WO2007086658A1

WO2007086658A1 - Fucoidan added kochujang, korean red pepper soybean paste, having increased anti-obesity effect

Info

Publication number: WO2007086658A1
Application number: PCT/KR2007/000035
Authority: WO
Inventors: Kun Young Park; Min Su Han; Chang Suk Kong; Su Ok Kim; In Sook Ahn; Moon Su Kim
Original assignee: Pusan National University Industry-University Cooperation Foundation; Cj Cheiljedang Corporation
Priority date: 2006-01-27
Filing date: 2007-01-03
Publication date: 2007-08-02
Also published as: KR20070078638A

Abstract

The present invention relates to fucoidan added Kochujang having an increased anti-obesity effect, more specifically, fucoidan added Kochujang having increased anti-obesity and anticancer effects.

Description

FUCOIDAN ADDED KOCHUJANG, KOREAN RED PEPPER SOYBEAN PASTE, HAVING INCREASED ANTI-OBESITY

EFFECT

Technical Field

[1] The present invention relates to fucoidan added Kochujang, Korean red pepper soybean paste, having an increased anti-obesity effect. More specifically, the present invention relates to fucoidan added Kochujang having increased anti-obesity and anticancer effects. Background Art

[2] Korean red pepper soybean paste (hereinafter, referred to as 'Kochujang' is paste food, which uses soybean, starch, powdered red pepper, and salt as essential ingredients, and features a harmony of taste, such as, a savory taste from amino acid (degradation product of soybean), a sweet, amylaceous sweet taste from glutinous rice, nonglutinous rice, barley and the like, a salty taste from salt, and a spicy taste from powdered red pepper, and visual perception of bright redness. Kochujang is one of important fermented foods and a seasoning that always shows up in Korean dishes. Unlike Doenjang, another Korean traditional fermented food, the red pepper soybean paste uses powdered red pepper in addition to meju (a starter culture mass of soybeans), glutinous rice powder such as rice, and malt. The origin of the red pepper soybean paste is not found in other parts of the world, yet it is a native typical Korean staple food. Kochujang is largely divided into two types depending on how it is made: traditional type and mass production type. In case of Kochujang prepared by the traditional method, many kinds of bacteria or fungi inhabit while boiling soybeans, and ingredients of Kochujang are decomposed, during the aging process (6-18 months), by treatment with enzymes secreted by those microorganism into organic acid, nucleic acid, alcohol, etc., which affect taste or flavor to a harmony of savor. On the other hand, mass-produced Kochujang is aged, diastatic product, using koji obtained with pure culture of Aspergillus oryzae in stead of meju. In preparation of koji, soybeans are used as a protein source, and rice or flour is used as glutinous rice powder. Apergillus oryzae is inoculated into soybeans and rice, respectively, and fermented for 3-4 days, and a 1 : 1 mixture of these ingredients is added in replacement of meju. Therefore, taste and flavor are enriched by an enzymic reaction of Apergillus oryzae and fermentation effects of yeast, and the aging period (15-60 days) is relatively short.

[3] In addition to the features described above, Kochujang and Doenjang of Korea have drawn a lot of attention as functional fermented foods effective for suppressing obesity. Obesity is a serious world- wide concern in health. There are many chronic, fatal diseases that are linked to obesity, such as, diabetes, arterial hypertension, cardiovascular diseases, arthrosis of backbone and of lower limbs due to excessive body weight, pulmonary diseases, and some cancers. Now, an obesity as a chronic disease epidemic has rapidly spread across all regions of the world, irrespective of social class and gender. A growing number of researches are actively in progress across the world on a natural element, rather than a chemical element in the long view, which has direct/indirect effects on losing weight or suppressing obesity yet is safe to intake. In this light, Kochujang together with Doenjang are adequate foods to meet such needs.

[4] Meanwhile, brown seaweeds contain a great quantity of laminaran, the neutral polysaccharide, and acid polysaccharides having a sulfate group. Typical examples of sulfate containing acid polysaccharide are fucoidan and alginate. In particular, fucoidan has been reported to have blood coagulation activities, anticancer activities and anti-AIDS activities. With growing interests in dietary fiber, a number of studies have been actively conducted on seaweeds as diet foods. Most of them are, however, in vivo studies, so additional in vitro studies into physiological actions of dietary fiber are much needed.

[5] Therefore, the present inventors put collaborative efforts to observe an increased in vitro anti-obesity effect of Kochujang that contains fucoidan extracted from seaweeds. Since anticancer effects or anticancer activities of Kochujang itself differ depending on preparation methods and sub materials used, the inventors added fucoidan to Kochujang to increase its anticancer effect. Disclosure of Invention

Technical Problem

[6] It is, therefore, an object of the present invention to provide Kochujang having increased anticancer function and anti-obesity function by adding fucoidan. Technical Solution

[7] The foregoing object was achieved by investigating the enhanced anti-obesity effect of fucoidan and fucoidan added Kochujang (Korean red pepper soybean paste) on adipocytes, and the in vitro anticancer effect in human cancer cells.

[8] That is, the present invention is largely divided into three steps: preparing fucoidan added Kochujang; experimentally investigating anti-obesity effects of fucoidan and fucoidan added Kochujang on adipocytes; and experimentally investigating in vitro anticancer effect in cancer cells.

[9] Accordingly, the present invention provides fucoidan added Kochujang having an increased anti-obesity effect.

[10] The Kochujang preferably includes, but is not limited to, 1 wt% or more of fucoidan. [11] The other objectives and advantages of the invention will be understood by the following description and will also be appreciated by the embodiments of the invention more clearly.

Advantageous Effects

[12] As mentioned above and will be described below, fucoidan added Kochujang of the present invention reveals enhanced anti-obesity and anticancer effects. Brief Description of the Drawings

[13] The above and other objects and features of the present invention will become apparent from the following description of the preferred embodiments given in conjunction with the accompanying drawings, in which:

[14] Fig. 1 is a graph showing a change in glycerol content, with fucoidan treatment;

[15] Fig. 2 is a graph showing leptin outputs in adipocytes, with fucoidan treatment; and

[16] Fig. 3 is a graph showing leptin outputs in mast cells treated with methanol extract from fucoidan added Kochujang. Best Mode for Carrying Out the Invention

[17] The above-mentioned objectives, features, and advantages will be more apparent by the following detailed description in association with the accompanying drawings, and thus, the invention will be readily conceived by those skilled in the art to which the invention pertains. Further, in the following description, well-known arts will not be described in detail if it seems that they could obscure the invention in unnecessary detail. Hereinafter, preferred embodiments of the present invention will be set forth in detail with reference to the accompanying drawings. Mode for the Invention

[18] Example 1 : Preparation of Fucoidan Added Kochujang

[19] Kochujang (Korean red pepper soybean paste) was prepared by a commonly used method, and Fucoidan (manufactured by MSC Co., Ltd.) was added thereto by 0.5%, 1%, and 3% by weight, respectively. Refined salt (manufactured by Hanjoo Co., Ltd.) and husked wheat cereals (80%) imported from Australia were used. Kochujang was prepared by mass production. That is, wheat cereals and flour were cooked, cooled, and inoculated with A, oryzae. Then, flour malt, cooked rice, table salt, and water were added, and the resultant mixture was inoculated with yeast (30°C, fermented for one week). Finally, powdered red pepper and fucoidan were added to fermented adding ingredients that was homogenized for 30 to 40 days.

[20] Fucoidan added Kochujang was freeze-dried, and samples were triturated to get powder. The powder samples were mixed with 20% (w/v) methanol, stirred for 12 hours two times, and filtered. After that, the resultant mixture was introduced into a rotatory vacuum evaporator to obtain methanol extracts. These methanol extracts were diluted in DMSO (dimethyl sulfoxide) to prepare 20% stocks. The stocks were then treated in primary cells having been diluted to the concentration of lmg/ml and fully differentiated into adipocytes.

[21] For an experimental investigation on anti-obesity effects of fucoidan, a methanol extract from Kochujang containing 1 wt% of fucoidan was used. In addition, for an experimental investigation on anticancer effects of fucoidan, methanol extracts from Kochujang containing 0.5 wt% and 3 wt% of fucoidan, respectively, were used.

[22]

[23] Incubation of Adipocytes

[24] 3T3-L1 preadopocyte cell line originated from a mouse embryo was obtained from

ATCC (ATCC, USA), and incubated in an incubator at 37°C under 5% CO , in use of DMEM with 10% fetal bovine serum and 100 unit/ml of streptomycin. When the cells were in a confluent phase 3 to 4 days later, they were treated with 0.05% trypsin- EDTA for subincubation. The mediums were refed every 2-3 days. To induce cell differentiation, a 3.3x10 cell/cm cell density suspension was prepared. ImI of the suspension was seeded per well in a 12- well plate for secondary incubation. When the cells were incubated to confluency up to 80%, the mediums were refed with 10% FBS- DMEM containing 5ms/ml insulin, 0.25mM dexamethazone, and 0.5mM IMBX to induce cell differentiation. The mediums were refed every other day with medium containing 5ms/ml insulin. After 10 days with medium differentiation, more than 90% of the cells were differentiated to adipocytes.

[25]

[26] Measurement of Leptin Outputs by ELISA

[27] An amount of leptin secreted from adipocytes was measured by ELISA (Enzyme

Linked Immonosolvent Assay, R&D systems) from the mediums collected. First, 100ml of anti-mouse leptin IgG (2mg/ml) was put in a Maxisorb ELISA plate (Nunc) and incubated overnight. Then, the plate was washed with PBS-T containing 0.05% of Tween 20 three times, and the leptin was incubated for another hour in 100ml of the medium collected in the plate. Again, the plate was washed with PBS-T three times, and 100ml of biotinylated anti-mouse leptin IgG (200ng/ml) was incubated at room temperature for 1 hour, followed by three-times of washing with PBS-T again. Extravidin-horse radish peroxidase (1:200 dilution ratio, R&D systems) was incubated at room temperature for 1 hour and washed three times. Immunoreactivity was revealed in each well, after a 30-min reaction with 100ml of TMB (tetramethyl benzidine dihydrochloride substrate, Amresco). This reaction was put to an end by addition of 50ml of 2M H SO into each well. After the reactions were over, optical density per well was measured by using plate spectrophotometer at 450nm. [28]

[29] Measurement of Glycerol Content

[30] Glycerol concentration was measured by using GPO-TRINDER (Sigma, USA) according to an enzymatic method. Glycerol assay reagents were dissolved in 50ml of ultra pure water, and ImI of the solution was heated at 37°C for 5 minutes. Approximately ImI of a pre- warmed glass glycerol reagent was added to 10ml of the collected medium, and incubation was continued at 37°C for 5 minutes. Quantitative assay of glycerol in the medium, 12.5mg and 25mg of glycerol standard solution (Sigma, USA) were reacted with the same method with the samples, respectively. 200ml of each was put into a 96-well plate, and optical density was measured at 540nm.

[31]

[32] Incubation of Cancer Cells

[33] An AGS human gastric adenocarcinoma cell and a HT-29 human colon adenocarcinoma cell provided from Korean Cell Line Bank (School of Medicine at Seoul National University) were incubated for an experiment. The AGS and the HT-29 cancer cells were incubated in RPMI 1640 containing 100 units/ml penicillin- streptomycin and 10% FBS (fetal bovine serum). Each of the incubated cancer cells was introduced into a new medium that was refed 2-3 times a week, and washed with PBS every 6-7 days. Confluent adherent cells were separated by 0.05% trypsin-0.02% EDTA, and centrifuged. Next, the integrated cancer cells were introduced into a medium and well mixed with a pippet for uniform distribution. Subincubation was performed every 6-7 days. At the time of subincubation, each passage number was recorded. If the passage number was greater than 10, a new cancer cell was taken from a liquid nitrogen tank and incubated again for an experiment.

[34]

[35] MTT Assay

[36] The incubated cancer cells were seeded per well (1x10 cells/ml for each) in a

96-well plate, and mixed with samples of different concentrations. The cells were incubated in an incubator at 37°C under 5% CO . After 72 hours, 20ml of MTT

2

(Sigma, USA) was added thereto for incubation for additional 4 hours. The resultant formazan crystal was dissolved in DMSO, and optical density was measured at 540nm. [37] Cytotoxicity (%) = (Optical density of the control group Optical density of the treated sample group) / (Optical density of the control group) x 100 [38]

[39] Statistical Analysis

[40] Significances of the experimental results obtained from the control group and from each sample were tested by t-test. Its results are shown in terms of (Mean)±(Standard Deviation, SD). [41] [42] Example 2: Anti- Obesity and Anticancer Effects of Fucoidan and Fucoidan

Added Kochujang

[43] Anti-obesity and anticancer effects of fucoidan and fucoidan added Kochujang of

Example 1 were experimentally investigated.

[44]

[45] Experimental Example 1: Anti- Obesity Effect of Fucoidan

[46] To find out obesity suppression effects of fucoidan on 3T3-L1 adipocytes, a difference in glycerol contents with/without fucoidan treatment was measured to further observe the degree of fat decomposition by the sample treatment (Fig. 1). Since neutral fat is decomposed into fatty acids and glycerol, an amount of glycerol secreted into a medium tells the degree of fat decomposition by the sample treatment. Differentiated adipocytes were treated with fucoidan of different concentrations, and glycerol secretion amount in each case was examined. It turned out that the glycerol secretion amount was significantly increased in proportion to an increase in the treatment concentration of fucoidan, and the decomposition rate was increased by 50% with 5mM treatment, compared with the control group.

[47] In addition, when 3T3-L1 adipocytes were treated with fucoidan, leptin outputs

(refer to Fig. 2) were decreased, compared with the leptin outputs in the fucoidan treated control group. Leptin is a protein secreted only from adipocytes, and combines with its receptors on terminals of neurons, delivering stimulus to the nervous system. This stimulus signal suppresses food intake and increases energy expenditure, to thereby control the induction of obesity. Increases in leptin secretion amount in adipocytes means fat accumulation in adipocytes. In other words, a decrease in leptin secretion indicates that fucoidan treatment suppressed differentiation of adipocytes, or fucoidan is effective for reducing number and size of adipocytes.

[48]

[49] Experimental Example 2: Anti-Obesity Effect of Fucoidan Added Kochujang

[50] To evaluate anti-obesity effects of fucoidan added Kochujang, leptin secretion by

3T3-L1 adipocytes treated with fucoidan added Kochujang was observed. In detail, methanol extracts from fucoidan added Kochujang were used for treatment of adipocytes and leptin secretion therefrom was observed (Fig. 3). Compared with the control group, leptin secretion was decreased with Kochujang treatment. That is, Kochujang treatment is associated with a decrease in the amount of leptin in adipocytes. Moreover, leptin secretion was significantly decreased with treatment of fucoidan added Kochujang. As mentioned before, leptin is a protein secreted only from adipocytes, and a decrease in leptin secretion is believed because fucoidan treatment has suppressed differentiation of adipocytes, or fucoidan was indeed effective for reducing number and size of adipocytes. A conclusion may be drawn out of these results that fucoidan is exceedingly effective in suppression of fat accumulation, and fucoidan added Kochujang may increase anti-obesity activities of Kochujang.

[51] [52] Experimental Example 3: Anticancer Effects of Fucoidan [53] To evaluate in vitro anticancer effects of fucoidan, toxicity of fucoidan in normal cells was examined. Fucoidan showed a high survival rate (higher than 97%) (not shown) even at 5mg/ml of concentration of fucoidan addition. Inhibitory effects on the growth of cancer cells were observed within this concentration range. In particular, Table 1 below shows inhibitory effects of fucoidan on the growth of AGS and HT-29 human cancer cells.

[54] Even at a low concentration of fucoidan addition such as lmg/ml, fucoidan showed 74% and 76% of high inhibition ratios on the AGS and HT-29 human cancer cells, respectively.

[55] Table 1 Inhibitory effects of fucoidan on the growth of cancer cells

[56] [57] Experimental Example 4: Anticancer Effects of Fucoidan Added Kochujang [58] For development of Kochujang having an increased anticancer function, fucoidan showing high anticancer activities was added during the preparation of Kochujang, at a concentration of 0.5% and 3%, respectively. In vitro anticancer effects of fucoidan added Kochujang were observed within a concentration range that does not cause any toxicity to normal healthy cells. Table 2 below shows an observation result of inhibitory effects of Kochujang with 0.5% fucoidan on the growth of cancer cells. As can be seen, the cancer cell growth inhibition ratios increased proportionally to the sample treatment concentrations. For instance, at 5mg/ml of the treatment concentration, fucoidan added Kochujang showed 59% and 66% of high inhibition ratios on the AGS and HT-29 human cancer cells, respectively.

[59] A similar trend is also shown in an observation result (Table 3) of inhibitory effects of Kochujang with 3% fucoidan on the growth of cancer cells. That is, the inhibition ratios of the AGS and HT-29 human cancer cell growth increased proportionally to the sample treatment concentrations. Moreover, the higher the concentration of fucoidan added to Kochujang, the higher the inhibition ratio of the cancer cell growth. That is, although Kochujang, by itself, has anticancer activities, its anticancer function was enhanced even more by addition of fucoidan.

[60] [61] Table 2 Inhibitory effects of Kochujang with 0.5% fucoidan on the growth of cancer cells

[62] [63] Table 3 Inhibitory effects of Kochujang with 3% fucoidan on the growth of cancer cells

[64]

Industrial Applicability

[65] Fucoidan added Kochujang of the present invention can be advantageously used as functional foods in improvement of anti-obesity and anticancer effects. [66] The present application contains subject matter related to Korean patent application No. 2006-0009097, filed with the Korean Intellectual Property Office on January 27, 2006, the entire contents of which are incorporated herein by reference. [67] While the present invention has been described with respect to certain preferred embodiments, it will be apparent to those skilled in the art that various changes and modifications may be made without departing from the scope of the invention as defined in the following claims.

[68]

Claims

[1] Kochujang (Korean red pepper soybean paste) containing fucoidan as an essential ingredient for increasing anti-obesity effects. [2] The Kochujang according to claim 1, wherein a content of the fucoidan is 1% or more by weight.