JP2005075787A - Use of extract of fermented glycine max(l.) in inhibition of 15-lipoxygenase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a pharmaceutical composition or a food composition used for inhibiting 15-lipoxygenase and treating or reducing risk of cardiovascular disorders. <P>SOLUTION: In the use of a fermented Glycine max(L.) extract, particularly a fermented black soybean extract, the fermented Glycine max (L.) extract is prepared by fermenting an aqueous Glycine max (L.) extract with at least one kind of lactic acid bacterium together with at least one kind of yeast. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention inhibits 15-lipoxygenase, prevents and / or prevents diseases in which inhibition of 15-lipoxygenase is intended in a subject (eg, cardiovascular disease, immune disorders (eg, asthma and / or inflammation)). Or the use of fermented Glycine max (L.) extract in treating as well as modulating the immune system. The present invention also relates to the use of fermented Glycine max (L.) extract in the prevention and / or treatment of microbial infection.

  Lipooxygenase (LOX) is a non-heme iron-containing enzyme that catalyzes the oxidation of certain polyunsaturated fatty acids such as lipoproteins. Several different lipooxygenase enzymes (eg, LOX-5, LOX-12 and LOX-15) are known, each having a characteristic oxidizing effect. LOX-15 catalyzes the oxidation of arachidonic acid and linolenic acid and is involved in the oxidative modification of low density lipoprotein (LDL). Numerous studies have shown that LOX-15 is associated with coronary artery disease and atherosclerosis (Shen et al., J. Clin. Invest. 1996, Vol. 98, No. 10, pp. 2201-2208; Timo et al., 1995, Vol. 92). (11), pp. 3297-3303; Ravalli et al., 1995, Arteriosclerosis, Thrombosis and Vascular Biology, Vol. 15, No. 3, pp. 340-348; .99, No. 5, pp. 888-893), cancer and inflammatory diseases (US Pat. No. 6,001,866; Mogul et al., 2001, Biochemistry, 40, 4391-497; Walter et al., 19 9, Molecular Pharmacology, 56: 196-203; and Kamitani et al., 1998, the Journal of Biological Chemistry, Vol. 126: 2-8). Therefore, a substance having an effect of inhibiting LOX is useful as a drug for preventing or treating LOX-related diseases.

  Soybean is one of the concentrated sources of isoflavones in the human diet. Soy also contains a number of compounds, including saponins, phytosterols, soy phytate, protease inhibitors, phenolic acids, complex sugars, boron, lecithin, omega-3 fatty acids and folic acid. Soybeans can confer health benefits. Many established traditional foods (eg, tembe and natto) are produced from soybean fermentation. For example, Tenbe is known as Rhizopus oligosporus, R.P. oryzae, R.A. arihizus and R.H. Produced by fermenting soybeans using stolonifer. Natto is produced by fermenting soybeans using Bacillus NATTO. Traditional fermented foods can be used as an excellent protein source. However, there is no prior art that discloses that any known fermented soy foods and soybeans are effective at inhibiting 15-lipoxygenase (LOX-15).

(Summary of the Invention)
(1) Use of a fermented Glycine max (L.) extract for the production of a composition for use in the inhibition of 15-lipoxygenase in a subject, wherein the fermented Glycine max (L.) extract is Prepared by fermenting an aqueous Glycine max (L.) extract using at least one lactic acid bacterium together with at least one yeast, and the inhibition of 15-lipoxygenase is Use, used to treat or reduce the risk.
(2) Use of item 1 whose said Glycine max (L.) is black soybean.
(3) The use according to item 1, wherein the lactic acid bacterium is a Lactobacillus species.
(4) The use according to item 1, wherein the yeast is a Saccharomyces species.
(5) The use according to item 1, wherein the cardiovascular disease is atherosclerosis.
(6) The use according to item 1, wherein the composition is a pharmaceutical composition or a food composition.

  The present invention relates to the use of a fermented Glycine max (L.) extract in inhibiting 15-lipoxygenase (LOX-15), which fermented Glycine max (L.) extract is an aqueous Glycine max (L.) extract. ) Prepared by fermenting the extract with at least one lactic acid bacterium and at least one yeast. In particular, Glycine max (L.) is soybean or black soybean. More specifically, the fermented Glycine max (L.) extract of the present invention comprises a fermented soy extract and a fermented black soy extract.

  The fermented Glycine max (L.) extract of the present invention prevents and / or prevents diseases in which inhibition of 15-lipoxygenase is intended in a subject (eg, cardiovascular disease, immune disorders (eg, asthma and inflammation)). It can be used in treating and in regulating the immune system.

  The fermented Glycine max (L.) extract of the present invention can also be used in preventing and / or treating microbial infections in a subject.

(Detailed description of the invention)
The present invention provides a novel use of fermented Glycine max (L.) extract to produce a composition for use in the inhibition of diseases involving 15-lipoxygenase (LOX-15) and 15-lipoxygenase. Here, the fermented Glycine max (L.) extract is prepared by fermenting an aqueous Glycine max (L.) extract with at least one lactic acid bacterium and at least one yeast. It has been unexpectedly found that this fermented Glycine max (L.) extract can effectively inhibit 15-lipoxygenase at low levels.

  In particular, a composition comprising a fermented Glycine max (L.) extract of the present invention may be used to treat diseases in which inhibition of 15-lipoxygenase is intended in a subject (eg, cardiovascular disease, immune disorders (eg, asthma and inflammation) ) In the prevention and / or treatment, and in regulating the immune system. Furthermore, the fermented soy extract of the present invention can also be used in treating and / or preventing microbial infections.

  The composition of the present invention may be a pharmaceutical composition or a food composition.

(Process for producing fermented soybean extract)
The fermented Glycine max (L.) extract was fermented with an aqueous Glycine max (L.) extract with at least one lactic acid bacterium and at least one yeast, and then filtered and concentrated as necessary. Made by sterilizing the fermentation broth (eg, by heat).

  According to the present invention, the preferred Glycine max (L.) used in the preparation of the fermented Glycine max (L.) extract is selected from the group consisting of soybean and black soybean. More specifically, the fermented Glycine max (L.) extract of the present invention is a fermented soybean extract or a fermented black soybean extract.

  The fermented Glycine max (L.) extract is a combination of at least one lactic acid bacterium (eg, one or more strains of Lactobacillus species or several strains of a number of Lactobacillus species) and at least one Glycine max (L.) extract. Produced by fermentation with a single yeast (eg, one or more strains of Saccharomyces species or several strains of multiple Saccharomyces species). Fermentation of aqueous Glycine max (L.) extract with one or more lactic acid bacteria and optionally Saccharomyces species can be carried out sequentially or simultaneously, preferably simultaneously, in any order.

  If more than one microorganism is used in the fermentation, the fermentation can be performed using the microorganisms sequentially or simultaneously. Preferably, a Glycine max (L.) aqueous extract of a selected grade of non-genetically modified organism is used as the starting material. Preferably, the fermentation is performed using a Lactobacillus heterogeneous culture (eg, a culture of 5, 10, 15, 20, 25 or 30 strains of Lactobacillus) and at least one yeast is Added to Lactobacillus heterogeneous cultures. Lactobacillus strains that can be used include, for example, Lactobacillus acidophilus CCRC 10695, 14026, 14064, 14065 and / or 14079, Lactobacillus delbrueckii bulgaricus CCRC 10696, 14007, 14069, 140, 140, 140, 140, 140, 140, 140, 140, 140, 140, 140, 140, 140, 140, 140, 140 16054, Lactobacillus lactis lactis CCRC 10791, 12267, 12306, 12312, 12315, 12323, 14016, 14015, and / or 14117, Lactobacillus kefir CCRC 14011 and / or Lact obacillus kefiranofaciens CCRC 16059. Yeasts that may be used include, for example: Saccharomyces cerevisiae CCRC 20577, 20578, 20581, 21494, 21550, 2197, 21805, 22138, 22234, 22337, 22728 and / or Candida kefyr CCRC 21269 21742 and / or 22057. After fermentation, the fermentation broth is sterilized by, for example, heat or irradiation (preferably by heat) to obtain a sterilized liquid. Preferably, the sterile solution is filtered or centrifuged, preferably filtered to remove most or all dead microorganisms to obtain a fermented Glycine max (L.) extract. More preferably, after this filtration step, some water is removed from the filtrate and the fermentation broth is concentrated to obtain a fermented Glycine max (L.) extract. Unless otherwise specified, tests performed in this application included fermented Glycine max (L.) extract after the concentration step. If necessary, the fermented Glycine max (L.) extract can be dried, for example by lyophilization, to obtain a fermented Glycine max (L.) extract in powder form.

  This process can be carried out by mixing (degreased) organic Glycine max (L.) with distilled water in a ratio of 1:10. The mixture is heated at 100 ° C. for 30 minutes and then filtered to obtain a Glycine max (L.) extract. Boil beef and kelp for 30 minutes in distilled water to obtain broth. Salt, sugar and agar are added to produce a special agar medium. Lactic acid bacterial strains and yeast strains are added to this special agar medium. This lactic acid bacterium, optionally with yeast, in the medium is transferred to a Glycine max (L.) extract and incubated at 36-43 ° C. for 45-50 hours. Preferably, the various microbial strains have similar growth characteristics (eg, any requirement for unique nutrient media, whether these microbial strains can produce a good odor after fermentation, and microorganisms classified under unique conditions. Whether or not can survive, so that the group of microorganisms is added separately to the Glycine max (L.) extract prior to incubation. The purpose of this process is to reduce any negative interactions between the various strains. Also preferably, an equal proportion of different groups of microbial strains are added to the Glycine max (L.) extract prior to incubation and the resulting extract is incubated at 40 ° C. for 45-47 hours. At the end of the incubation period, the heterogeneous culture is then transferred again to the Glycine max (L.) extract and incubated at 36-43 ° C. for 100-150 hours. The final fermentation extract is heat sterilized and filtered; and 95% of the water content filtrate is removed in the concentration to obtain a concentrated or condensed form of fermented Glycine max (L.) extract. The upper layer is then filtered through porcelain and then dispensed into a container and sealed.

  A fermented extract of Chinese herb prepared by replacing soybean with Chinese herb and prepared in a process similar to that described above is within the scope of the present invention. The fermented extracts of Chinese medicinal herbs can be: Glycyrrhiza uralensis Fish, Lycium barbarum, Coix lacryma-jobi L var. Ma-yune Stapf, Sophora tokinensis gapnep. Cassia btusifolia. Scutellaria baicalensis Georgi, Artemisia capillaries Thunb. , Coptis chinensis Frsnch. , Gentiana scabra Bge. Nelumbo nucifera Gaertn. , Chrysantheifera mumorifolium Ramat. , Gardenia jasminoides Ellis, Hordeum vulgare L .; Cinnamum cassia Presl, Raph, anus sativus L .; Dioscorea opposita Thunb. , Angelica sinensis (Olib.), Ligusticum chunxiong Hort. , Notopterygium incisum, Paeonia lactiflora Pall. Allium satium L .; Schizandra chinensis (Turcz.) Baill, Rehmannia glutinosa Libosch. Acanthopanax graciltylus W .; W. Smith, Equus asinus L.M. Ligustrum lucidum Ait. , Phaseolus radiatas L .; , Triticum aestivum L. Dolichos lablab L. , Actylodes macrocephala Koidz. Saposhinikova divaricata, Lonicera japonica Thund. Cinnamum Cassia Presl, Zingiber officinal Rosc. Gastrodia elata Bl. Asparagus cochinchinensis (Liur.) Merr. Dendrobien lodgesiii Rolfe. And Sesamum indicum L.

(Use in inhibition of 15-lipoxygenase)
15-lipoxygenase is a non-heme iron-containing enzyme that catalyzes the oxidation of certain polyunsaturated fatty acids such as lipoproteins. Compounds that inhibit the action of 15-lipoxygenase enzyme are known to be useful in the treatment or alleviation of inflammatory, allergic, cardiovascular and immune diseases in mammals (including humans).

  Studies in the present invention have demonstrated that the fermented Glycine max (L.) extract of the present invention can inhibit 15-lipoxygenase more favorably than known fermented soy foods and non-fermented soy.

(Use of antioxidants)
Fermented Glycine max (L.) extract has significant antioxidant and free radical scavenger activity. The fermented Glycine max extract, superoxide free radical ^{_{(e.g., O 2 · -, H 2}} O 2 and ROO ^·) (L.) Obtained was removed, and act as an antioxidant to unsaturated fatty acids and fatty Can do. This fermented Glycine max (L.) extract transforms free radicals into harmless substances using a remarkable ability to remove excess oxygen anions and an energy reduction procedure to prevent cells from oxidative damage Has remarkable ability.

(Use of drugs for cardiovascular disease)
A number of studies have shown inhibitors of LOX-15 in the treatment and prevention of inflammation and atherosclerosis (Cornicelli et al., US Pat. No. 6,001,866; Bocan et al., Atherosclerosis, 136, 203-216). 1998, and Timo et al., Circulation, col. 92 (1), December 1, 1995, 3297-3303). Fermented Glycine max (L.) extract is expected to be useful for the prevention and / or treatment of cardiovascular diseases such as atherosclerosis.

(Use to enhance immune function)
In vitro studies have shown that the fermented Glycine max (L.) extract of the present invention improves immune function. The effect of fermented Glycine max (L.) extract on the regulation of immunity in animals (Bala / c mice) is combined or combined with a challenge with various mitogens including lipopolysaccharide, concanavalin A and phytohemagglutinin aggregation No fermented Glycine max (L.) extract was studied by treating the animals. Splenocyte proliferation assays showed that the fermented Glycine max (L.) extract was associated with T cell and B cell interactions in immunomodulation. The fermented Glycine max (L.) extract may also be associated with an anti-immune response. Glycine max (L.) extract also increased macrophage phagocytic activity by 71%. Similar results were found in in vivo studies of mice. It was also demonstrated that the anti-tumor effect of fermented Glycine max (L.) extract is mediated by cytokine release. Conditioned media derived from peripheral blood mononuclear cells stimulated with fermented Glycine max (L.) extract is 45-56%. The levels of interleukin-1β, interleukin-β and tumor necrosis factor-α were much higher than the levels of untreated controls. Presumed that anti-tumor activity is derived from enhanced levels of cytokines because naïve macrophages and T lymphocytes produced little or no cytokines and normal mononuclear cells suppressed white blood cell proliferation. Is done.

  Fermented Glycine max (L.) extract is beneficial for children with asthma. For example, daily administration of 3 ml of fermented soy extract for 4 months resulted in significant weight gain in a group of children with asthma. Blood tests showed that ingestion of fermented Glycine max (L.) extract increased RBC and Hb levels in these asthmatic children.

(Use as anti-inflammatory agent)
The present invention also relates to a method for treating and / or preventing inflammation in a subject, wherein the method administers to a subject in need thereof an effective amount of fermented Glycine max (L.) extract. Comprising fermenting the aqueous Glycine max (L.) extract with at least one lactic acid bacterium together with at least one yeast, wherein the fermented Glycine max (L.) extract comprises Prepared by

  Fermented Glycine max (L.) extract is anti-inflammatory at a dose of 10 ml / kg against the reduction of carrageenin-induced hind limb edema in rats and the anti-inflammatory effect against acute and chronic arthritis in adjuvant arthritis studies. Inflammatory effect was demonstrated.

(Use to prevent or treat infection)
Fermented Glycine max (L.) extract has demonstrated antimicrobial activity in vitro and in vivo. This is due to Helicobacter pylori, ampicillin and methicillin resistant Staphylococcus aureus, Salmonella typhimurium, Bacillus subtilis, E. et al. Inhibits the growth of E. coli, Proteus vulgaris and enterococci resistant to vancomycin. Preferably, the enterococcus resistant to vancomycin is E. coli. avium, E.I. caseliflavus, E.I. durans, E .; faecalis and E.I. Selected from the group consisting of faecium. More preferably, vancomycin-resistant Enterococcus is E. coli. avium, E.I. caseliflavus, E.I. durans and E.M. Selected from the group consisting of faecali. More preferably, vancomycin-resistant Enterococcus is E. coli. durans, E .; faecalis and E.I. Selected from the group consisting of faecium. More preferably, vancomycin-resistant Enterococcus is E. coli. avium, E.I. faecalis and E.I. Selected from the group consisting of faecium. More preferably, vancomycin-resistant Enterococcus is E. coli. faecalis and E.I. Selected from the group consisting of faecium. Most preferably, the enterococcus resistant to vancomycin is E. coli. faecalis.

  Effective concentrations of fermented Glycine max (L.) extract are generally in the range of 1-10%. Selective antibacterial decontamination of fermented Glycine max (L.) extract for prevention of bacterial infection in patients (at risk of developing neutropenia due to combined treatment with anti-cancer chemotherapy) The effect has also been demonstrated in 100 patients. Preferably, the enterococcus resistant to vancomycin is E. coli. avium, E.I. caseliflavus, E.I. durans, E .; faecalis and E.I. Selected from the group consisting of faecium. More preferably, vancomycin-resistant Enterococcus is E. coli. avium, E.I. caseliflavus, E.I. durans and E.M. Selected from the group consisting of faecali. More preferably, vancomycin-resistant Enterococcus is E. coli. durans, E .; faecalis and E.I. Selected from the group consisting of faecium. More preferably, vancomycin-resistant Enterococcus is E. coli. avium, E.I. faecalis and E.I. Selected from the group consisting of faecium. More preferably, vancomycin-resistant Enterococcus is E. coli. faecalis and E.I. Selected from the group consisting of faecium. Most preferably, the enterococcus resistant to vancomycin is E. coli. faecalis.

(Administration of fermented Glycine max (L.) extract)
In the present invention, the fermented Glycine max (L.) extract can be used alone or a composition comprising the fermented Glycine max (L.) extract and a pharmaceutically acceptable carrier, diluent and / or excipient. Can be administered in a product. Preferably, the fermented Glycine max (L.) extract is a fermented soy extract or a fermented black soy extract. The fermented Glycine max (L.) extract can be administered at a dose of about 0.001 to 40 ml / kg body weight (2000 ml maximum dose per person per dose). Preferably, the dose of fermented Glycine max (L.) extract is 0.01-20 ml / kg, more preferably 0.1-5 ml / kg body weight of the subject. These doses are based on fermented Glycine max (L.) extract in concentrated form, but the appropriate dose of fermented Glycine max (L.) extract in unconcentrated or dry powder form is Can be calculated. This dose can be adjusted based on the health condition of the subject or the disease to be prevented or treated.

  Fermented Glycine max (L.) extract has demonstrated high safety for daily intake of 1-10 ml on a long-term basis in a 6-month chronic toxicity study in rodents. Mice receiving doses of 10 ml / kg and 1 ml / kg over 28 days did not show any significant differences or abnormal symptoms in subacute oral toxicity studies. In a rodent subacute oral toxicity study, no signs of severe toxicity or mortality were observed in two groups of test animals dosed with 20 ml / kg and 1 ml / kg. Fermented Glycine max (L.) extract is non-mutagenic in the Ames test, does not cause chromosomal damage in mammalian cells in vitro, and does not induce micronuclei in bone marrow cells of ICR mice tested Proved.

  When fermented Glycine max (L.) extract is administered to a pregnant woman, the dosage of fermented Glycine max (L.) extract is increased during pregnancy until the daily intake reaches 12 ml. obtain. The fermented Glycine max (L.) extract can be administered during early and mid-gestation and at birth. This result indicated that the fermented Glycine max (L.) extract could improve symptoms commonly seen in pregnancy, including constipation, nausea, vomiting and gastrointestinal discomfort. Furthermore, administration of fermented Glycine max (L.) extract can reduce gestational and birth abnormalities. The fermented Glycine max (L.) extract is not only good for improving health during pregnancy, but also does not produce harmful effects as a long-term dietary supplement. Daily administration of fermented Glycine max (L.) extract to a newborn or infant increases the weight of the newborn or infant. Similarly, increased body weight can be achieved in a nursing mother's infant who is continually ingesting fermented Glycine max (L.) extract.

  The fermented Glycine max (L.) extract was also added to 1 ml fermented Glycine max (L) along with other treatment products for women who had surgery after hospitalization excluding the day of surgery and several days after surgery. .) Can enhance post-surgical hematopoietic and liver function as demonstrated through daily administration of the extract.

  The invention will now be described with reference to the following non-limiting examples.

  LOX-15 is a major metabolic enzyme in arachidonic acid (AA) metabolism. One of the metabolic pathways of AA relates to LOX-5, a 15-lipoxygenase, which leads to the formation of HETE (hydroxyeicosatetraenoic acid). HETE has been reported to play an important role in cancer cell metastasis. HETE can induce protein kinase C activity that results in cancer cell metastasis. HETE is also a mitogenic factor that causes angiogenesis of cancer cells. LOX-15 was isolated from rabbit reticulocytes. Linoleic acid was used as a substrate for LOX-15 with or without fermented soy extract. The amount of HETE formed was determined with a spectrophotometer. This data indicates that the published soy extract has an inhibitory effect on LOX-15 (see FIG. 1). This result indicated that the fermented soy extract inhibits neovascularization and metastasis of cancer cells and induces apoptosis of cancer cells.

  Further LOX-15 inhibition tests were performed according to the method described above to compare the inhibitory effect of LOX-15 between unfermented soy extract and fermented soy extract. These results indicate that the inhibitory effect of the fermented soybean extract was more than four times that of the unfermented soybean extract.

  Fermented black soybean extract was tested in rabbit reticulocytes for inhibitory effect on LOX-15. This test method refers to the method described in Analytical Biochemistry, 1992, 201, 375-380. 1%, 0.5%, 0.1%, 0.05% and 0.01% (v / v) black soybean extracts were used in reticulocyte incubation at 4 ° C. for 10 minutes. The incubation buffer is 0.05M potassium phosphate buffer (pH 5.9). The resulting 13-HPODE is quantified with a spectrophotometer.

  The relationship between the concentration of black soybean and its inhibition rate against LOX-15 is shown in FIG.

  Further LOX-15 inhibition tests were performed according to the method described above to compare the inhibitory effect of LOX-15 between unfermented black soybean extract and fermented black soybean extract. The LOX-15 inhibition rates of unfermented black soybeans are shown in Table 1 below:

The LOX-15 inhibition rate of the fermented black soybean extract is shown in Table 2 below.

The results shown in the table above show that the fermented black soybean extract has an unexpectedly superior effect in inhibiting LOX-15.

The fermented soy extract functioned as an antioxidant and in the removal of free radicals. Several previously published models were used to study the antioxidant capacity of fermented soy extracts (vitamin C and Trolox were used as positive controls). The following methods were used to determine antioxidant activity: (1) NBT method (2) H ₂ O ₂ reduction method (3) DPPH reduction (4) TRAP reduction method (5) conjugated diene (6 ) Lipid peroxidation (7) Chemiluminescence in the presence of active oxygen (see Figures 3, 4, and 5). All the results demonstrated that the fermented soy extract has the highest antioxidant activity against unsaturated fatty acids and peroxides compared to vitamin C and Trolox.

  Experiments have demonstrated that the fermented soy extract functions as an antioxidant and free radical acceptor in the Okubo test system for chemiluminescent acceptors in the presence of active oxygen. This experiment was carried out by measuring chemiluminescence in hydrogen peroxide liquid with acetaldehyde or hydrogen peroxide liquid without acetaldehyde. Known antioxidants (eg, gallic acid (see FIG. 3 (b)), EGC, tea, and vitamin C (see FIGS. 4 (a)-(c))) or fermented soy extract (See Figure 5) was added in 200 seconds. This data is shown in FIGS. FIG. 3 (b) shows that chemiluminescence increased in 200 seconds when gallic acid was added to a mixture of hydrogen peroxide and acetaldehyde. FIG. 4 shows that when EGC, tea, or vitamin C was added at 200 seconds, chemiluminescence increased when acetaldehyde was present. However, chemiluminescence also increased in the absence of acetaldehyde when vitamin C was added at 200 seconds (see FIG. 4 (c)). This demonstrates that the antioxidant mechanism of vitamin C is probably different from that of EGC and tea. FIG. 5 shows that chemiluminescence increased after the fermented soy extract was added in 200 seconds, indicating that the fermented soy extract is a powerful antioxidant. The antioxidant activity of the fermented soy extract means that the fermented soy extract can function in removing free radicals. With antioxidant and free radical scavenging functions, fermented soy extracts are useful in promoting the overall health of an individual or in improving the health of a subject having a need for improved health. This is because oxidative stress (eg, excessive presence of reactive oxygen species and lipid peroxides) is known to be harmful to the body.

  The antimicrobial activity of the fermented soybean extract was demonstrated by determining using in vitro methods. In a first experiment, three strains of Salmonella typhimurium, Bacillus subtilis, Helicobacter pylori (TMU-C74, TMU-D16, and TMU-E86), and vancomycin-resistant Enterococcus fecalis in broth or B Transfer to Mueller Hinton agar plate or chocolate agar plate. On this agar plate, the fermented soy extract was placed on a paper disk and the size of the inhibition zone was measured after incubation at 37 ° C. These data are shown in Table 3 below.

In another experiment, the minimum inhibitory concentration (MIC) of fermented soy extract was determined in Salmonella typhimurium (ATCC 14028), Bacillus subtilis (CRCC 10447), Staphylococcus aureus (ATCC 25923), and vancomycin-resistant Enterococcus. The suspension of these bacteria was adjusted to 3 × 10 ⁵ CFU / ml. Adjusted bacterial suspensions with various concentrations (ie 10%, 5%, 2.5%, 1.25%, 0.65%, or 0.32%) of fermented soy extract or fermented The soy extract was not added to the 96 well plate. The plate was incubated at 37 ° C. for 15 hours. The MIC was determined after incubation and is shown in Table 4 below.

Fermented black soy extract was also used to test antimicrobial activity. The MIC was determined and is shown in Table 5 below.

The effect of fermented soy extract on immune regulation was studied.
(A) In vitro study (spleen cell proliferation assay (MTT method))
Spleen cells isolated from mice and RPMI medium containing one of several mitogens (ie lipopolysaccharide (LPS), concavalin A (Con A), and plant hemagglutinin (PHA)) or 2 × 10 ⁶ cells / ml in RPMI medium without aliquots in culture flasks. Spleen cell cultures were incubated overnight for MTT assay.

  Suboptimal concentrations of 5 μg / ml LPS mixed with 1%, 0.5%, 0.1%, 0.05%, or 0.01% fermented soy extract can result in spleen cells (particularly B cells) Has no effect on proliferation. PHA at a concentration of 5 μg / ml mixed with 0.05% fermented soy extract increased the number of spleen cells (especially T cells), which is 2.32 of the number of spleen cells obtained with PHA alone. It was twice. According to this result, the fermented soy extract has an effect on T cell and B cell interactions in immunomodulation. Con A at a concentration of 5 μg / ml mixed with 0.05% fermented soy extract resulted in a spleen cell count that was approximately 20% less than the spleen cell count obtained with Con A alone. According to this result, fermented soy extract may play a role in the anti-inflammatory response.

(Macrophage activity assay)
Balb / c mice were injected with thiogallate. Three to four days after injection, macrophages were isolated from the peritoneal cavity of mice and incubated with or without fermented soy extract for 30 minutes at 37 ° C. E. Fluorescent probe bound. E. coli cells were added to the macrophage suspension and incubated for 2 hours at 37 ° C. Phagocytosis assay was performed using flow cytometry. This data indicated that 0.05% fermented soy extract enhanced macrophage phagocytic activity by approximately 71% compared to macrophages not treated with fermented soy extract.

(B) In vivo studies Male ICR albino mice were treated with vehicle, 0.8 ml 1% fermented soy extract per mouse, 0.8 ml 0.1% fermented soy extract per mouse, 30 mg / kg. Of levamisole or 100 mg / kg azimexone was injected intraperitoneally. One hour after intraperitoneal injection, 1.5-2 × 10 ⁷ CFU / mouse of Candida albican (ATCC 10231) were injected intraperitoneally. Mice mortality was determined daily for 10 days (see Table 6).

  As shown in Table 6, fermented soy extract reduced Candida albican mortality in mice. The mortality reduction effect of the fermented soybean extract was more prominent than that of levamisole.

  Male ICR albino mice were pretreated with 30 mg / kg cyclophosphamide 5 days, 3 days, and 1 day before intraperitoneal injection of Candida albican. Mice were treated with vehicle, 0.1% fermented soy extract, 1% fermented soy extract, or 100 mg / kg azimexone 6 days, 4 days, and 2 days before intraperitoneal injection of Candida albican. Mice mortality was determined daily for 10 days (see Table 7). As shown in Table 7, with pretreatment with cyclophosphamide, fermented soy extract reduced Candida albican mortality in mice. The mortality reduction effect of this fermented soybean extract was comparable to that of azimexone.

I. p. One hour after dosing, animals were infected intraperitoneally with 1.5-2 × 10 ⁷ CFU / mouse Candida albicans (ATCC 10231). Statistical significance was determined using Fisher's exact test: ^* P <0.05, ^** P <0.01.
Note: 1% in 0.8 ml / mouse corresponds to 0.4 mg / kg in the first fermented soy extract (100%), 0.1% in 0.8 ml / mouse is the first fermented soy extract Equivalent to 0.04 mg / kg in (100%).

a: Vehicle: PBS not pretreated with cyclophosphamide (pH = 7.4)
b: CY: p. 5 days, 3 days, and 1 day before injection o. Administered cyclophosphamide 30 mg / kg. Fermented soy extract or azimexone i.v. with 0.8 ml 6 days, 4 days and 2 days before injection. p. Administered.
On day 0, mice were i.d. with 6-8 × 10 ⁶ CFU / mouse Candida albicans (ATCC 10231). v. I challenged. Statistical significance was determined using Fisher's exact test: ^* P <0.05, ^** P <0.01.

  To evaluate the effect of fermented soy extract in the treatment of cardiovascular disease. Tested in apoE deficient mice, which simultaneously have human-like hypercholesterolemia and atherosclerotic injury. ApoE deficient mice were given 10 ml / g fermented soy extract (concentration 15%) for 3 months, and then the level of plasma cholesterol and the extent of atherosclerotic injury were measured. Treatment using the fermented soy extract of the present invention was found to have no significant effect on plasma cholesterol levels, but atherosclerosis in mice fed the fermented soy extract of the present invention. Injury was significantly reduced compared to controls (see FIGS. 6, 7 and 8). According to these results, the effect of the fermented soy extract of the present invention on treating cardiovascular disease (eg, atherosclerosis) is a mechanism that inhibits 15-lipoxygenase rather than reducing plasma cholesterol levels. Achieved by:

  The present invention relates to a fermented Glycine max (L. cerevisiae) prepared by fermenting an aqueous Glycine max (L.) extract with at least one lactic acid bacterium with at least one yeast in inhibiting 15-lipoxygenase. ) Regarding the use of the extract. In particular, fermented Glycine max (L.) extract prevents diseases associated with inhibition of 15-lipoxygenase in a subject (eg, cardiovascular disease, immune disorders (eg, asthma and inflammation) and / or It can be used in treating as well as in regulating the immune system The present invention also relates to the use of fermented Glycine max (L.) extract in treating and / or preventing bacterial infections.

(References)
Adlercreuz, H.C. et al. , Evaluation nu- lation, intestinal microflora and prevention of cancer: a hypothesis, Proc. Soc. Exp. Biol. Med. , 217: 241-246 (1998).
Breimer LH. Ionizing Radiation-Induced Mutagenesis, Br J Cancer, 57: 6-18 (1998).
Briehl, M.M. M.M. et al. Modulation of the antioxidant defense as a factor in apoptosis, Cell Death Differ. 3: 63-70 (1996).
Chemoprevention Working Group to the American Association for Cancer Research, Cancer Res. 59: 4743-4758 (1999).
Cohen, L. A. et al. , Effect of intact and isoflavone-depleted soybean protein on NMU-induced rat mammary tumorigenesis, Carcinogenesis, 2: 929-935 (2000).
Dwyer, J .; T.A. et al. , Tofu and soybean drinks containers phytoestrogens, J. et al. Am. Diet Assoc. 94: 739-743 (1994).
Ghibelli, L .; et al. Rescue of cells from apoptosis by active GSH extension, FASEB J. 12: 479-486 (1998).
Greenwald, P.M. et al. Chemovention, CA-Cancer J., Chem. Clin. 45: 31-49 (1995).
Hong, W .; K. et al. , Recent advancements in chemoprevention of cancers, Science, 278: 1073-1077 (1993).
Hutchins, A.M. M.M. et al. , Uriary isoflavoneoid phytoestrogen and lignan excretion after consumption of fermented and unsupported soybean products, J. Am. Diet Assoc. 95: 545-551 (1995).
Ikeda, Y .; et al. , The molecular basis of brain injury and brain edema: the role of oxygen free radicals, Neurosurgery, 27: 1-11 (1990).
Keisari, Y. et al. et al. , A simple colorimetric method for the measurement of hydrogen produced by cells in culture, J. Am. Immunol Methods. 38: 161-170 (1980).
Kelloff, G.M. J. et al. , Approaches to the development and marketing approval of drugs that present cancer, Cancer Epidormiol. Biomarkers Pre. , 4: 1-10 (1995).
Kontos HA et al. Oxygen radicals in brain injury, CNS Trauma, 3: 257-63 (1986).
Messina, M .; et al. , Soybean intake and cancer risk: a review of the in vitro and in vivo data, Nutr. Cancer, 21: 113-131 (1994).
Nout, M .; J. et al. R. et al. , Reent development in template research, J. et al. Appl. Bacteriol. 69: 609-633 (1990).
Programmer, H .; J. et al. et al. , Reactive oxygen specifications and antioxidants in signal transduction and gene expression, Nutr. Rev, 55: 353-361 (1997).
Robak J.M. et al. , Flavonoids are scavengers of superoxide anions, Biochemical Pharmacology, 37 (5): 837-41 (1988).
Shao, Z .; M.M. et al. Genistin extremes multisuppressive effects on human breast carcinoma cells, Cancer Res. 58: 4851-4857 (1998).
Steinberg D.M. et al, Beyond cholesterol: modification of low-density lipoprotein that increase it aerogenicity, N Engl J Med, 320: 915-24 (1989).
Toshiki, Y. et al. et al. , Mechanism of catechin chemiluminescence in the presence of active oxygen, J. et al. Biolumin. Chemilumin. 11: 131-136 (1996).
Wang, H .; et al. , Isoflavone content of commercial soy foods, J. Agric. Food Chem. 42: 1666-1673 (1994).

FIG. 1 shows inhibition of LOX-15 using fermented soy extract. FIG. 2 shows the relationship between the concentration of black soybean and the rate of inhibition against LOX-15. FIG. 3 shows the antioxidant effect of gallic acid. After adding H ₂ O ₂ as a peroxide (X), gallic acid as an antioxidant (Y), and / or acetaldehyde as a radical receptor (Z) at a temperature of 37 ° C., the CL count of chemiluminescence is measured did. In FIG. 3 (a), curve A represents chemiluminescence after adding X and Y together; curve B represents chemiluminescence after adding Y and Z together; and curve C is Represents chemiluminescence after addition of X, Z and Z together. FIG. 3 (b) shows that chemiluminescence increased only when gallic acid was added to the mixture of X and Z at 200 seconds (ie, when all of X, Y and Z were present together). Is happening). FIG. 3 shows the antioxidant effect of gallic acid. After adding H ₂ O ₂ as a peroxide (X), gallic acid as an antioxidant (Y), and / or acetaldehyde as a radical receptor (Z) at a temperature of 37 ° C., the CL count of chemiluminescence is measured did. In FIG. 3 (a), curve A represents chemiluminescence after adding X and Y together; curve B represents chemiluminescence after adding Y and Z together; and curve C is Represents chemiluminescence after addition of X, Z and Z together. FIG. 3 (b) shows that chemiluminescence increased only when gallic acid was added to the mixture of X and Z at 200 seconds (ie, when all of X, Y and Z were present together). Is happening). FIG. 4 shows the antioxidant effect of tea and vitamin C. At a temperature of 37 ° C., chemiluminescence (CL count) was measured as H ₂ O ₂ as a peroxide when acetaldehyde (Z) was present as a radical receptor or when acetaldehyde (Z) was not present. And one of several antioxidants (ie EGC, tea and vitamin C) was added at 200 seconds and measured. FIG. 4 (a) shows the chemiluminescence determined when the polyphenol EGC (ie epigallocatechin) was added with or without acetaldehyde in 200 seconds as an antioxidant. Indicates. FIG. 4 (b) shows that chemiluminescence emitted in the absence of acetaldehyde (+ Z) is 5.79% of chemiluminescence in the presence of acetaldehyde when tea is added as an antioxidant in 200 seconds. Indicates that there is. FIG. 4 (c) shows that when vitamin C was used as an antioxidant, the chemiluminescence intensity in the absence of acetaldehyde was 64.13% of the detected intensity in the presence of acetaldehyde. Is shown. FIG. 4 shows the antioxidant effect of tea and vitamin C. At a temperature of 37 ° C., chemiluminescence (CL count) was measured as H ₂ O ₂ as a peroxide when acetaldehyde (Z) was present as a radical receptor or when acetaldehyde (Z) was not present. And one of several antioxidants (ie EGC, tea and vitamin C) was added at 200 seconds and measured. FIG. 4 (a) shows the chemiluminescence determined when the polyphenol EGC (ie epigallocatechin) was added with or without acetaldehyde in 200 seconds as an antioxidant. Indicates. FIG. 4 (b) shows that chemiluminescence emitted in the absence of acetaldehyde (+ Z) is 5.79% of chemiluminescence in the presence of acetaldehyde when tea is added as an antioxidant in 200 seconds. Indicates that there is. FIG. 4 (c) shows that when vitamin C was used as an antioxidant, the chemiluminescence intensity in the absence of acetaldehyde was 64.13% of the detected intensity in the presence of acetaldehyde. Is shown. FIG. 4 shows the antioxidant effect of tea and vitamin C. At a temperature of 37 ° C., chemiluminescence (CL count) was measured as H ₂ O ₂ as a peroxide when acetaldehyde (Z) was present as a radical receptor or when acetaldehyde (Z) was not present. And one of several antioxidants (ie EGC, tea and vitamin C) was added at 200 seconds and measured. FIG. 4 (a) shows the chemiluminescence determined when the polyphenol EGC (ie epigallocatechin) was added with or without acetaldehyde in 200 seconds as an antioxidant. Indicates. FIG. 4 (b) shows that chemiluminescence emitted in the absence of acetaldehyde (+ Z) is 5.79% of chemiluminescence in the presence of acetaldehyde when tea is added as an antioxidant in 200 seconds. Indicates that there is. FIG. 4 (c) shows that when vitamin C was used as an antioxidant, the chemiluminescence intensity in the absence of acetaldehyde was 64.13% of the detected intensity in the presence of acetaldehyde. Is shown. FIG. 5 shows the antioxidant effect at various concentrations of fermented soybean extract (FSE). At a temperature of 37 ° C., chemiluminescence was measured using H ₂ O ₂ as a peroxide in the presence or absence of acetaldehyde as a radical receptor (Z) and fermented soy extract at various concentrations. In 200 seconds. FIG. 5 (a) shows that at a 1: 1 FSE concentration, the chemiluminescence intensity in the absence of acetaldehyde was 44.13% of the emission intensity in the presence of acetaldehyde. FIG. 5 (b) shows that at an FSE concentration of 1:10, the chemiluminescence intensity in the absence of acetaldehyde was 63.64% of the intensity in the presence of acetaldehyde. FIG. 5 (c) and FIG. 5 (d) show that at an FSE concentration of 1: 100 or 1: 500, the chemiluminescence intensity in the absence of acetaldehyde is at least 90% of the emission intensity in the presence of acetaldehyde. It shows that there was. FIG. 5 shows the antioxidant effect at various concentrations of fermented soybean extract (FSE). At a temperature of 37 ° C., chemiluminescence was measured using H ₂ O ₂ as a peroxide in the presence or absence of acetaldehyde as a radical receptor (Z) and fermented soy extract at various concentrations. In 200 seconds. FIG. 5 (a) shows that at a 1: 1 FSE concentration, the chemiluminescence intensity in the absence of acetaldehyde was 44.13% of the emission intensity in the presence of acetaldehyde. FIG. 5 (b) shows that at an FSE concentration of 1:10, the chemiluminescence intensity in the absence of acetaldehyde was 63.64% of the intensity in the presence of acetaldehyde. FIG. 5 (c) and FIG. 5 (d) show that at an FSE concentration of 1: 100 or 1: 500, the chemiluminescence intensity in the absence of acetaldehyde is at least 90% of the emission intensity in the presence of acetaldehyde. It shows that there was. FIG. 5 shows the antioxidant effect at various concentrations of fermented soybean extract (FSE). At a temperature of 37 ° C., chemiluminescence was measured using H ₂ O ₂ as a peroxide in the presence or absence of acetaldehyde as a radical receptor (Z) and fermented soy extract at various concentrations. In 200 seconds. FIG. 5 (a) shows that at a 1: 1 FSE concentration, the chemiluminescence intensity in the absence of acetaldehyde was 44.13% of the emission intensity in the presence of acetaldehyde. FIG. 5 (b) shows that at an FSE concentration of 1:10, the chemiluminescence intensity in the absence of acetaldehyde was 63.64% of the intensity in the presence of acetaldehyde. FIG. 5 (c) and FIG. 5 (d) show that at an FSE concentration of 1: 100 or 1: 500, the chemiluminescence intensity in the absence of acetaldehyde is at least 90% of the emission intensity in the presence of acetaldehyde. It shows that there was. FIG. 5 shows the antioxidant effect at various concentrations of fermented soybean extract (FSE). At a temperature of 37 ° C., chemiluminescence was measured using H ₂ O ₂ as a peroxide in the presence or absence of acetaldehyde as a radical receptor (Z) and fermented soy extract at various concentrations. In 200 seconds. FIG. 5 (a) shows that at a 1: 1 FSE concentration, the chemiluminescence intensity in the absence of acetaldehyde was 44.13% of the emission intensity in the presence of acetaldehyde. FIG. 5 (b) shows that at an FSE concentration of 1:10, the chemiluminescence intensity in the absence of acetaldehyde was 63.64% of the intensity in the presence of acetaldehyde. FIG. 5 (c) and FIG. 5 (d) show that at an FSE concentration of 1: 100 or 1: 500, the chemiluminescence intensity in the absence of acetaldehyde is at least 90% of the emission intensity in the presence of acetaldehyde. It shows that there was. FIG. 6 shows the effect of fermented soy extract on total plasma cholesterol levels in apoE deficient mice. FIG. 7 shows the effect of fermented soy extract on lesion formation in ApoE deficient mice. FIG. 8 shows the staining results of aortic damage derived from ApoE-deficient mice.

Claims (6)

Use of a fermented Glycine max (L.) extract for the production of a composition for use in the inhibition of 15-lipoxygenase in a subject, the fermented Glycine max (L.) extract comprising at least 1 Prepared by fermenting an aqueous Glycine max (L.) extract using at least one lactic acid bacterium together with at least one yeast, and inhibition of the 15-lipoxygenase reduces the risk of cardiovascular disease Use, used to treat or reduce. The use according to claim 1, wherein the Glycine max (L.) is black soybean. The use according to claim 1, wherein the lactic acid bacterium is a Lactobacillus species. Use according to claim 1, wherein the yeast is a Saccharomyces species. The use according to claim 1, wherein the cardiovascular disease is atherosclerosis. The use according to claim 1, wherein the composition is a pharmaceutical composition or a food composition.