5) control Metformin Mean 1.70 3.48 4.54 5.88 6.42 7.04 8.22 8.90 (MET) Deviation 0.30 1.10 0.99 1.19 1.11 1.14 1.29 1.44
TABLE-US-00029 TABLE 29 group 1 week 2 weeks 3 weeks 4 weeks 5 weeks 6 weeks 7 weeks 8 weeks HFD vs DT20 0.7082 0.7623 1.0000 0.7485 0.5373 0.0498 0.0774 0.0118 HFD vs DT21 0.2521 0.6032 0.3923 0.8377 0.0330 0.0072 0.0042 0.0012 DT20 vs DT21 0.0154 0.8733 0.3409 0.8829 0.2028 0.9263 0.7659 0.6847
[0180] From Tables 27 to 29 above, it could be seen that the DT20 group administered with the modified taurine (TauAlc) and xylose (Ara) and the DT21 group administered with taurine (Tau) and xylose (Ara) showed a significant reduction in body weight gain compared to the group administered with high-fat diet (HFD) alone, at 8 weeks after high-fat diet (HFD) feeding.
TABLE-US-00030 TABLE 30 Body weight (g) Before admis. 1 week 2 weeks 3 weeks 4 weeks 5 weeks 6 weeks 7 weeks 8 weeks control Reference Mean 22.894 23.692 24.138 24.285 24.507 24.416 24.720 24.782 25.684 diet Deviation 0.599 0.363 0.442 0.283 0.183 0.221 0.321 0.362 0.170 (RD) control High fat Mean 22.322 24.642 26.136 27.642 29.472 31.176 32.494 34.478 38.764 diet Deviation 0.666 0.741 0.754 0.775 0.945 0.938 0.803 1.344 1.468 (HFD) Example DT7 Mean 20.834 22.456 22.744 24.094 25.884 27.388 29.236 29.638 31.662 2-4-1 (TauAlc Deviation 0.660 0.542 0.519 0.545 0.701 0.934 1.114 1.383 1.727 8.6 + Cat 3 + Bet 4) Comparative DT11 Mean 20.420 24.418 25.360 27.388 29.178 32.794 35.460 36.012 38.370 Example (Tau Deviation 0.353 0.498 0.608 0.630 0.853 0.843 1.101 0.950 0.771 2-5-1 8.6 + Cat 3 + Bet 4) control Metformin Mean 21.105 23.550 23.608 25.125 25.705 27.198 28.355 27.368 29.343 (MET) Deviation 0.568 0.727 0.794 0.829 1.034 1.022 1.101 1.319 1.516
TABLE-US-00031 TABLE 31 Body weight gain (g) 1 week 2 weeks 3 weeks 4 weeks 5 weeks 6 weeks 7 weeks 8 weeks control Reference Mean 0.798 1.244 1.391 1.613 1.522 1.826 1.888 2.790 diet Deviation 0.444 0.473 0.475 0.448 0.507 0.504 0.499 0.517 (RD) control High fat diet Mean 2.320 3.814 5.320 7.150 8.854 10.172 12.156 16.442 (HFD) Deviation 0.351 0.518 0.458 0.601 0.899 0.737 1.438 1.296 Example DT7 Mean 1.622 1.910 3.260 5.050 6.554 8.402 8.804 10.828 2-4-1 (TauAlc Deviation 0.251 0.200 0.259 0.269 0.334 0.486 0.733 1.120 8.6 + Cat 3 + Bet 4) Comparative DT11 Mean 3.998 4.940 6.968 8.758 12.374 15.040 15.592 17.950 Example (Tau Deviation 0.605 0.789 0.869 1.066 1.017 1.162 1.013 0.583 2-5-1 8.6 + Cat 3 + Bet 4) control Metformin Mean 2.445 2.503 4.020 4.600 6.093 7.250 6.263 8.238 (MET) Deviation 0.442 0.383 0.637 0.910 0.795 1.090 0.944 1.090
TABLE-US-00032 TABLE 32 Group 1 week 2 weeks 3 weeks 4 weeks 5 weeks 6 weeks 7 weeks 8 weeks HFD vs DT7 0.1444 0.0090 0.0045 0.0128 0.0434 0.0799 0.0715 0.0112 HFD vs DT11 0.0433 0.2672 0.1320 0.2253 0.0320 0.0077 0.0865 0.3197 DT7 vs DT11 0.0067 0.0059 0.0035 0.0097 0.0006 0.0008 0.0006 0.0005
[0181] From Tables 30 to 32 above, it could be seen that the DT7 group administered with the modified taurine (TauAlc), catechin (Cat) and betaine (Bet) showed a significant reduction in body weight gain compared to the group administered with high-fat diet (HFD) alone, at 8 weeks after high-fat diet (HFD) feeding, whereas the DT1 group administered with taurine (Tau), catechin (Cat) and betaine (Bet) showed no significant reduction in body weight gain. In addition, it was considered that the body weight control effect of DT7 was significantly greater than that of DT11.
TABLE-US-00033 TABLE 33 Body weight (g) Before 2 3 4 5 6 7 8 admis. 1 week weeks weeks weeks weeks weeks weeks weeks control Reference Mean 22.894 23.692 24.138 24.285 24.507 24.416 24.720 24.782 25.684 diet Deviation 0.599 0.363 0.442 0.283 0.183 0.221 0.321 0.362 0.170 (RD) control High fat Mean 22.322 24.642 26.136 27.642 29.472 31.176 32.494 34.478 38.764 diet Deviation 0.666 0.741 0.754 0.775 0.945 0.938 0.803 1.344 1.468 (HFD) Example DT10 Mean 21.956 25.356 25.458 25.998 27.062 29.836 31.732 30.562 32.740 2-4-2 (TauAlc Deviation 0.483 0.640 0.524 0.740 0.666 0.811 0.946 0.975 1.135 8.6 + EGCG 1.5 + Bet 4) Comparative DT14 Mean 21.440 24.750 26.114 27.556 29.456 31.968 34.020 32.874 37.470 Example (Tau Deviation 0.638 0.998 1.142 1.400 1.654 2.005 2.181 2.012 2.186 2-5-2 8.6 + EGCG 1.5 + Bet 4) control Metformin Mean 21.105 23.550 23.608 25.125 25.705 27.198 28.355 27.368 29.343 (MET) Deviation 0.568 0.727 0.794 0.829 1.034 1.022 1.101 1.319 1.516
TABLE-US-00034 TABLE 34 Body weight gain (g) 1 week 2 weeks 3 weeks 4 weeks 5 weeks 6 weeks 7 weeks 8 weeks control Reference Mean 0.798 1.244 1.391 1.613 1.522 1.826 1.888 2.790 diet Deviation 0.444 0.473 0.475 0.448 0.507 0.504 0.499 0.517 (RD) control High fat diet Mean 2.320 3.814 5.320 7.150 8.854 10.172 12.156 16.442 (HFD) Deviation 0.351 0.518 0.458 0.601 0.899 0.737 1.438 1.296 Example DT10 Mean 3.400 3.502 4.042 5.106 7.880 9.776 8.606 10.784 2-4-2 (TauAlc Deviation 0.274 0.253 0.330 0.441 0.683 0.929 1.126 1.204 8.6 + EGCG 1.5 + Bet 4) Comparative DT14 Mean 3.310 4.674 6.116 8.016 10.528 12.580 11.434 16.030 Example (Tau Deviation 0.407 0.580 0.850 1.077 1.412 1.596 1.414 1.577 2-5-2 8.6 + EGCG 1.5 + Bet 4) control Metformin Mean 2.445 2.503 4.020 4.600 6.093 7.250 6.263 8.238 (MET) Deviation 0.442 0.383 0.637 0.910 0.795 1.090 0.944 1.090
TABLE-US-00035 TABLE 35 Group 1 week 2 weeks 3 weeks 4 weeks 5 weeks 6 weeks 7 weeks 8 weeks HFD vs DT10 0.0416 0.6033 0.0536 0.0254 0.4134 0.7471 0.0879 0.0126 HFD vs DT14 0.1029 0.3012 0.4338 0.5024 0.3467 0.2079 0.7296 0.8451 DT10 vs DT14 0.8592 0.1013 0.0526 0.0369 0.1299 0.1673 0.1564 0.0295
[0182] From Tables 33 to 35 above, it could be seen that the DT10 group administered with the modified taurine (TauAlc), epigallocatechin gallate (EGCG) and betaine (Bet) showed a significant reduction in body weight fat compared to the group administered with high-fat diet (HFD) alone, at 8 weeks after high-fat diet (HFD) feeding, whereas DT14 administered with taurine (Tau), epigallocatechin gallate (EGCG) and betaine (Bet) showed no significant reduction in body weight gain. In addition, it was considered that the body weight control effect of DT10 was significantly greater than that of DT14.
TABLE-US-00036 TABLE 36 Body weight (g) Before 2 3 4 5 6 7 8 admis. 1 week weeks weeks weeks weeks weeks weeks weeks control Reference Mean 25.706 25.614 27.006 26.814 28.744 29.442 29.258 29.476 27.442 diet Deviation 0.940 0.698 0.939 0.789 0.843 0.828 1.037 0.813 0.838 (RD) control High fat Mean 24.920 27.448 29.878 34.452 36.850 38.748 40.524 43.686 42.344 diet Deviation 0.547 0.453 0.635 0.725 0.523 0.548 0.941 1.164 0.591 (HFD) Example DT4 Mean 25.422 27.170 30.180 32.488 33.968 35.218 36.214 38.048 36.188 2-5 (TauAlc Deviation 0.237 0.565 0.800 0.744 0.642 0.781 1.038 1.053 1.044 8.6 + EGCG 1.5 + Bet 4 + Xyl 3.5) Comparative DT6 Mean 23.088 26.216 29.082 32.910 32.922 38.018 39.414 40.820 40.618 Example (Tau Deviation 0.690 1.032 1.372 2.118 1.931 2.000 1.890 2.036 1.784 2-6 8.6 + EGCG 1.5 + Bet 4 + Xyl 3.5) control Metformin Mean 23.938 25.094 26.344 27.430 27.708 28.572 28.772 28.468 27.608 (MET) Deviation 0.612 0.551 0.792 0.710 0.915 0.853 0.784 0.836 0.979
TABLE-US-00037 TABLE 37 Body weight gain (g) 1 week 2 weeks 3 weeks 4 weeks 5 weeks 6 weeks 7 weeks 8 weeks control Reference Mean -0.092 1.300 1.108 3.038 3.736 3.552 3.770 1.736 diet Deviation 0.325 0.259 0.270 0.414 0.339 0.389 0.333 0.369 (RD) control High fat diet Mean 2.528 4.958 9.532 11.930 13.828 15.604 18.766 17.424 (HFD) Deviation 0.326 0.461 0.473 0.440 0.960 1.156 1.228 0.689 Example DT4 Mean 1.748 4.758 7.066 8.546 9.796 10.792 12.626 10.766 2-5 (TauAlc Deviation 0.372 0.625 0.580 0.465 0.589 0.835 0.846 0.849 8.6 + EGCG 1.5 + Bet 4 + Xyl 3.5) Comparative DT6 Mean 3.128 5.994 9.822 9.834 14.930 16.326 17.732 17.530 Example (Tau Deviation 0.619 0.933 1.724 1.487 1.526 1.382 1.553 1.357 2-6 8.6 + EGCG 1.5 + Bet 4 + Xyl 3.5) control Metformin Mean 1.156 2.406 3.492 3.770 4.634 4.834 4.530 3.670 (MET) Deviation 0.180 0.185 0.223 0.350 0.398 0.398 0.936 0.463
TABLE-US-00038 TABLE 38 Group 1 week 2 weeks 3 weeks 4 weeks 5 weeks 6 weeks 7 weeks 8 weeks HFD vs DT4 0.1538 0.8033 0.0109 0.0007 0.0072 0.0097 0.0034 0.0003 HFD vs DT6 0.4161 0.3485 0.8751 0.2135 0.5580 0.6992 0.6156 0.9462 DT4 vs DT6 0.0924 0.3030 0.1682 0.4324 0.0138 0.0090 0.0203 0.0029
[0183] From Tables 36 to 38 above, it could be seen that the DT4 group administered with the modified taurine (TauAlc), epigallocatechin gallate (EGCG), betaine (Bet) and xylose (Xyl) showed a significant reduction in body weight gain compared to the group administered with high-fat diet (HFD) alone, at 8 weeks after high-fat diet (HFD) feeding, whereas the DT6 group administered with taurine (Tau), epigallocatechin gallate (EGCG), betaine (Bet) and xylose (Xyl) showed no significant reduction in body weight gain. In addition, it was considered that the body weight control effect of DT4 was significantly greater than that of DT6.
[0184] (2) Glucose Tolerance Test (GTT)
[0185] GTT results and T-Test results for the GTT results are shown in
TABLE-US-00039 TABLE 39 Glucose level (mg/dl) 0 min 30 min 60 min 90 min 120 min control Reference Mean 123.60 328.60 324.00 235.60 151.20 diet Deviation 6.44 12.80 30.00 16.84 6.57 (RD) control High fat diet Mean 188.70 479.70 461.60 458.40 250.10 (HFD) Deviation 10.67 26.87 39.06 33.42 27.98 Example 1-2 DT15 Mean 121.25 335.25 295.25 193.75 126.25 (3) (TauAlc) Deviation 3.73 3.77 7.73 11.43 0.56 Comparative DT19 Mean 111.80 298.00 295.60 189.40 119.00 Example 1-1 (Tau) Deviation 11.08 26.76 11.52 9.75 6.47 control Metformin Mean 144.20 386.40 301.40 194.40 135.20 (MET) Deviation 15.92 17.73 24.43 21.21 17.23
TABLE-US-00040 TABLE 40 Before After After After After Group administration 30 min 60 min 90 min 120 min HFD vs DT15 0.0023 0.0062 0.0224 0.0004 0.0182 HFD vs DT19 0.0006 0.0010 0.0119 0.0001 0.0065 DT15 vs DT19 0.4936 0.2625 0.9822 0.7904 0.3569
[0186] From Tables 39 and 40 above, it could be seen that the fasting glucose levels of the DT15 group administered with the modified taurine (TauAlc) and DT19 administered with taurine (Tau) were all lower than that of the mice administered with high-fat diet (HFD), and were similar to the blood glucose levels of normal mice.
[0187] The results of the GTT indicated that the glucose control abilities of the mouse groups administered with DT15 and with DT19 all showed were significantly higher than that of the group administered with HFD alone, and the glucose control ability was similar between DT15 and DT19.
TABLE-US-00041 TABLE 41 Glucose level (mg/dl) 0 min 30 min 60 min 90 min 120 min control Reference Mean 123.60 328.60 324.00 235.60 151.20 diet Deviation 6.44 12.80 30.00 16.84 6.57 (RD) control High fat Mean 188.70 479.70 461.60 458.40 250.10 diet Deviation 10.67 26.87 39.06 33.42 27.98 (HFD) Example DT16 Mean 117.00 289.20 302.20 196.20 121.40 2-1 (TauAlc Deviation 4.86 20.15 35.60 31.33 14.44 8.6 + Ara 2.5) Comparative DT 18 Mean 169.33 367.00 343.00 236.33 172.67 Example (Tau Deviation 9.21 22.27 32.94 28.55 23.62 2-3-2 8.6 + Ara 2.5) control Metformin Mean 144.20 386.40 301.40 194.40 135.20 (MET) Deviation 15.92 17.73 24.43 21.21 17.23
TABLE-US-00042 TABLE 42 Before After After After After Group administration 30 min 60 min 90 min 120 min HFD vs DT16 0.0005 0.0005 0.0219 0.0003 0.0084 HFD vs DT18 0.3740 0.0549 0.1488 0.0060 0.1848 DT16 vs DT18 0.0029 0.0626 0.4990 0.4502 0.1322
[0188] From Tables 41 and 42 above, the fasting blood glucose level of the DT16 group administered with the modified taurine (TauAlc) and arabinose (Ara) was significantly lower than that of the mice administered with high-fat diet (HFD) alone, and was comparable with that of normal mice.
[0189] The results of the GTT indicated that the glucose control ability of the mice administered with DT16 was significantly higher than that of the group administered with HFD alone, and thus the blood glucose level of the mice administered with DT16 was comparable with that of normal mice. In addition, it could be seen that the glucose control effect of DT18 was better than that of DT16 and that the fasting glucose level of DT18 was significantly lower than that of DT16.
TABLE-US-00043 TABLE 43 Glucose level (mg/dl) 0 min 30 min 60 min 90 min 120 min control Reference diet Mean 123.60 328.60 324.00 235.60 151.20 (RD) Deviation 6.44 12.80 30.00 16.84 6.57 control High fat diet Mean 188.70 479.70 461.60 458.40 250.10 (HFD) Deviation 10.67 26.87 39.06 33.42 27.98 Example 2-1 DT20 Mean 115.00 306.40 330.40 240.40 142.80 (TauAlc Deviation 7.00 33.04 35.07 32.68 15.94 8.6 + Xyl 3.5) Comparative DT 21 Mean 134.20 340.60 331.40 240.40 155.40 Example (Tau 8.6 + Xyl Deviation 13.04 27.41 29.45 35.59 19.60 2-3-2 3.5) control Metformin Mean 144.20 386.40 301.40 194.40 135.20 (MET) Deviation 15.92 17.73 24.43 21.21 17.23
TABLE-US-00044 TABLE 44 Before After After After After group administration 30 min 60 min 90 min 120 min HFD vs DT20 0.0005 0.0019 0.0513 0.0012 0.0233 HFD vs DT21 0.0089 0.0065 0.0483 0.0014 0.0440 DT20 vs DT21 0.2306 0.4486 0.9831 1.0000 0.6314
[0190] From Tables 43 and 44 above, it could be seen that the fasting glucose levels of the DT20 group, administered with the modified taurine (TauAlc) and xylose (Ara), and the DT21 group administered with taurine (Tau) and xylose (Ara), were all lower than that of the mice administered with high-fat diet (HFD) alone, and were similar to the blood glucose level of normal mice.
[0191] The results of the GTT indicated that the glucose control abilities of the mouse group administered with DT20 and the mouse group administered with DT21 were significantly higher than that of the group administered with HFD alone. In addition, it could be seen that although there was no significant difference between DT20 and DT21, DT20 group showed better effects than DT21.
TABLE-US-00045 TABLE 45 Glucose level (mg/dl) 0 min 30 min 60 min 90 min 120 min control Reference diet Mean 120.60 392.80 296.00 203.20 158.60 (RD) Deviation 2.16 58.61 40.72 27.48 17.28 control High fat diet Mean 210.80 528.20 527.40 448.80 342.20 (HFD) Deviation 13.79 23.68 17.28 37.78 44.72 Example DT4 Mean 121.20 418.75 347.25 265.25 182.75 2-5 (TauAlc Deviation 7.81 25.89 10.69 25.07 9.81 8.6 + EGCG 1.5 + Bet 4 + Xyl 3.5) Comparative DT6 Mean 199.60 505.80 471.40 417.40 318.40 Example (Tau Deviation 13.55 33.81 65.31 75.23 61.39 2-6 8.6 + EGCG 1.5 + Bet 4 + Xyl 3.5) control Metformin Mean 104.40 438.40 296.40 178.00 146.20 (MET) Deviation 3.72 8.68 23.15 14.35 3.02
TABLE-US-00046 TABLE 46 Before After After After After Group administration 30 min 60 min 90 min 120 min HFD vs DT4 0.0005 0.0211 0.0001 0.0075 0.0176 HFD vs DT6 0.5783 0.6021 0.4312 0.7189 0.7620 DT4 vs DT6 0.0010 0.1003 0.1409 0.1303 0.0949
[0192] From Tables 45 and 46 above, it could be seen that the fasting glucose level of the DT4 group administered with the modified taurine (TauAlc), epigallocatechin gallate (EGCG), betaine (Bet) and xylose (Xyl) was significantly lower than that of the mice administered with high-fat diet (HFD) alone, and was similar to that the blood glucose level of normal mice.
[0193] The results of the GTT indicated that the glucose control ability of the mouse group administered with DT4 was significantly higher than that of the group administered with HFD alone, but the glucose control ability of the group administered with DT6 was lower than that of the DT4 group.
[0194] (3) Results of Blood Biochemistry
[0195] The results of measurement of triglyceride, AST and ALT levels and the T-test results for the measurement results are shown in Tables 47 to 56.
TABLE-US-00047 TABLE 47 Triglyc- eride AST ALT Group (mg/dl) (IU/L) (IU/L) control Reference Mean 76.02 91.34 10.85 diet(RD) S.D. 14.42 7.23 6.20 control High fat Mean 148.78 129.70 24.71 diet(HFD) S.D. 32.47 6.48 3.75 control Metformin Mean 62.99 110.32 22.80 (MET) S.D. 10.70 16.00 7.16 Example 1-2 DT15 Mean 53.62 98.96 14.02 (3) (TauAlc) S.D. 7.22 6.11 3.82 Comparative DT19 Mean 116.20 118.63 25.47 Example 1-1 (Tau) S.D. 13.34 12.02 8.60
TABLE-US-00048 TABLE 48 Group Triglyceride AST ALT HFD vs DT15 0.0383 0.0118 0.0890 HFD vs DT19 0.3805 0.4408 0.9372 DT15 vs DT19 0.0066 0.2211 0.3034
[0196] As can be seen in Tables 47 and 48, DT15 (modified taurine) showed triglyceride, AST and ALT levels which are similar to or lower than those of normal mice. In addition, the triglyceride level of DT15 was significantly lower than that of DT19 (taurine), and the AST and ALT levels thereof were also lower than those of DT19.
TABLE-US-00049 TABLE 49 Triglyc- eride AST ALT Group (mg/dl) (IU/L) (IU/L) control Reference Mean 76.02 91.34 10.85 diet(RD) S.D. 14.42 7.23 6.20 control High fat Mean 148.78 129.70 24.71 diet(HFD) S.D. 32.47 6.48 3.75 control Metformin Mean 62.99 110.32 22.80 (MET) S.D. 10.70 16.00 7.16 Example 2-1 DT16 Mean 54.30 96.48 10.68 (TauAlc S.D. 8.66 3.52 4.15 8.6 + Ara 2.5) Comparative DT18 Mean 66.97 94.51 7.02 Example (Tau 8.6 + Ara 2.5) S.D. 18.17 3.02 7.09 2-3-2
TABLE-US-00050 TABLE 50 Group Triglyceride AST ALT HFD vs DT16 0.0402 0.0042 0.0407 HFD vs DT18 0.1208 0.0076 0.0492 DT16 vs DT18 0.5202 0.7015 0.6546
[0197] As can be seen in Tables 49 and 50 above, DT16 (modified taurine+arabinose) and DT18 (taurine+arabinose) showed AST and ALT levels which are similar to those of normal diet (RD) and which are significantly lower than those of high-fat diet (HFD). In addition, DT16 showed a triglyceride level lower than that of reference diet (RD).
TABLE-US-00051 TABLE 51 Triglyc- eride AST ALT Group (mg/dl) (IU/L) (IU/L) control Reference Mean 76.02 91.34 10.85 diet(RD) S.D. 14.42 7.23 6.20 control High fat Mean 148.78 129.70 24.71 diet(HFD) S.D. 32.47 6.48 3.75 control Metformin Mean 62.99 110.32 22.80 (MET) S.D. 10.70 16.00 7.16 Example 2-1 DT20 Mean 60.81 100.44 8.67 (TauAlc S.D. 23.96 8.57 3.04 8.6 + Xyl 3.5) Comparative DT21 Mean 42.35 85.41 6.76 Example (Tau 8.6 + Xyl 3.5) S.D. 5.26 2.97 0.71 2-3-2
TABLE-US-00052 TABLE 52 Group Triglyceride AST ALT HFD vs DT20 0.0609 0.0261 0.0105 HFD vs DT21 0.0120 0.0003 0.0015 DT20 vs DT21 0.4732 0.1363 0.5582
[0198] As can be seen in Tables 51 and 52 above, DT20 (modified taurine+xylose) and DT18 (taurine+xylose) showed AST and ALT levels which are similar to those of reference diet (RD) and which are significantly lower than those of high-fat diet (HFD). In addition, DT20 and DT21 showed triglyceride levels lower than that of reference diet (RD).
TABLE-US-00053 TABLE 53 Triglyc- AST ALT Group eride (IU/L) (IU/L) control Reference Mean 113.61 10.30 5.32 diet(RD) S.D. 22.21 1.41 0.33 control High fat Mean 135.37 21.60 13.31 diet(HFD) S.D. 22.74 4.33 3.96 control Metformin Mean 107.14 16.06 8.05 (MET) S.D. 10.29 0.99 0.72 Example DT7 Mean 100.68 11.41 7.01 2-4-1 (TauAlc S.D. 7.23 1.71 1.07 8.6 + Cat 3 + Bet 4)
TABLE-US-00054 TABLE 54 Group Triglyceride AST ALT HFD vs DT7 0.451 0.071 0.176
[0199] As can be seen in Tables 53 and 54 above, DT7 (modified taurine+catechin+betaine) showed AST and ALT levels which are similar to those of reference diet (RD) and which are smaller than those of high-fat diet (HFD). In addition, DT7 showed a triglyceride level lower than that of reference diet (RD).
TABLE-US-00055 TABLE 55 Triglyc- AST ALT Group eride (IU/L) (IU/L) control Reference Mean 81.39 10.09 0.99 diet(RD) S.D. 17.61 3.35 0.38 control High fat Mean 133.80 26.92 14.79 diet(HFD) S.D. 8.13 4.44 5.31 control Metformin Mean 46.75 14.10 3.50 (MET) S.D. 16.82 5.08 1.20 Example DT4 Mean 59.85 13.52 1.03 2-5 (TauAlc 8.6 + EGCG S.D. 7.61 2.66 0.43 1.5 + Bet 4 + Xyl 3.5) Comparative DT6 Mean 86.72 14.10 11.22 Example (Tau 8.6 + EGCG S.D. 17.42 5.08 4.19 2-6 1.5 + Bet 4 + Xyl 3.5)
TABLE-US-00056 TABLE 56 Group Triglyceride AST ALT HFD vs DT4 0.003 0.0465 0.0565 HFD vs DT6 0.0386 0.0940 0.6119 DT4 vs DT6 0.2458 0.9284 0.0695
[0200] As can be seen in Tables 55 and 56, DT4 (modified+EGCG+betaine+xylose) showed AST and ALT levels which are similar to those of reference diet (RD) and which are significantly lower than those of high-fat diet (HFD). In addition, DT4 showed a triglyceride level lower than that of reference diet (RD).
[0201] (4) Results of Histological Examination
[0202] The results of examination of the liver, white adipose tissue (WAT), brown adipose tissue (BAT) and kidney tissue of the test mice shown in
[0203] As can be seen in
[0204] As can be seen in
[0205] As can be seen in
[0206] As can be seen in
[0207] Although the present disclosure has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only of a preferred embodiment thereof, and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.