Method of Reducing Uric Acid with Fucoxanthin-Containing Composition

Abstract

The present invention provides a method of reducing uric acid in a subject in need thereof, comprising administering to the subject a composition comprising an effective amount of fucoxanthin, wherein the fucoxanthin has the effect of reducing uric acid and preventing gout.

Claims

1. A method of reducing uric acid in a subject in need thereof, comprising administering to the subject a composition comprising an effective amount of fucoxanthin.

2. The method as claimed in claim 1, wherein the fucoxanthin is obtained by the extraction of seaweed with water at a predetermined temperature for a period of time.

3. The method as claimed in claim 2, wherein the predetermined temperature is 50□ to 100□.

4. The method as claimed in claim 2, wherein the period of time is 30 minutes to 120 minutes.

5. The method as claimed in claim 2, wherein the extraction of seaweed with water is conducted at a solid:liquid ratio of 1:5 to 1:20.

6. The method as claimed in claim 2, wherein the seaweed is Ascophyllum nodosum.

7. The method as claimed in claim 3, wherein the seaweed is Ascophyllum nodosum.

8. The method as claimed in claim 4, wherein the seaweed is Ascophyllum nodosum.

9. The method as claimed in claim 5, wherein the seaweed is Ascophyllum nodosum.

10. The method as claimed in claim 2, wherein the fucoxanthin inhibits xanthine oxidase activity to reduce uric acid.

11. The method as claimed in claim 3, wherein the fucoxanthin inhibits xanthine oxidase activity to reduce uric acid.

12. The method as claimed in claim 4, wherein the fucoxanthin inhibits xanthine oxidase activity to reduce uric acid.

13. The method as claimed in claim 5, wherein the fucoxanthin inhibits xanthine oxidase activity to reduce uric acid.

14. The method as claimed in claim 1, wherein the composition is a pharmaceutical composition and/or a food composition.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1 is a graph showing the fucoxanthin content of the Ascophyllum nodosum extract of the present invention.

[0011] FIG. 2 is a graph showing the effect of the Ascophyllum nodosum extract of the present invention in inhibiting xanthine oxidase activity.

[0012] FIG. 3 shows the results of co-treatment of ADTC5 cells with monosodium urate and Ascophyllum nodosum extract for 24 hours.

[0013] FIG. 4 shows the results of co-treatment of ADTC5 cells with monosodium urate and Ascophyllum nodosum extract for 48 hours.

DETAILED DESCRIPTION

[0014] Values used herein are approximations and all experimental data values are expressed in the range of 20%, preferably in the range of 10%, and most preferably in the range of 5%.

[0015] The present invention provides a method of using a composition comprising fucoxanthin for reducing uric acid in a subject in need thereof, wherein the fucoxanthin is obtained by the extraction of seaweed with water at a predetermined temperature for a period of time, and the seaweed is preferably Ascophyllum nodosum. The molecular formula of the fucoxanthin is C.sub.42H.sub.58O.sub.6, and its structural formula is shown in formula (I) below.

##STR00001##

[0016] In one embodiment of the present invention, the seaweed is preferably dried seaweed, the predetermined temperature is 50□ to 100□, the period of time is 30 to 120 minutes, and the extraction of seaweed with water is conducted at a solid: liquid ratio of 1:5 to 1:20.

[0017] Fucoxanthin, the special compound with uric acid-reducing effect in seaweed, is extracted from seaweed, preferably Ascophyllum nodosum, and purified using extraction technology in the present invention. The uric acid-reducing effect of the extract comprising fucoxanthin was evaluated in terms of its inhibitory efficiency against xanthine oxidase activity. It was further confirmed that the extract comprising fucoxanthin can be used as a health material for reducing uric acid or alleviating gout in general foods or health foods. The present invention will be described in detail by the following Examples 1 to 4.

EXAMPLE 1

Seaweed Extraction

[0018] The seaweeds used in the present invention include, but are not limited to, Ascophyllum nodosum, Dictyota divaricata, Sargassum cristaefolium, Laminaria japonica, and Hincksia mitchellae. Fresh Ascophyllum nodosum was adopted here and dried for use in the subsequent extraction process. The extraction of dried Ascophyllum nodosum with water was conducted at a solid:liquid ratio of 1:5 to 1:20, at 50□ to 100□, respectively, for 30 to 120 minutes; the mixture was then cooled to room temperature and then filtrated with a 200 μm mesh to obtain Ascophyllum nodosum extract.

[0019] In the following Examples, the Ascophyllum nodosum extract will be used to evaluate its xanthine oxidase activity inhibition ability and determine its fucoxanthin content.

EXAMPLE 2

Determination of the Fucoxanthin Content

[0020] The Ascophyllum nodosum extract obtained from Example 1 was filtered through a 0.45 μm filter, and the content of fucoxanthin was analyzed by HPLC. The analysis conditions are shown as follows: [0021] Instruments: Waters e2695 & Waters 2998 Photodiode Array Detector [0022] Column: Inertsil ODS-3V (5 μm 4.6×250 mm)+guard column [0023] Flow rate: 0.7 ml/min [0024] Injection volume: 20 μL [0025] Column temperature: 35° C. [0026] Detection wavelength: 445 nm [0027] Mobile phase: 75% acetonitrile [0028] Time: 40 minutes [0029] Standard: fucoxanthin

[0030] Analysis was performed by drawing a standard curve according to the fucoxanthin standard, then calculating the fucoxanthin content in test sample by interpolation.

[0031] As shown in FIG. 1, with the increase in extraction time, the content of fucoxanthin increases significantly. This shows that the longer the extraction time, the more fucoxanthin from Ascophyllum nodosum that can be extracted by the present method. Therefore, longer extraction times can effectively increase the fucoxanthin content in the extracts.

EXAMPLE 3

Evaluation of the Inhibition of XANTHINE OXIDASE ACTIVITY

[0032] 50 μL of the test sample, 35 μL of phosphate buffer (70 mM, pH 7.5) and 30 μL of xanthine oxidase (0.02 units/mL) were mixed and shaken, and allowed to stand at room temperature for 15 minutes. Then, 60 μL of xanthine (150 μM) was added and allowed to react at room temperature for 30 minutes; the reaction was then stopped by the addition of 100 μL of the HCl (1N), followed by measuring absorbance at 290 nm. The inhibitory effect of the test sample against xanthine oxidase activity was evaluated and compared with the control group.

[0033] Evaluation results are shown in FIG. 2. Ascophyllum nodosum extract being extracted for 120 minutes showed 40% inhibition rate against xanthine oxidase. The fucoxanthin with the same concentration of the Ascophyllum nodosum extract showed 44% inhibition rate against xanthine oxidase, indicating that inhibitory effect of the Ascophyllum nodosum extract against xanthine oxidase is derived from fucoxanthin.

EXAMPLE 4

Cell Experiment

[0034] ADTC5 mouse cartilaginous cells (Mouse 129 teratocarcinoma AT805 derived) were seeded into a culture plate at a density of 5×10.sup.5 cells/well and cultured at 37□ under 5% CO.sub.2 for 24 hours. The culture medium was DMEM/F12 supplemented with 5% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco). Next, the cells were treated with monosodium urate (MSU) (0.125 mg/ml, in medium) to induce uric acid crystallization. Cells were divided into three groups: Group 1 was treated with Ascophyllum nodosum extract extracted for 120 minutes (2 mg/ml, in medium); group 2 was treated with fucoxanthin (1 ppm, in medium); group 3 was untreated (the control group). After 24 and 48 hours of treatment, the cells were harvested and lysed, then washed once with PBS, and the supernatant was removed and 350 μl of the RNA lysate was added for a further RNA extraction process (using GeneMark's RNA purification kit, operated according to the manufacturer's instructions). Next, reverse transcription was performed using a SuperScript™ reverse transcription kit (Invitrogen). The NOS2 (inducible nitric oxide synthase) gene was quantified using the ABI StepOnePlus™ System (Applied Biosystems), and β-actin was used as a reference gene for normalizing each sample. It is known that accumulation of uric acid crystals in the joints causes inflammation in joint cavities, which leads to the increase of NOS2 gene expression in chondrocytes. Therefore, NOS2 gene expression levels were used to evaluate the degree of inflammation of cells caused by uric acid crystallization. The relative quantification of gene expression was performed using the 2.sup.−ΔΔct method. Statistical analysis was performed by Student's t-test (Microsoft Excel software).

[0035] FIG. 3 shows the results of ADTC5 cells co-treated with MSU and Ascophyllum nodosum extract for 24 hours. Ascophyllum nodosum extract can reduce the NOS2 gene expression level in ATDC5 cells. The NOS2 gene expression level in the MSU-induced group was expressed as 1, then the NOS2 gene expression levels in the Ascophyllum nodosum extract-treated group and the fucoxanthin-treated group could be reduced to 0.71 and 0.24, respectively. The results indicate that Ascophyllum nodosum extract can alleviate the inflammation of the joint cavity induced by MSU in the joints. Also, the effect of fucoxanthin can be significantly greater than that of Ascophyllum nodosum extract, indicating that the anti-inflammatory effect ofAscophyllum nodosum extract may come from fucoxanthin.

[0036] FIG. 4 shows the results of ADTC5 cells co-treated with MSU and Ascophyllum nodosum extract for 48 hours. Ascophyllum nodosum extract can reduce the NOS2 gene expression level in ATDC5 cells. The NOS2 gene expression level in the MSU-induced group was expressed as 1, then the NOS2 gene expression levels in the Ascophyllum nodosum extract-treated group and the fucoxanthin-treated group could be reduced to 0.57 and 0.12, respectively. The results indicate that Ascophyllum nodosum extract can alleviate the inflammation of the joint cavity induced by MSU in the joints. Also, the effect of fucoxanthin can be significantly greater than that of Ascophyllum nodosum extract, indicating that the anti-inflammatory effect of Ascophyllum nodosum extract may come from fucoxanthin. The results also show that the effect to NOS2 gene by the co-treatment of MSU and Ascophyllum nodosum extract in ATDC5 cells for 48 hours was better than that for 24 hours, indicating that the NOS2 gene inhibitory effect resulted from Ascophyllum nodosum extract and fucoxanthin are long-lasting.

[0037] From the above Examples, it can be seen that the fucoxanthin described in the present invention does provide the effect of reducing uric acid. In particular, because the Ascophyllum nodosum extract (extracted for 120 minutes) in the preferred embodiment of the present invention was rich in fucoxanthin, it could be used as a health material for reducing uric acid or alleviating gout in general foods or health foods.