Patent Application: US-19253402-A

Abstract:
the present invention relates to the preparation of therapeutic compositions comprising of fermented soy extracts made with lactic acid bacteria and the optional addition of at least a yeast . the invention also relates to therapeutic uses of such extracts in promoting general health , improving the health of subjects , preventing and / or treating cancer , preventing infections , reducing the incidence of infections , treating infections , treating asthma , treating inflammation , modulating the immune system and treating immune disorders .

Description:
the fermented soy extract is produced by fermentation of soy bean extract with at least one lactic acid bacteria , e . g . one or more strains of a lactobacillus species or several strains of a number of lactobacillus species , optionally together with at least one yeast , e . g . a strain of a saccharomyces species . if more than one microbe is used in the fermentation , the fermentation can be conducted with the microbes sequentially or simultaneously . preferably , an aqueous extract of non - genetically modified organic soybeans of selected grade is used as a starting material . preferably , the fermentation is carried out using a heterogeneous culture of lactobacillus , for example , a culture of 5 , 10 , 15 , 20 , 25 or 30 strains of lactobacillus . more preferably , at least one yeast is added to the heterogeneous culture of lactobacillus . the strains of lactobacillus that can be used include , for examples , lactobacillus acidophilus ccrc 10695 , 14026 , 14064 , 14065 and / or 14079 , lactobacillus delbrueckii bulgaricus ccrc 10696 , 14007 , 14009 , 14010 , 14069 , 14071 , 14098 and / or 16054 , lactobacillus lactis lactis ccrc 10791 , 12267 , 12306 , 12312 , 12315 , 12323 , 14016 , 14015 and / or 14117 , lactobacillus kefir ccrc 14011 , and / or lactobacillus kefiranofaciens ccrc 16059 . the yeast that can be used include , for example , saccharomyces cerevisiae ccrc 20577 , 20578 , 20581 , 21494 , 21550 , 21797 , 21805 , 22138 , 22234 , 22337 , 22731 and / or 22728 , and / or candida kefyr ccrc 21269 , 21742 and / or 22057 . after fermentation , the fermented liquid is sterilized , e . g . by heat or irradiation , preferably by heat , to obtain a sterilized liquid . preferably , the sterilized liquid is filtered or centrifuged , preferably filtered , to remove most or all of the dead microbes to obtain the fermented soy extract . more preferably , the filtration step is followed by removal of some of the water from the filtrate to concentrate the fermented liquid to obtain the fermented soy extract . unless otherwise specified , the tests performed in this application involved the fermented soy extract after the concentration step . optionally , the fermented soy extract can be dried , e . g . via lyophilization , to obtain the fermented soy extract in a powder form . the process can be carried out by mixing organic soybean ( with fat removed ) with distilled water at a ratio of 1 : 10 . the mixture is heated at 100 ° c . for 30 minutes and then filtered to obtain a soy extract . beef and kelp are boiled in distilled water for 30 minutes to obtain a broth . salt , sugar and agar are added to produce a special agar medium . the lactic acid bacteria and yeast strains are added to the special agar medium . the lactic acid bacteria with the optional inclusion of the yeast in the medium are transferred to the soy extract and incubated at 36 - 43 ° c . for 45 - 50 hours . preferably , the various strains of the microbes are grouped according to similar growth characteristics , e . g . any requirements of unique nutrient medium , whether the microbial strains could produce a good smell after fermentation and whether the grouped microbes can survive in the unique condition , so that groups of the microbes are added to the soy extract separately before the incubation . the purpose of this step is to reduce any negative interaction among the various strains . also preferably , equal proportion of the different groups of microbial strains are added to the soy extract before the incubation and the resulting extract is incubated at 40 ° c . for 45 - 47 hours . upon completion of the incubation period , the heterogeneous culture is then transferred to the soy extract again and incubated at 36 - 43 ° c . for 100 - 150 hours . the final fermented extract is heat sterilized and filtered ; and 95 % of the water content of the filtrate is removed in a concentrator to obtain a fermented soy extract in a concentrated or condensed form . the upper layer is then filtered through porcelain , and thereafter dispensed in containers and sealed . a fermented soy extract was prepared as described above . the specific gravity of the fermented soy extract was 1 . 136 g / ml with 71 . 49 % moisture , 5 . 15 % ash , 0 . 16 % crude fat , 5 . 45 % crude protein , 0 . 15 % crude fiber , and carbohydrate . it also contained several vitamin and minerals : vitamin b1 , 0 . 004 mg / 100 g ; vitamin b2 , 0 . 12 mg / 100 g ; iron , 2 . 17 mg / 100 g ; calcium , 113 . 55 mg / 100 g and phosphorous , 379 . 19 mg / 100 g . in this invention , the fermented soy extract may be administered alone or in a composition comprising the fermented soy extract and a pharmaceutically acceptable carrier , diluent and / or excipient . the fermented soy extract may be administered at a dose of about 0 . 001 to 40 ml / kg body weight , with a maximum dose of 2000 ml per person per administration . preferably , the dose of the fermented soy extract is 0 . 01 to 20 ml / kg , more preferably 0 . 1 to 5 ml / kg , body weight of the subject . these doses are based on the fermented soy extract in the concentrated form , but appropriate doses of the fermented soy extract in the unconcentrated form or dry powder form can be calculated accordingly . the dose can be adjusted based on the health condition of the subject or the disease to be prevented or treated . the fermented soy extract was demonstrated to be highly safe for daily intake of 1 - 10 ml on a long - term basis in a 6 months chronic toxicity study of rodents . mice receiving a dose of 10 ml / kg and 1 ml / kg for 28 days did not exhibit any significant difference or abnormal symptom in a subacute oral toxicity study . no signs of gross toxicity or mortality were observed in two groups of tested animals administered 20 ml / kg and 1 ml / kg in an acute oral toxicity study of rodents . the fermented soy extract was demonstrated to be non - mutagenic in ames test , to not cause chromosomal damage in mammalian cells in vitro and to not induce micronuclei in bone marrow cells in icr mice tested . when the fermented soy extract is administered in pregnant women , the dosage of the fermented soy extract can be increased during pregnancy until the daily intake reaches 12 ml . the fermented soy extract can be administered at early and midstage pregnancy , as well as delivery . results showed that the fermented soy extract could improve symptoms , including constipation , nausea , vomiting , and gastrointestinal discomfort , commonly found in pregnancy . in addition , the administration of the fermented soy extract can reduce abnormalities during pregnancy and at delivery . the fermented soy extract is not only good for health improvement during pregnancy , but it also produces no adverse effect as a long - term dietary supplement . daily administration of the fermented soy extract to newborns or infants daily increases weight gain of the babies or infants . similarly , increased weight gain can be achieved in infants of nursing mothers continuously taking the fermented soy extract . the fermented soy extract can also enhance hemopoeitic and liver functions after a surgical operation as demonstrated through daily administration of 1 ml of the fermented soy extract along with other therapeutic products to women undergoing operation after hospital admission except for the surgery day and several post - surgery days . the fermented soy extract has prominent antioxidant and free radical scavenger activities . the fermented soy extract can remove superoxide free radicals , e . g . o 2 — h 2 o 2 , roo , and can act as an antioxidant for unsaturated fatty acid and fat . the fermented soy extract has a prominent ability to eliminate hyper oxygen anions to protect the cell from oxidative injury and change free radicals to harmless substances with an energy decreasing procedure . the fermented soy extract has demonstrated antimicrobial activity in vitro . it inhibits the growth of helicobacter pylori , ampicillin and methycillin resistant staphylococcus aureus , salmonella typhimurium , bacillus subtilis , e . coli , proteus vulgaris and vancomycin resistant enterococcus feacalis . the effective concentration is generally in the range of 1 - 10 %. the selective antimicrobial decontamination effect of fermented soy extract for prophylaxis of bacterial infection in patients who are under risk of developing neutropenia due to the concurrent treatment of anti - cancer chemotherapy is also demonstrated in 100 patients . the fermented soy extract has demonstrated anti - inflammatory effect at dosage of 10 ml / kg on the reduction of carrageenan induced hind paw edema in rats and anti - inflammatory effect on acute and chronic arthritis in adjuvant arthritis test . the fermented soy extract is beneficial to asthmatic children . results obtained also showed significant body weight gain in a group of children with asthma when administered with 3 ml of the fermented soy extract daily for 4 months . blood tests showed that taking fermented soy extract can increase the rbc and hb levels in these asthmatic children . in vitro study indicated that the fermented soy extract improved immune function . the effect of the fermented soy extract on modulation of the immunity of animals ( bala / c mice ) was studied by treating the animal with the fermented soy extract combined with or without a challenge with various mitogens including lipopolysachrride , concanavalin a and phytohaemagglutilin . spleen cell proliferation assay indicated that the fermented soy extract could be related with t & amp ; b cell interaction in immunity modulation . the fermented soy extract can also be correlated with anti - inflammation reaction . the soy extract also enhanced phagocytosis activity of macrophages by 71 %. similar results were found with in vivo studies in mice . it was also demonstrated that the anti - tumor effect of fermented soy extract is mediated by cytokines released . conditioned medium from fermented soy extract - stimulated peripheral blood mononuclear cells by 45 - 56 %. levels of interleukin - 1b , interleukin - b and tumor necrosis factor - a were much higher than those of untreated control . since untreated macrophages and t lymphocytes produced little or no cytokine and normal mononuclear cells did not suppress leukemic cell growth , the anti - tumor activity is speculated to be derived from elevated level of cytokine . studies have demonstrated that the fermented soy extract can inhibit lipoxygenases which are highly expressed in most of malignant cancer cells . the lipoxygenase that can be inhibited by the fermented soy extract includes lox - 5 , lox - 12 and / or lox - 15 . the inhibition of lipoxygenases by the fermented soy extract can have antiproliferative effects by modulating signal transduction , modulating growth factor activation and inhibiting oncogene expression . the inhibition of lipoxygenases by the fermented soy extract can also induce apoptosis . the induction of apoptosis by the fermented soy extract can be due to the anti - oxidant activity of the fermented soy extract . the inhibition of lipoxygenases by the fermented soy extract can also inhibit angiogenesis resulting in inhibition of membrane degradation , decreased tumor cell adhesion and motility , and inhibition of metastasis . the inhibition of lipoxygenases by the fermented soy extract can also result in anti - inflammatory activities leading so that the fermented soy extract can prevent tissue damage and modulate immune responses . with the inhibition of lipoxygenase , the fermented soy extract is useful in preventing or treating cancer , asthma , coronary heart disease , cardiac failure , inflammation , allergy , ulcerative colitis , pruritis and dermatitis , and also useful in immunomodulation . arachidonic acid ( aa ) is an essential component of the cell membrane phospholipids , and lox is the main metabolizing enzyme in aa ( arachidonic acid ) metabolism . aa metabolism can result in the generation of mutagens capable of damaging dna and inducing mutations . aa is metabolized via two major biochemical pathways : ( i ) the cyclooxygenase ( cox ) pathway leading to the generation of prostaglandins ( ii ) the lipoxygenase ( lox ) pathway leading the generation of hydroxy ( hetes ) fatty acids . hetes have been reported to play a significant role in cancer cell metastasis , induction of protein kinase c activity , and angiogenesis . therefore the reduced synthesis of lox can result in suppression of tumor growth . the fermented soy extract can be a potential lox inhibitor . the fermented soy extract also contain genestein & amp ; other components that have been reported to inhibit lox . the fermented soy extract of the present invention has anti - cancer activity for the treatment and / or prevention of cancer , whilst overcoming one or more disadvantages of prior art chemotherapeutic agents available for the treatment cancer . the cancer that can be treated with the fermented soy extract includes the most prevalent types of cancer in the human population , namely breast cancer , colon cancer , cervix , prostate , kidney , lung , colon and liver cancers . in cancer cells , the fermented soy extract of the present invention can induce one or more effects of inhibition of cell proliferation , induction of cell differentiation , induction of apoptosis ( programmed cell death ), and / or cell cycle blocking . as a consequence , the compositions of the present invention have wide ranging activity against cancer cells and are accordingly effective in the treatment and / or prevention of cancers including benign prostatic hypertrophy , prostatic cancer , breast cancer , uterine cancer , leukemia , ovarian cancer , endometrial cancer , cervical cancer , colon cancer , testicular cancer , lymphoma , rhabdosarcoma , neuroblastoma , pancreatic cancer , lung cancer , brain tumor , skin cancer , gastric cancer , oral cancer , liver cancer , laryngeal cancer , bladder cancer , thyroid cancer , liver cancer , kidney cancer and nasoharyngeal carcinoma . within the scope of the present invention is a fermented extract of a chinese herb prepared in a process similar to the one described above with the substitution of the soy bean with the chinese herb . the fermented extract of the chinese herbs can be glycyrrhiza uralensis fish , lycium barbarum , coix lacryma - jobi l var ., ma - yine stapf sophora tonkinensis gapnep ., cassia btusifolia ., scutellaria baicalensis georgi , artemisia capillaries thunb ., coptis chinensis frsnch ., gentiana scabra bge ., nelumbo nucifera gaertn ., chrysantheiferamum morifolium ramat ., gardenia jasminoides ellis , hordeum vulgare l ., cinnamomum cassia presl , raph , anus sativus l ., dioscorea opposita thunb ., angelica sinensis ( oliv . ), ligusticum chuanxiong hort ., notopterygium incisum , paeonia lactiflora pall ., allium satium l ., schisandra chinensis ( turcz .) baill , rehmannia glutinosa libosch ., acanthopanax gracilistylus w . w . smith , equus asinus l ., ligustrum lucidum ait ., phaseolus radiatus l ., triticum aestivum l ., dolichos lablab l ., atractylodes macrocephala koidz ., saposhnikovia divaricata , lonicera japonica thund ., cinnamomum cassia presl , zingiber officinale rosc ., gastrodia elata bl ., asparagus cochinchinensis ( liur .) merr ., dendrobiun loddigesii rolfe ., and sesamum indicum l . this invention will now be described with reference to the following non - limiting examples . the fermented soy extract functioned as an antioxidant and in the removal of free radicals . several models published previously were used to study the antioxidant capacity of the fermented soy extract , with vitamin c and trolox used as positive controls . the following methods were used for determining the 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 ( fig1 fig2 fig3 ) in the presence of active oxygen . all results demonstrated that the fermented soy extract has the highest antioxidant activity against unsaturated fatty acid and peroxidation compared with vitamin c and trolox . experiments demonstrated that the fermented soy extract functions both as a antioxidant and free radical acceptor in the okubo test system for chemiluminescence acceptor in the presence of active oxygen . the experiments were performed by measuring chemiluminescence in a liquid of hydrogen peroxide with or without acetaldehyde . known antioxidants , e . g . gallic acid ( fig1 ( b )), egc , tea and vitamin c ( fig2 ( a )-( c )) or the fermented soy extract ( fig3 ) was added at 200 seconds . the data are shown in fig1 - 3 . fig1 ( b ) shows that chemiluminescence was increased at 200 seconds when gallic acid was added to a mixture of hydrogen peroxide and acetaldehyde . fig2 shows that when egc , tea or vitamin c was added at 200 seconds , the chemiluminescence was increased when acetaldehyde was present . however , the chemiluminescence was also increased in the absence of acetaldehyde when vitamin c was added at 200 seconds ( fig2 ( c )) demonstrating that the anti - oxidant mechanism of vitamin c probably differs from that of egc and tea . fig3 shows that , after the addition of the fermented soy extract at 200 seconds , the chemiluminescence increased indicating that the fermented soy extract was a powerful anti - oxidant . the anti - oxidant activity of the fermented soy extract means that the fermented soy extract can function in removing free radicals . with anti - oxidant and free radical removing functions , the fermented soy extract is useful in promoting the general health of individuals or improving the health of subjects in need of health improvement because oxidative stresses , such as excessive presence of reactive oxygen species and lipid peroxidation , are known to be harmful to the body . a human breast cancer cell line mcf - 7 ( atcc htb - 22 ) was used to study the anti - cancer activity of the fermented soy extract . the cytotoxic effects of the fermented soy extract was demonstrated in the cancer cell line ( see fig4 ). compared with the control group of each cell , the value of fermented soy extract treatments was then normalized to reflect cell viability . the result showed that treatment with fermented soy extract at various concentrations ( 0 . 8 mg / ml , 1 . 6 mg / ml , 3 . 2 mg / ml , 8 mg / ml , 16 mg / ml ) for 48 hours caused significant reduction in the viability of mcf - 7 cells . experiments conducted show that strong cytotoxic activities on breast ( mcf7 ), lung ( h460 ) and liver ( hep g2 ) cell lines were detected at low ( 0 . 8 mg / ml ) concentration of the fermented soy extract . maximal cytotoxicities of cervix ( hela ) and lung ( h1299 ) cancer cells were achieved at 3 . 2 mg / ml , whereas kidney ( 293 ) and colon ( ht - 29 ) cells were at 8 mg / ml . among the cell lines tested , mcf - 7 ( breast cancer cell line ) showed the most sensitive response . characterized by electrophoresis as well as quantified by tunel assay and flow cytometry , the fermented soy extract was demonstrated to trigger apoptosis in mcf - 7 cells ( see fig5 ). the apoptotic nuclei increased from 1 . 57 % to 34 . 11 % when the mcf - 7 cells were treated with the fermented extract . the results demonstrated that the reduction of cell viability by fermented soy extract was cause by a successfully triggering of apoptotic cell death , at least , in the case of mcf - 7 cells . the fermented soy extract was diluted with distilled water to make a 2 % solution . severe combined immune deficiency ( scid ) female mice were transplanted with mcf - 7 cells via a subcutaneous injection of 1 × 10 7 mcf - 7 cells into the dorsal side of the mouse ( this day was denoted as day 1 ). estradiol benzoate was injected subcutaneously weekly at a dose of 50 ug / mouse for 4 weeks . the scid mice were administered daily with a carrier or 2 % fermented soy extract by oral gavage in a dosing volume of 10 ml / kg body weight for 7 days before tumor cells implantation and then daily for 41 days after tumor cells implantation ( the dose of 10 ml of the 2 % solution per kg body weight was equivalent to a dose of 0 . 2 ml of the fermented soy extract in the concentrated form per kg body weight ). the tumor size , body weight , and the signs of overt animal toxicity after fermented soy extract treatment were observed and recorded . according to the results obtained ( see fig6 ), the fermented soy extract significantly inhibit the tumor growth from day 25 to day 41 . body weight of the tested animals did not have significant difference compared with the control group ( see fig7 ). no signs of overt animal toxicities were seen throughout the experiment . daily oral intake of the fermented soy extract was considered to have anti - tumor effects . the anti - microbial acitivities of the fermented soy extract were demonstrated by determining with in vitro methods . in the first experiment , salmonella typhimurium , bacillus subtilis , three strains ( tmu - c74 , tmu - d 16 and tmu - e86 ) of helicobacter pylori and vancomycin resistant enterococcus feacalis were cultured in nutrient broth or bhi broth and transferred to mueller hinton agar plates or chocolate agar plates . the fermented soy extract was put on a paper disk on the agar plate and the size of an inhibition zone was measured after incubation at 37 ° c . the data are shown in the table below . microbe fermented soy extract inhibition zone ( mm ) salmonella typhimurium undiluted 11 bacillus subtilis undiluted 14 h pylori tmu - c74 undiluted 15 h pylori tmu - d16 undiluted 16 h pylori tmu - e86 undiluted 15 v . r . e . feacalis undiluted 25 v . r . e . feacalis diluted 50 % 15 in another experiment , the minimal inibitory concentrations ( mics ) of the fermented soy extract were determined in salmonella typhimurium ( atcc 14028 ), bacillus subtilis ( crcc 10447 ), staphylococcus aureus ( atcc 25923 ) and vancomycin resistant enterococcus feacalis . suspensions of these bacteria were adjusted to 3 × 10 5 cfu / ml . the adjusted bacteria suspensions were added to a 96 - well plate with or without various concentrations , i . e . 10 %, 5 %, 2 . 5 %, 1 . 25 %, 0 . 65 %, or 0 . 32 %, of the fermented soy extract . the plate was incubated at 37 ° c . for 15 hours . the mics were determined after incubation and shown in the table below . microbe mic of fermented soy extract salmonella typhimurium 2 . 5 % bacillus subtilis 2 . 5 % staphylococcus aureus 2 . 5 % v . r . enterococcus feacalis 1 . 25 % the effects of the fermented soy extract on immunity modulation were studied . spleen cells were isolated from mice and put in a culture flask at 2 × 10 6 cells / ml in a rpmi medium with or without one of several mitogens , i . e . lipopolysaccharide ( lps ), concavalin a ( con a ) and phytohemagglutinin ( pha ). the spleen cell cultures were incubated overnight for mtt assay . a sub - optimal concentration of 5 ug / ml of lps combined with the fermented soy extract at 1 %, 0 . 5 %, 0 . 1 %, 0 . 05 % or 0 . 01 %, had no effect on spleen cell proliferation , especially for b cells . a concentration of 5 ug / ml of pha combined with 0 . 05 % of the fermented soy extract increased the spleen cell number , especially for t cells , which was 2 . 32 fold of the spleen cell number obtained with pha alone . according to this result , the fermented soy extract has an effect on t and b cell interaction in immunity modulation . a concentration of 5 ug / ml of con a combined with 0 . 05 % of the fermented soy extract produced a spleen cell number which was about 20 % less than the spleen cell number obtained with con a alone . according to this result , the fermented soy extract could play a role in anti - inflammation reactions . macrophage activity assay . balb / c mice were injected with thiogllate . three to four days after the injection , macrophages were isolated from the peritoneal cavity of the mouse and incubated with or without the fermented soy extract at 37 ° c . for 30 minutes . e . coli cells conjugated with a fluorescence probe were added to the macrophage suspension and incubated at 37 ° c . for 2 hours . a phagocytosis assay was conducted with flow cytometry . the data showed that the fermented soy extract at 0 . 05 % enhanced the phagocytosis activity of the macrophage by about 71 % compared with macrophages not treated with the fermented soy extract . male icr albino mice were injected intraperitoneally with vehicle , 0 . 8 ml of 1 % of the fermented soy extract per mouse , 0 . 8 ml of 0 . 1 % of the fermented soy extract per mouse , levamisole at 30 mg / kg , or azimexone at 100 mg / kg . one hour after the intraperitoneal injection , candida albican ( atcc 10231 ) was injected intravenously into the mouse at 1 . 5 to 2 × 10 7 cfu per mouse . the mortality of the mouse was determined daily for 10 days ( see table 1 ). as shown in table 1 , the fermented soy extract reduced the mortality of candida albican in the mouse . the mortality reductive effect of the fermented soy extract was more pronounced than that of levamisole . male icr albino mice were pretreated with cyclophosphamide at 30 mg / kg on days 5 , 3 and 1 before injected intravenously with candida albican . on days 6 , 4 and 2 before the intravenous injection of candida albican , the mouse was treated with vehicle , 0 . 1 % of the fermented soy extract , 1 % of the fermented soy extract or azimexone at 100 mg / kg . the mortality of the mouse was determined daily for 10 days ( see table 2 ). as shown in table 2 , with cyclophosphamide pretreatment , the fermented soy extract reduced the mortality of candida albican in the mouse . the mortality reductive effect of the fermented soy extract was comparable to that of azimexone . lox is the main metabolizing enzyme in arachidonate acid ( aa ) metabolism . one of the metabolic pathways of aa involves lipoxygenase , lox , 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 to result in cancer cell metastasis . hete is also a mitogenic factor , which results in angiogenesis of cancer cells . lox - 15 was isolated from rabbit reficulocytes . linoleic acid was used as a substrate of lox - 15 with or without the fermented soy extract . the amount of hete formed was determined spectrophotometrically . the data show that the fermented soy extract had an inhibitory effect on lox - 15 ( see fig8 ). the result indicated that the fermented soy extract can inhibit angiogenesis and metastasis of cancer cells and induce apoptosis of cancer cells . adlercreuz , h . et al ., evaluation nutrition , intestinal microflora and prevention of cancer : a hypothesis , proc . soc . exp . biol . med ., 217 : 241 - 246 ( 1998 ). breimer l h . ionizing radiation - induced mutagenesis , br j cancer , 57 : 6 - 18 ( 1998 ). briehl , 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 - 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