Main Isoflavones Research Articles

Isoflavone profile and protein molecular weight distribution of soy protein concentrates after soaking treatments

The effects of soybean soaking at 40, 50, and 60°C, during 4, 8, 12, and 16 hr time periods, on the isoflavone profile and molecular weight distribution of proteins in their soy protein concentrate were evaluated. Results indicated that soaking treatments increased aglycone content (daidzein, genistein, and glycitein) and decreased β-glucosylated and malonyl glucosylated isoflavone contents. Soy protein concentrate obtained after soaking at 50°C for 16 hr yielded the greatest total aglycone content (5,993.6 µmol 100 g−1), which was 75% greater than the total aglycone content found in soy protein concentrate obtained from non-treated soybeans (3,636.9 µmol 100 g−1). Glycitein was the main isoflavone aglycone present in soy protein concentrates. Acidic and basic subunits of 11S globulins are released at low and intermediate soaking periods (4, 8, and 12 hr), while longer soaking time (16 hr) causes a protein rearrangement of partially cleaved proteins into more hydrophobic core. Practical applications Soy-based food products can be considered functional foods because of their isoflavone content, mainly the aglycone content (daidzein, genistein, and glycitein). The utilization of soaking treatment in soybeans may increase the isoflavone aglycone content in soy protein concentrate. Our results indicated that soaked soybeans had more isoflavone aglycone content than untreated ones. The highest isoflavone aglycone content was obtained from soaked soybeans at 50°C for 16 hr and glycitein was the predominant isoflavone aglycone found in soy protein concentrates. The obtained soy protein concentrate can be used in a wide range of products, such as bread, cakes, cereals, beverages, and meat products, and contributes to increasing the nutritional and functional value of the product, mainly due to the quantity and quality of proteins and isoflavones.

Genistein protects intervertebral discs from degeneration via Nrf2-mediated antioxidant defense system: An in vitro and in vivo study.

Oxidative stress has been reported to be closely associated with the development of intervertebral disc degeneration (IDD). IDD is one of the major causes of low back pain. Genistein (GES), one of the main isoflavones of soybean, has been shown to exert multiple biological functions on different diseases. Here, we tested the therapeutic potential of GES for IDD. In vitro experiments, we confirmed GES was nontoxic to rat nucleus pulposus cells (NPCs) within the concentration of 100 μM. Furthermore, GES was able to suppress apoptosis in tert-butyl hydroperoxide (TBHP)-treated NPCs. In the aspect of extracellular matrix (ECM), GES not only reduced metalloproteinase-13 (MMP-13) and a disintegrin-like and MMP thrombospondin type 1 motif 5 expression, but also increased aggrecan and type II collagen levels. Also, we found GES might rescue TBHP-induced NPCs degeneration by enhancing Nrf2-mediated antioxidant defense system. Silencing Nrf2 partly abolished the protective effects of GES on apoptosis and ECM disruption in TBHP-treated NPCs. Correspondingly, GES ameliorated IDD in a rat model by preserving morphology of degenerative intervertebral discs and promoting Nrf2 expression. To sum up, our study suggests that GESexerts protective effects in NPCs against degeneration and reveals the underlying mechanism of GES on Nrf2 activation in NPCs.

Variety, time of harvest and conditions during growing season have impact on red clover isoflavone content

Red clover (Trifolium pratense L.) is the predominant legume used in northern European agriculture. Official red clover variety trials are conducted by Natural Resources Institute Finland (Luke) to determine the value of field crop varieties. The trials used for the current analysis were conducted in Luke units in southern Finland (Mikkeli) and northern Finland (Ruukki) in two consecutive years. Plant samples for isoflavone analyses were collected from four varieties grown as four replicates and harvested twice during both growing seasons. The four main isoflavones biochanin A, genistein, daidzein and formononetin were analysed using high performance liquid chromatography. Total phytoestrogen content in the varieties varied in the range of 11.2−14.8 mg g-1 dry matter (DM). The variety and the time of harvest had most effect on the isoflavone, especially formononetin, contents of red clover. A more northern growing area and challenging weather conditions were associated with increased isoflavone concentrations.

Variability of Isoflavone Content in Soy Milk Products Commercially Available in Thailand

Objective: To determine the isoflavone content in soy milk products commercially available in Thailand.Material and Methods: Two flavors of each of 4 brands of soy milk were obtained from retail outlets in Chiang Mai, Thailand. The isoflavone content in soy milk was determined using high-performance liquid chromatography.Results: Genistin and daidzin, both β-glycoside conjugates, were found to be the main isoflavone components in soy milk. The total amount of isoflavones varied considerably among different products, ranging from 25.5 to 63.5 mg per serving (250 mL) of soy milk. Marked variation in total isoflavones was also found even among different lots of the same product.Conclusion: The isoflavone content in soy milk products available in Thailand varies substantially among different products as well as among different lots of the same product. This suggests that standardizing or, at least, declaring the isoflavone content in soy milk products is needed to facilitate the optimal consumption of soy milk for health benefits.

Variability of Isoflavone Content in Soy Milk Products Commercially Available in Thailand

Objective: To determine the isoflavone content in soy milk products commercially available in Thailand.Material and Methods: Two flavors of each of 4 brands of soy milk were obtained from retail outlets in Chiang Mai, Thailand. The isoflavone content in soy milk was determined using high-performance liquid chromatography.Results: Genistin and daidzin, both β-glycoside conjugates, were found to be the main isoflavone components in soy milk. The total amount of isoflavones varied considerably among different products, ranging from 25.5 to 63.5 mg per serving (250 mL) of soy milk. Marked variation in total isoflavones was also found even among different lots of the same product.Conclusion: The isoflavone content in soy milk products available in Thailand varies substantially among different products as well as among different lots of the same product. This suggests that standardizing or, at least, declaring the isoflavone content in soy milk products is needed to facilitate the optimal consumption of soy milk for health benefits.

Main Isoflavones Found in Dietary Sources as Natural Anti-inflammatory Agents.

Isoflavones are natural compounds belonging to the class of isoflavonoids, molecules with a chemical structure based on the 3-phenyl chromen-4-one backbone. As a particular element, the isoflavones are also called phytoestrogens, due to the structural similarity with 17-β estradiol, the primary female sex hormone, presenting both estrogen-agonist and estrogen-antagonist properties. This class of secondary plant metabolites has been extensively reported in the literature for a wide range of therapeutic activities including antioxidant, chemopreventive, anti-inflammatory, antiallergic, antibacterial, and cardio preventive effects. To sum up the latest information regarding the main isoflavones found in dietary sources as natural anti-inflammatory agents. The review updates with the recent research about genistein, daidzein, glycitein, biochanin A, formononetin and equol as anti-inflammatory phytocompounds. As a particular element, the implications of these isoflavones in the link between inflammation and angiogenesis are also discussed. The review concludes that the main isoflavones in dietary sources display in vitro and/or in vivo anti inflammatory potential by activating various biochemical and molecular mechanisms. The depicted findings translated into practical aspects strengthen the idea that the isoflavones genistein, daidzein, glycitein, biochanin A, formononetin and equol can be used in various inflammation based diseases as an alternative source for classical medication and that consummation of foods rich in isoflavones may represent prerequisite use in order to prevent the evolution of inflammation based diseases.

Extract from Astragalus membranaceus inhibit breast cancer cells proliferation via PI3K/AKT/mTOR signaling pathway

BackgroundAstragalus membranaceus (AM) is a commonly used herb in traditional Chinese medicine (TCM), which has been used as an essential tonic to treat various diseases for more than 2000 years. In this study, we aimed to investigate the biological effects of extract from AM on breast cancer cell and its mechanism.MethodsTo prepare the extract, dried AM were ground and extracted with water extraction-ethanol supernatant method. Then the main isoflavones in the extract was detect by HPLC analysis. Furthermore, the anti-proliferative activity of AM extract was examined by MTT assay and morphological observation. Cell apoptosis was evaluated with flow cytometric analysis. The expressions of total and phosphorylated PI3K, GS3Kβ, Akt and mTOR were determined by western blot analysis.ResultsHPLC analysis demonstrated that AM extract contained with four kinds of isoflavones, campanulin, ononin, calycosin and formononetin. The MTT test and morphological observation indicated that cells proliferation of MCF-7, SK-BR-3 and MDA-MB-231were inhibited by AM extract in a dose dependent manner. Furthermore, flow cytometric analysis displayed that after treated with 25 μg/ml and 50 μg/ml AM extract, apoptosis of breast cancer cells was significantly increased as compared with DMSO and blank control group (all p < 0.05). Western blot analysis found that the level of p-PI3K, p-GS3Kβ, p-Akt, and p-mTOR were significantly decreased, but the level of total-mTOR was observably increased as compared with DMSO control group.ConclusionsTaken together, the inhibited cell proliferation and induced cell apoptosis effect of AM extract via PI3K/AKT/mTOR pathway confirmed the anti-tumor potential of AM. Therefore, our findings provide a new insight into anti-cancer effect of AM extract as a promising agent in breast cancer treatment.

Effect of germination with sodium selenite on the isoflavones and cellular antioxidant activity of soybean (Glycine max)

Isoflavones profiles and cellular antioxidant activity (CAA) of soybeans (Glycine max) germinated for different times with several sodium selenite (Na2SeO3) concentrations were assessed. The experiments were performed using central composite design and isoflavones evaluated by HPLC–UV and HPLC-MS-TOF. Malonylated genistein and malonylated daidzein glycosides were the main isoflavones associated to the soybean sprouts. Soybeans germinated for 48 h at 20 °C with a frequent spraying of water containing less than 32 mg/L of Na2SeO3 per liter contained the optimal concentrations of selenium (Se), total isoflavones and cellular antioxidant activity. This research demonstrated that Se-enriched soybean sprouts are a good source of both dietary Se and isoflavones and potentially can be used to formulate new functional foods.

Extraction of isoflavones from Puerariae lobata using subcritical water.

As an alternative to organic solvents, subcritical water was employed for the first time as an effective solvent for the extraction of isoflavones from Puerariae lobata. Optimum experimental conditions for the extraction of the four main isoflavones were established by single factor experiments, and the optimum experimental conditions for total isoflavone extraction were established further by response surface methodology. With an extraction time of 45 min and a liquid/solid ratio of 1 : 20, the extraction yields of puerarin, 3′-methoxypuerarin, and daidzin reached maxima at extraction temperatures of 120 °C, 140 °C and 200 °C, respectively. Moreover, puerarin, 3′-methoxypuerarin and daidzin were degraded and produced various byproducts due to hydrothermal reactions at higher temperatures. The maximum extraction yields of the total isoflavones were obtained by response surface methodology (extraction time of 45 min, solid/liquid ratio of 1 : 15 and extraction temperature of 120 °C). Compared to conventional solvents, subcritical water utilized less solvent and required a shorter extraction time.

A comparative study on the effect of promoting the osteogenic function of osteoblasts using isoflavones from Radix Astragalus.

Radix Astragalus has been shown to exert beneficial effects regarding the prevention postmenopausal osteoporosis. However, its mechanism of action remains to be investigated. Calycosin, formononetin, and calycosin-7-O-β-d-glucoside are the main isoflavone constituents of Astragalus. In this study, the abilities of these 3 compounds to promote osteogenic function of osteoblasts were compared, and the structure-activity relationships of these osteotrophic isoflavones were determined. Calycosin exhibited a greater effect than formononetin and calycosin-7-O-β-d-glucoside regarding improvements in osteogenic function of osteoblasts, as demonstrated by cell proliferation, alkaline phosphatase activity, collagen I and osteocalcin secretion, and the number and area of mineralized bone nodules. This suggests that calycosin may be better than formononetin and calycosin-7-O-β-d-glucoside at preserving bone mass. In addition, calycosin, formononetin, and calycosin-7-O-β-d-glucoside stimulate the expression of bone morphogenetic protein 2 and runt-related transcription factor 2 proteins, which indicates that all 3 agents may promote the osteogenesis of osteoblasts via regulation of bone morphogenetic protein 2 expression. In conclusion, calycosin may be the best candidate, with higher osteogenic activity than formononetin and calycosin-7-O-β-d-glucoside. The higher osteogenic activity of calycosin could be attributable to the superiority of its chemical structure (a hydroxyl group at position C3 of Ring B and no glucosyl group).

Daidzein down-regulates ubiquitin-specific protease 19 expression through estrogen receptor β and increases skeletal muscle mass in young female mice

Ubiquitin-specific protease 19 (USP19) is a key player in the negative regulation of muscle mass during muscle atrophy. Loss-of-function approaches demonstrate that 17β-estradiol (E2) increases USP19 expression through estrogen receptor (ER) α and consequently decreases soleus muscle mass in young female mice under physiological conditions. Daidzein is one of the main isoflavones in soy, and activates ERβ-dependent transcription. Here, we investigated the effects of daidzein on E2-increased USP19 expression and E2-decreased soleus muscle mass in young female mice. Daidzein stimulated the transcriptional activity of ERβ in murine C2C12 cells and down-regulated USP19 expression. Consistently, daidzein inhibited E2-induced USP19 expression in a reporter activity using a functional half-estrogen response element (hERE) from Usp19. Daidzein inhibited E2-induced recruitment of ERα and promoted recruitment of ERβ to the Usp19 hERE. Dietary daidzein down-regulated the expression of USP19 at the mRNA and protein levels and increased soleus muscle mass in female mice, but not in males. In soleus muscle from ovariectomized (OVX) female mice, dietary daidzein inhibited E2-increased USP19 mRNA expression and E2-decreased muscle mass. Furthermore, E2 induced the recruitment of ERα and ERβ to the hERE, whereas daidzein inhibited E2-induced recruitment of ERα, and enhanced E2-increased recruitment of ERβ, to the Usp19 hERE. These results demonstrate that dietary daidzein decreases USP19 mRNA expression through ERβ and increases soleus muscle mass in young female mice, but not in male mice, under physiological conditions.

Genistein Ameliorates Ischemia/Reperfusion-Induced Renal Injury in a SIRT1-Dependent Manner.

Renal ischemia/reperfusion (I/R) injury continues to be a complicated situation in clinical practice. Genistein, the main isoflavone found in soy products, is known to possess a wide spectrum of biochemical and pharmacological activities. However, the protective effect of genistein on renal I/R injury has not been well investigated. In the current study, we explore whether genistein exhibits its renal-protective effects through SIRT1 (Sirtuin 1) in I/R-induced mice model. We found the treatment of genistein significantly reduced renal I/R-induced cell death, simultaneously stimulating renal cell proliferation. Meanwhile, SIRT1 expression was up-regulated following the administration of genistein in renal region. Furthermore, pharmacological inhibition or shRNA-mediated depletion of SIRT1 significantly reversed the protective effect of genistein on renal dysfunction, cellular damage, apoptosis, and proliferation following I/R injury, suggesting an indispensible role of the increased SIRT1 expression and activity in this process. Meanwhile, the reduced p53 and p21 expression and increased PCNA (Proliferating Cell Nuclear Antigen) expression were blocked after the depletion of SIRT1 compared with the genistein treatment group in the renal I/R process. Hence, our results provided further experimental basis for the potential use of genistein for the treatment of kidney disease with deficiency of SIRT1 activity.

Enhancement of antioxidant activity of Radix Puerariae and red yeast rice by mixed fermentation with Monascus purpureus.

In this work, a new functional food combined Radix Puerariae and red yeast rice was explored. The pigment intensity, antioxidant activities and the main isoflavones of it were evaluated and compared with traditional red yeast rice and Radix Puerariae. The fermented mixture showed higher contents of isoflavones and pigment intensities than red yeast rice and Radix Puerariae. The DPPH, OH, FRAP and total antioxidant activity results of fermented mixture also showed higher antioxidant potential than those of Radix Puerariae and red yeast rice, owing to the higher pigment intensity and total phenolic contents. It is concluded that the fermented mixture of Radix Puerariae and rice could be widely used as a source of polyphenols with high antioxidative potential, thus introducing numerous health benefits for the consumer.

Isoflavones, their Glycosides and Glycoconjugates. Synthesis and Biological Activity.

Glycosylation of small biologically active molecules, either of natural or synthetic origin, has a profound impact on their solubility, stability, and bioactivity, making glycoconjugates attractive compounds as therapeutic agents or nutraceuticals. A large proportion of secondary metabolites, including flavonoids, occur in plants as glycosides, which adds to the molecular diversity that is much valued in medicinal chemistry studies. The subsequent growing market demand for glycosidic natural products has fueled the development of various chemical and biotechnological methods of glycosides preparation. The review gives an extensive overview of the processes of the synthesis of isoflavones and discusses recently developed major routes towards isoflavone-sugar formation processes. Special attention is given to the derivatives of genistein, the main isoflavone recognized as a useful lead in several therapeutic categories, with particular focus on anticancer drug design. The utility of chemical glycosylations as well as glycoconjugates preparation is discussed in some theoretical as well as practical aspects. Since novel approaches to chemical glycosylations and glycoconjugations are abundant and many of them proved suitable for derivatization of polyphenols a new body of evidence has emerged, indicating that sugar moiety can play a much more significant role, when attached to a pharmacophore, then being a mere “solubilizer”. In many cases, it has been demonstrated that semisynthetic glycoconjugates are much more potent cytostatic and cytotoxic agents than reference isoflavones. Moreover, the newly designed glycosides or glycoside mimics can act through different mechanisms than the parent active molecule.

Influence of testosterone on phase II metabolism and availability of soy isoflavones in male Wistar rats.

Genistein and daidzein are the main isoflavones in soy. Their potential beneficial or adverse effects in males like the prevention of prostate cancer or the impact on reproductive functions are controversially discussed. Major determinants of their bioactivity are the absorption and biotransformation of isoflavones. In this study, we focused on the influence of testosterone on plasma availability and phase II metabolism of isoflavones. Male Wistar rats, receiving an isoflavones rich diet, were randomized into three groups: Two groups were orchiectomized (ORX) at postnatal day (PND) 80 and treated for 11days with testosterone propionate (TP) (ORX TP group) or a vehicle (ORX group) after a 7days lasting hormonal decline. The third group served as control and remained intact. Rats were sacrificed at PND 98. ORX rats had reduced isoflavones plasma levels. Differently regulated mRNA expressions of transporters relevant for transport of phase II metabolites in liver and kidney may be responsible for this reduction, more precisely Slc10a1 and Slc21a1 in kidney as well as Slc22a8 in liver. While main phase II metabolites in intact rats were disulfates and sulfoglucuronides, the amount of sulfate conjugates was significantly diminished by ORX. In accordance with that, mRNA expression of different sulfotransferases was reduced in liver by ORX. The observed effects could be almost restored by TP treatment. In conclusion, testosterone, and likely further androgens, has a huge impact on phase II metabolism and availability of isoflavones by influencing the expression of different sulfotransferases and transporters.

Phytoestrogens in milk: Overestimations caused by contamination of the hydrolytic enzyme used during sample extraction

Isoflavones are natural phytoestrogens with antioxidant and endocrine-disrupting potencies. Monitoring of their levels is important to ensure the high quality and safety of food, milk, and dairy products. The efficiency and accuracy of phytoestrogen analyses in complex matrices such as milk depend on the extraction procedure, which often uses hydrolysis by means of the β-glucuronidase/sulfatase enzyme originating from Helix pomatia. The present study reveals that the commercially available hydrolytic enzyme is contaminated by several phytoestrogen isoflavones (genistein, daidzein, formononetin, and biochanin A) and their metabolite equol, as well as flavones (naringenin and apigenin) and coumestrol. We show that the concentrations of daidzein and genistein in the enzyme could have impaired the results of analyses of the main isoflavones in several previously published studies. Of 8 analyzed compounds, only equol was confirmed in the present study and it serves as a reliable marker of phytoestrogens originating from cow feed. Critical reassessment of phytoestrogen concentrations in milk is needed because several previously published studies might have overestimated the concentrations depending on the extraction procedure used.

Rapid, sensitive separation of the three main isoflavones in soybean using immunoaffinity chromatography.

Daidzin, genistin, and glycitein are major isoflavone compounds in soybean that are indispensable nutrients in traditional Chinese foods. Generally, strategies for detecting and separating soy isoflavones have been based on HPLC and chromatographic techniques, which are tedious and time-consuming procedures. In the present study, we developed an ELISA-based approach for daidzin detection using a broad-specificity monoclonal antibody (clone number: AA9) with an effective detection range of 10-10 000 ng/mL. Subsequently, we prepared an immunoaffinity column by coupling the monoclonal antibody AA9 to CNBr-activated Sepharose 4B. Our results demonstrate that the immunoaffinity column can efficiently and specifically extract daidzin, glycitein, and genistin from numerous structurally similar soy isoflavones in leguminous plants, thereby providing a new method for the extraction of target components from similar compounds in natural products.

Puerarin Exerts a Delayed Inhibitory Effect on the Proliferation of Cardiomyocytes Derived from Murine ES Cells via Slowing Progression through G2/M Phase

Objective: Puerarin, which shows beneficial and protective effects on cardiovascular diseases, is the main isoflavone extracted from Pueraria lobata (kudzu) root. The aim of this study was to investigate the effects of puerarin on in vitro myocardial proliferation and its underlying mechanism. Methods: Myocardial differentiation of transgenic embryonic stem (ES) cells was performed by embryoid body-based differentiation method. The proliferation assay of cardiomyocytes (CMs) derived from ES cells (ES-CMs) was performed by EdU (5-Ethynyl-2'-deoxyuridine) staining. Flow cytometry was employed to determine the cell cycle distribution and apoptosis of purified ES-CMs. Quantitative real-time PCR was utilized to study the transcription of genes related to cell cycle progression. Signaling pathways relating to proliferation were studied by western blot analysis and application of specific inhibitors. Results: Puerarin exerted a delayed inhibitory effect on the proliferation of ES-CMs at the early-stage differentiation. Meanwhile, puerarin slowed progression through G2/M phase without inducing apoptosis of ES-CMs. Further assays showed that puerarin up-regulated the transcription of Cyclin A2, Cyclin B1 and Cdk1 in ES-CMs. The ERK1/2 specific inhibitor PD0325901 and the PI3K specific inhibitor Wortmannin successfully reversed puerarin-induced up-regulation of Cdk1 but not Cyclin A2 and B1. Conclusion: These findings suggest that puerarin inhibits CM proliferation via slowing progression through G2/M phase during early-stage differentiation.

Puerarin attenuates severe burn-induced acute myocardial injury in rats

BackgroundPuerarin, the main isoflavone glycoside extracted from the root of Pueraria lobata, is widely prescribed for patients with cardiovascular disorders in China. This study investigates the effect of puerarin on severe burn-induced acute myocardial injury in rats and its underlying mechanisms. Materials and methodsHealthy adult Wistar rats were divided into three groups: (1) sham group, sham burn treatment; (2) burn group, third-degree burns over 30% of the total body surface area (TBSA) with lactated Ringer's solution for resuscitation; and (3) burn plus puerarin group, third-degree burns over 30% of TBSA with lactated Ringer's solution containing puerarin for resuscitation. The burned animals were sacrificed at 1, 3, 6, 12, and 24h after burn injury. Myocardial injury was evaluated by analyzing serum creatine kinase MB fraction (CK-MB) activity and cardiac troponin T (cTNT) level. Changes in cardiomyocyte ultrastructure were also determined using a transmission electron microscope. Tumor necrosis factor (TNF)-α concentration in serum was measured by radioimmunoassay. Cardiac myeloperoxidase (MPO) activity and malondialdehyde (MDA) concentration were measured to determine neutrophil infiltration and oxidative stress in the heart, respectively. The expression of p38 mitogen-activated protein (MAP) kinase in the heart was determined by Western blot analysis. ResultsAfter the 30% TBSA full-thickness burn injury, serum CK-MB activities and cTnT levels increased markedly, both of which were significantly decreased by the puerarin treatment. The level of serum TNF-α concentration in burn group at each time-point was obviously higher than those in sham group (1.09±0.09ng/ml), and it reached the peak value at 12h post burn. Burn trauma also resulted in worsen ultrastructural condition, elevated MPO activity and MDA content in heart tissue, and a significant activation of cardiac p38 MAP kinase. Administration of puerarin improved the ultrastructural changes in cardiomyocytes, decreased TNF-α concentration in serum as well as suppressed cardiac MPO activity and reduced MDA content, and abolished the activation of p38 MAP kinase in heart tissue after severe burn. ConclusionsThese results suggest that puerarin attenuates inflammatory responses, reduces neutrophil infiltration and oxidative stress in the heart, and protects against acute myocardial injury induced by severe burn.

Study of the Biotransformation of Tongmai Formula by Human Intestinal Flora and Its Intestinal Permeability across the Caco-2 Cell Monolayer.

Tongmai formula (TMF) is a well-known Chinese medicinal preparation that contains isoflavones as its major bioactive constituents. As traditional Chinese medicines (TCMs) are usually used by oral administration, their fate inside the intestinal lumen, including their biotransformation by human intestinal flora (HIF) and intestinal absorption deserves study. In this work TMF extract was incubated with human intestinal bacteria under anaerobic conditions and the changes in the twelve main constituents of TMF were then investigated. Their intestinal permeabilities, i.e., the transport capability across the intestinal brush border were investigated with a human colon carcinoma cell line (Caco-2) cell monolayer model to predict the absorption mechanism. Meanwhile, rapid HPLC-DAD methods were established for the assay. According to the biotransformation curves of the twelve constituents and the permeability coefficients, the intestinal absorption capacity of the typical compounds was elevated from the levels of 10−7 cm/s to 10−5 cm/s from those of the original compounds in TMF. Among them the main isoflavone glycosides puerarin (4), mirificin (6) and daidzin (7) were transformed into the same aglycone, daidzein (10). Therefore it was predicted that the aglycone compounds might be the real active ingredients in TMF. The models used can represent a novel path for the TCM studies.