In vitro mitochondrial apoptosis of melanoma cells via immature Poncirus trifoliata fruit extract.

OBJECTIVE: Poncirus trifoliata (P. trifoliata) fruits exert phytotherapeutic effects, depending on their maturity level. However, the mechanism by which these phytotherapeutic effects are exerted remains undefined - especially in cancers. Therefore, in this study, we investigated the effects of the immature fruit extract of P. trifoliata on a B16 melanoma cell line. MATERIALS AND METHODS: The effect of immature P. trifoliata extract on B16 cells was evaluated by MTT assay, cell proliferation, FACScan analysis of cell cycles, confocal imaging analysis, nuclear (Hoechst) staining, apoptosis assay (Annexin V-fluorescein isothiocyanate/propidium iodide staining), and Western blot assay. The capacity of immature P. trifoliata extract to inhibit the invasion and migration of B16 cells was assessed using the scratch-wound assay and Matrigel migration assay. The effect of immature P. trifoliata extract on mitochondrial function was determined via the mitochondrial membrane potential assay, activity, and fraction and cytosol proteins. RESULTS: Treating B16 cells with a methanol extract of immature P. trifoliata (MEPT) significantly inhibited cell viability, migration, and invasiveness in a dose- (p<0.01) and time (p<0.01)- dependent manner. MEPT arrested the cells in the G1 phase of the cell cycle and led to the activation of the PI3K/AKT/p21 pathway. Furthermore, MEPT dose-dependently induced apoptosis in B16 cells by increasing the expression of the pro-apoptotic proteins Bax and Apaf-1, while decreasing the expression of the anti-apoptotic protein, Bcl-2. MEPT treatment also decreased mitochondrial membrane potential. CONCLUSIONS: Immature P. trifoliata extract inhibited the growth of melanoma cells by inducing cell apoptosis through mitochondrial pathways. Therefore, further research into immature P. trifoliata extract as a potential therapeutic compound for melanoma treatment is warranted.

Lacticaseibacillus paracasei NK112 mitigates Escherichia coli-induced depression and cognitive impairment in mice by regulating IL-6 expression and gut microbiota.

The gut microbiota communicates with the brain through microbiota-gut-brain (MGB) and hypothalamus-pituitary-adrenal (HPA) axes and other pathways. Excessive expression of interleukin (IL)-6 is closely associated with the occurrence of the psychiatric disorders depression and dementia. Therefore, to understand whether IL-6 expression-suppressing probiotics could alleviate psychiatric disorders, we isolated IL-6 expression-inhibiting Lacticaseibacillus paracasei (formerly Lactobacillus paracasei) NK112 from the human faecal bacteria strain collection (Neurobiota Research Center, Seoul, Korea) and examined its therapeutic effect for the depression and cognitive impairment in mice. C57 BL/6J mice with depression and cognitive impairment were prepared by exposure to Escherichia coli K1. Oral gavage of NK112 significantly alleviated K1-induced anxious, depressive, and memory-impaired behaviours in the elevated plus maze, tail-suspension and Y-maze tasks, IL-1ß, IL-6, and tumour necrosis factor (TNF)-α expression, and nuclear factor kappa beta (NF-κB) activation in the hippocampus, while K1-suppressed brain-derived neurotrophic factor (BDNF) expression increased. Treatment with NK112 also improved K1-induced myeloperoxidase activity, IL-6 and TNF-α expression, and NF-κB activation in the colon and reduced K1-induced Proteobacteria population in the gut microbiota. Heat-killed NK112 and its lysate supernatant, and precipitate fractions also improved anxiety/depression, cognitive impairment, and colitis in mice. In conclusion, NK112, even if heat-killed or lysed, alleviated K1 stress-induced colitis, anxiety/depression, and cognitive impairment by suppressing IL-6, TNF-α, and BDNF expression through the regulation of gut microbiota and NF-κB activation.

Gastrointestinal inflammation by gut microbiota disturbance induces memory impairment in mice.

In this study, we tested our hypothesis regarding mechanistic cross-talk between gastrointestinal inflammation and memory loss in a mouse model. Intrarectal injection of the colitis inducer 2,4,6-trinitrobenzenesulfonic acid (TNBS) in mice caused colitis via activation of nuclear factor (NF)-κB and increase in membrane permeability. TNBS treatment increased fecal and blood levels of lipopolysaccharide (LPS) and the number of Enterobacteriaceae, particularly Escherichia coli (EC), in the gut microbiota composition, but significantly reduced the number of Lactobacillus johnsonii (LJ). Indeed, we observed that the mice treated with TNBS displayed impaired memory, as assessed using the Y-maze and passive avoidance tasks. Furthermore, treatment with EC, which was isolated from the feces of mice with TNBS-induced colitis, caused memory impairment and colitis, and increased the absorption of orally administered LPS into the blood. Treatment with TNBS or EC induced NF-κB activation and tumor necrosis factor-α expression in the hippocampus of mice, as well as suppressed brain-derived neurotrophic factor expression. However, treatment with LJ restored the disturbed gut microbiota composition, lowered gut microbiota, and blood LPS levels, and attenuated both TNBS- and EC-induced memory impairment and colitis. These results suggest that the gut microbiota disturbance by extrinsic stresses can cause gastrointestinal inflammation, resulting in memory impairment.

Anxiolytic-like effect of Bifidobacterium adolescentis IM38 in mice with or without immobilisation stress.

To better understand the role of gut microbiota in the anxiety, we isolated bifidobacteria and lactobacilli from the human faecal microbiota, investigated their inhibitory effects on the expression of interleukin (IL)-6 and tumour necrosis factor (TNF)-α in lipopolysaccharide-stimulated macrophages, and examined the anxiolytic-like effect of Bifidobacterium adolescentis IM38 in mice treated with or without immobilisation stress using the elevated plus maze (EPM) task. Oral administration of IM38 at a dose of 1×109 cfu/mouse showed a significant anxiolytic-like effect both in mice exposed to immobilisation stress and in control mice using the EPM test (P<0.05). Moreover, IM38 treatment significantly increased the amount of time spent on open arms and open arm entries. The anxiolytic-like effect of IM38 was comparable to that of buspirone (1 mg/kg). Moreover, this anxiolytic-like effect was blocked by treatment with flumazenil (3 mg/kg, i.p.), a benzodiazepine receptor antagonist, but was not affected by treatment with bicuculine or WAY-100635. IM38 treatment also reduced the blood levels of corticosterone and IL-6 in mice with or without immobilisation stress, whereas this effect was abolished by treatment with flumazenil. IM38 treatment also reduced the blood TNF-α level in mice subjected to immobilisation stress but not in normal control mice. Treatment with flumazenil also significantly increased TNF-α and IL-6 levels in immobilisation stress-free mice treated with IM38. These findings suggest that IM38 may attenuate anxiety through modulation of the benzodiazepine site on the GABAA receptor and modulate stress-related cytokine expression.

Lactobacillus fermentum IM12 attenuates inflammation in mice by inhibiting NF-κB-STAT3 signalling pathway.

In the present study, we isolated Lactobacillus fermentum IM12 from human gut microbiota, which strongly inhibited interleukin (IL)-6 expression and STAT3 activation in lipopolysaccharide (LPS)-stimulated murine peritoneal macrophages, and examined its anti-inflammatory effect in mice with carrageenan-induced hind-paw oedema (CIE) or 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis (TIC). Oral administration of IM12 (0.2×109, 1×109 or 5×109 cfu/mouse, once a day for 3 days) in mice with CIE significantly suppressed the increase of oedema volume and thickness, as well as myeloperoxidase activity and IL-6, IL-17, NO, and prostaglandin E2 levels in the carrageenan-stimulated paw. Treatment with IM12 (1×109 cfu/mouse, once a day for 3 days) in mice with TIC significantly suppressed colon shortening, and myeloperoxidase activity and IL-6 and IL-17 levels. Treatment with IM12 in mice with CIE or TIC also suppressed the expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2, as well as activation of nuclear factor kappa beta (NF-κB) and signal transducer and activator of transcription 3 (STAT3). Furthermore, IM12 significantly inhibited the expression of iNOS, and COX-2, as well as activation of NF-κB in LPS-stimulated mouse peritoneal macrophages. The inflammatory effect of heat-inactivated IM12 was significantly different to that of live IM12 in mice with TIC, although anti-inflammatory effect of IM12 was reduced by heat treatment. Based on these findings, IM12 may attenuate inflammation by inhibiting NF-κB-STAT3 signalling pathway.

Metabolites from the co-culture of nigranoic acid and Umbelopsis dimorpha SWUKD3.1410, an endophytic fungus from Kadsura angustifolia.

The ability of the endophytic fungus Umbelopsis dimorpha SWUKD3.1410 to transform the triterpene nigranoic acid (3, 4-secocycloarta-4(28), 24-(Z)-diene-3, 26-dioic acid; 1) was investigated. Co-culture of nigranoic acid with U. dimorpha SWUKD3.1410 yielded six major products, including a new triterpene (2) and 5 known compounds (3-7). Their structures were identified by the extensive 1D, 2D NMR and HR-ESI-MS Data analysis, and by comparison with those reported data. Among them, 2-4 were transformed from nigranoic acid (1), while 5-7 were derived from the culture medium stimulated by the substrate. On the basis of the structures of 2-4, it was proposed that the transformative process probably involved isomerase and hydroxylase, in which the former was rare in fungi. Additionally, all the compounds (1-7) were evaluated for their cytotoxic activity. As a result, compounds 1, 3-5 exhibited weak cytotoxicity against the tested cell lines, while others showed no cytotoxicity.

Anti-inflammaging effects of Lactobacillus brevis OW38 in aged mice.

In the present study, lactic acid bacteria (LAB) strains were collected from kimchi and were screened to isolate strains that inhibit lipopolysaccharide (LPS) production by Escherichia coli and p16 expression and nuclear factor-kappa B (NF-κB) activation in LPS-stimulated macrophages. Oral administration of Lactobacillus brevis OW38 (1×109 cfu/mouse) to aged mice (male, 18 months old) for 8 weeks reduced the LPS level in colon fluid and blood. In addition, OW38 treatment also reduced the ratio of Firmicutes or Proteobacteria to Bacteroidetes, which was significantly higher in aged mice than in young mice. Treatment with OW38 in aged mice inhibited the expression of inflammatory markers, such as myeloperoxidase, tumour necrosis factor (TNF), and interleukin (IL)-1ß, and inhibited NF-κB activation. Furthermore, it induced the expression of colonic tight junction proteins zonula occludens-1, occludin, and claudin-1. OW38 treatment also suppressed the expression of senescence markers p16, p53, and SAMHD1 in the colon and the hippocampus of aged mice. In addition, it significantly restored spontaneous alternation as well as the expression of brain-derived neurotrophic factor and doublecortin in aged mice compared to that in young mice (P<0.05). Based on these findings, we conclude that OW38 treatment may ameliorate aging-associated colitis and memory impairment by inhibiting gut microbiota LPS production, NF-κB activation, and p16 expression.

Lactobacillus pentosus var. plantarum C29 ameliorates age-dependent memory impairment in Fischer 344 rats.

UNLABELLED: To understand the anti-inflammaging effect of lactic acid bacteria, we selected NF-κB activation-inhibitory Lactobacillus pentosus var. plantarum C29 and investigated its memory-enhancing and anti-inflammatory effects in aged Fischer 344 rats. C29 (2 × 10(9) CFU rat(-1) ), which was orally administered once a day (6 days per week) for 8 weeks, significantly restored age-reduced spontaneous alternation to 95.2% of that seen in young rats (P < 0.05). C29 treatment also shortened the escape latency on the 4th day to 53.8% of that seen in young rats (P < 0.05). Twenty hours after the last training session, C29 significantly increased the swimming time within the platform quadrant, which was shortened in the aged control rats. Oral administration of C29 restored age-reduced doublecortin (DCX) and brain-derived neurotrophic factor (BDNF) expression and cAMP response element binding protein (CREB) activation in aged rats. Treatment of aged rats with C29 suppressed the expression of p16, cyclooxygenase-2, and inducible nitric oxide synthase, as well as the activation of Akt, mTOR, and NF-κB in the hippocampus. These findings suggest that C29 ameliorates ageing-dependent memory impairment by inhibiting NF-κB signalling pathway, inducing DCX and BDNF expression and activating CREB. SIGNIFICANCE AND IMPACT OF THE STUDY: The anti-inflammatory Lactobacillus pentosus var. plantarum C29 had the memory-enhancing effect in aged Fischer 344 rats by restoring doublecortin and brain-derived neurotrophic factor expression and suppressing p16 expression and NF-κB activation in the brain. Therefore, C29 may be useful in ameliorating age-related degenerative dementia.

MR imaging prediction of local control of nasopharyngeal carcinoma treated with radiation therapy and chemotherapy.

OBJECTIVE: To retrospectively evaluate the association of MRI findings with local control of nasopharyngeal carcinoma (NPC) treated with radiation therapy and chemotherapy (chemoradiotherapy). METHODS: Pre-treatment MRIs of 101 patients (78 males and 23 females, 23-79 years of age) who had NPC treated with chemoradiotherapy were retrospectively reviewed to evaluate tumour involvement of nasopharyngeal anatomic subsites, tumour volume and MRI appearance. Local control rates were evaluated with respect to these MRI findings. RESULTS: Univariate analysis (using the Kaplan-Meier method) showed that invasion of the skull base as determined by MRI was a significant predictor of local control. In terms of clinical characteristics, T stage and pathological subtype were significant predictors of local control. Multivariate analysis (Cox regression model) of the radiologic findings and clinical characteristics revealed that invasion of the skull base (p = 0.003) and pathological subtype (p < 0.001) were independent prognostic factors for local control. CONCLUSION: Invasion of the skull base as determined by MRI predicts the likelihood of local failure and may be helpful in identifying a subset of patients with tumours at risk of local recurrence within 3 years after primary chemoradiotherapy. ADVANCES IN KNOWLEDGE: It has now become common practice to use MRI for pre-treatment evaluation of patients with NPC. The potential role for MRI findings in predicting local control and prognosis in patients with NPC has implications for treatment planning.

Lactobacillus brevis G-101 ameliorates colitis in mice by inhibiting NF-κB, MAPK and AKT pathways and by polarizing M1 macrophages to M2-like macrophages.

AIM: We isolated Lactobacillus brevis G-101 from kimchi lactic acid bacteria (LAB) strains, which induced IL-10 expression in lipopolysaccharide (LPS)-stimulated peritoneal macrophages. To evaluate the inflammatory effect of G-101, we examined its inhibitory effect in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitic mice. MATERIALS AND RESULTS: The colitic mice were prepared by intrarectal injection of TNBS. We measured intestinal mucosal cytokines by enzyme-linked immunosorbent assay; activation of transcription factors, by immunoblotting; and macrophage polarization markers, by real-time polymerase chain reaction. Of 200 LAB strains tested, Lact. brevis G-101 showed most potent activity for induction of IL-10 expression in LPS-stimulated peritoneal macrophages. However, it significantly inhibited the expression of TNF-α, IL-1ß and IL-6 and the phosphorylation of IRAK1 and AKT, and activated NF-κB and MAPKs. Treatment with TNBS caused colon shortening; increased myeloperoxidase activity; and increased IL-1ß, IL-6 and TNF-α expression in mice. Oral administration of Lact. brevis G-101 significantly inhibited these activities. Lactobacillus brevis G-101 inhibited TNBS-induced IRAK-1 phosphorylation and NF-κB activation, as well as the expression of COX-2 and iNOS. Lactobacillus brevis G-101 inhibited the expression of M1 macrophage markers, but increased the expression of M2 macrophages in the colons of TNBS-treated mice. CONCLUSIONS: Lactobacillus brevis G-101 may improve colitis by inhibiting the IRAK1/NF-κB, MAPK and AKT pathways and by polarizing M1 macrophages to M2-like macrophages. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that IL-10 expression-inducing LAB can ameliorate colitis by inhibiting NF-κB activation and macrophage polarization.

Metabolism of cyanidin-3-O-beta-D-glucoside isolated from black colored rice and its antiscratching behavioral effect in mice.

Black-colored rice (BCR), the main constituent of which is cyanidin-3-O-beta-D-glucoside (C3G), exhibits an anti-allergic effect, and orally administered C3G is mainly metabolized to protocatechuic acid in rats. Therefore, to understand the relationship between the metabolism of C3G and its pharmacological effect, we isolated C3G from BCR, anaerobically incubated it with fecal microflora, investigated its metabolite(s) by LC-MS/MS, and measured the antiscratching behavioral effects of C3G and its metabolites. C3G was metabolized to protocatechuic acid via cyanidin. Protocatechuic acid and cyanidin were identified as the metabolites. The activities transforming C3G to protocatechuic acid and cyanidin were 28.2 +/- 11.7 and 21.8 +/- 5.2 nmol/h/mg fecal microflora, respectively. C3G and its metabolites showed inhibitory effects against histamine- or compound 48/80-induced scratching behaviors in mice. C3G more potently inhibited scratching behaviors following oral administration than following intraperitoneal administration. However, protocatechuic acid and cyanidin showed more potent inhibition when administered intraperitoneally than when administered orally. These metabolites also inhibited the expression of allergic cytokines, IL-4 and TNF-alpha, and the activation of their transcription factor, NF-kappaB, in RBL-2H3 cells stimulated with IgE-antigen. These findings suggest that C3G-rich BCR may be a beneficial food for diseases involving scratching behaviors, such as chronic dermatitis, rhinitis, and psoriasis.

Phase II trial of irinotecan plus oxaliplatin and 5-fluorouracil/leucovorin in patients with untreated metastatic gastric adenocarcinoma.

BACKGROUND: This nonrandomized open label phase II study evaluated the efficacy and safety of FOLFOXIRI in metastatic or recurrent gastric cancer patients. PATIENTS AND METHODS: Patients with histologically proven, metastatic gastric adenocarcinoma, aged 18-70 years, performance status zero to two, no prior chemotherapy, and with signed written informed consent were eligible. Treatment consisted of irinotecan 150 mg/m2 day 1, oxaliplatin 85 mg/m2 day 1, leucovorin 100 mg/m2 day 1, and 5-fluorouracil 2000 mg/m2 as a 48-h continuous infusion starting on day 1, which was repeated every 2 weeks. RESULTS: From August 2004 to August 2005, 48 patients were prospectively enrolled. The median age was 54 years (24-69). In total, 386 cycles were administered with a median of nine cycles per patient (range 1-12 cycles) and 45 of 48 patients were assessable for treatment response. An independent review of tumor responses resulted in overall response rate of 66.7% (95% confidence interval=53.4% to 80.0%) by intent-to-treat analysis with one complete response and 31 partial responses. The median survival of all patients was 14.8 months and the median time to progression was 9.6 months. Most common grade 3/4 toxic effects were neutropenia (12% of all cycles) and emesis (8% of all cycles). Grade 2 peripheral neuropathy occurred in five patients. One (2%) patient had severe tumor bleeding and five (10%) patients experienced grade 3 diarrhea. CONCLUSIONS: The modified FOLFOXIRI combination chemotherapy showed a very promising preliminary antitumor activity and was generally well tolerated as a first-line treatment of patients with metastatic gastric cancer.

In vitro anti-Helicobacter pylori activity of panaxytriol isolated from ginseng.

This study investigated the effect that some polyacetylenes and protopanaxatriol, which were isolated from heated ginseng (family Araliaceae), have on inhibiting Helicobacter pylori (HP) growth. Among the compounds tested, panaxytriol was quite effective in inhibiting HP growth with an MIC of 50 microg/ml. Ginsenoside Rh1 and protopanaxatriol weakly inhibited H+/K+-ATPase from a rat stomach.

In vitro anti-Helicobacter pylori activity of irisolidone isolated from the flowers and rhizomes of Pueraria thunbergiana.

The inhibitory effect of isoflavones isolated from the flowers and rhizomes of Pueraria thunbergiana (Leguminosae) on the growth of Helicobacter pylori (HP) was investigated. Isoflavone glycosides did not inhibit the growth of HP. However, their aglycones, irisolidone, tectorigenin and genistein, inhibited HP growth. Among them, irisolidone had the most potent inhibitory activity against HP and its MIC was 12.5-25 micrograms/ml. Genistein only weakly inhibited the urease of HP and H+/K(+)-ATPase of rat stomach: its IC50 were 0.43 and 0.89 mg/ml, respectively.

Antitumor activity of Bifidobacterium spp. isolated from a healthy Korean.

The antitumor activity of Bifidobacterium breve K-110, and K-111, and B. infantis K-525 was investigated. These Bifidobacterial cells and their cell wall preparations (WPG) significantly increased the survival rate of mice who had been intraperitoneally implanted with sarcoma 180 cells. Solid tumor growth was inhibited even when the sarcoma 180 cells were implanted into the groins of the mice. However, the Bifidobacterial cells did not show in vitro cytotoxicity against tumor cell lines. Cell kinetic studies revealed that these WPGs induced neutrophils, which were followed by macrophages, at the site of peritoneal injection. The WPGs directly activated these cells to inhibit the growth of tumor cells in in vitro assays. Our results suggest that Bifidobacterial WPGs induce and activate nonspecific phagocytes in situ to reject growing tumor cells in the mouse peritoneal cavity.

Biotransformation of glycyrrhizin by human intestinal bacteria and its relation to biological activities.

The relationship between the metabolites of glycyrrhizin (18beta-glycyrrhetinic acid-3-O-beta-D-glucuronopyranosyl-(1-->2)-beta-D-glucuronide, GL) and their biological activities was investigated. By human intestinal microflora, GL was metabolized to 18beta-glycyrrhetinic acid (GA) as a main product and to 18beta-glycyrrhetinic acid-3-O-beta-D-glucuronide (GAMG) as a minor product. The former reaction was catalyzed by Eubacterium L-8 and the latter was by Streptococcus LJ-22. Among GL and its metabolites, GA and GAMG had more potent in vitro anti-platelet aggregation activity than GL. GA also showed the most potent cytotoxicity against tumor cell lines and the potent inhibitory activity on rotavirus infection as well as growth of Helicobacter pylori. GAMG, the minor metabolite of GL, was the sweetest.

Metabolism of quercitrin by human intestinal bacteria and its relation to some biological activities.

When quercitrin was anaerobically incubated with human intestinal bacteria, quercetin, 3,4-dihydroxyphenylacetic acid and 4-hydroxybenzoic acid were found as metabolites. The main metabolite was quercetin. The bacterium transforming quercitrin to quercetin was Fusobacterium K-60. However, Bacteroides JY-6, which produced alpha-L-rhamnosidase, did not transform quercitrin to quercetin. Among quercitrin and its metabolites, 3,4-dihydroxyphenylacetic acid and 4-hydroxylphenylacetic acid had more potent activity than quercitrin on in vitro anti-platelet aggregation activity, and quercetin and 3,4-dihydroxyphenylacetic acid showed more potent cytotoxicity against tumor cell lines than quercitrin and 4-hydroxylphenylacetic acid.

Metabolism of liriodendrin and syringin by human intestinal bacteria and their relation to in vitro cytotoxicity.

When liriodendrin or syringin was incubated for 24 h with human intestinal bacteria, two metabolites, (+)-syringaresinol-beta-D-glucopyranoside and (+)-syringaresinol, from liriodendrin and one metabolite, synapyl alcohol, from syringin were produced. The metabolic time course of liriodendrin was as follows: at early time, liriodendrin was converted to (+)-syringaresinol-beta-D-glucopyranoside, and then (+)-syringaresinol. The in vitro cytotoxicities of these metabolites, (+)-syringaresinol and synapyl alcohol, were superior to those of liriodendrin and syringin.

Metabolism of 6"-O-xylosyltectoridin and tectoridin by human intestinal bacteria and their hypoglycemic and in vitro cytotoxic activities.

6"-O-Xylosyltectoridin and tectoridin isolated from the flowers of Pueraria thunbergiana (Leguminosae), are metabolized to tectorigenin by human intestinal bacteria. Although tectoridin is metabolized to tectorigenin by most intestinal bacteria, 6"-O-xylosyltectoridin is metabolized to tectorigenin via tectoridin by only a few intestinal bacteria, such as Bifidobacterium breve K-110 and Eubacterium A-44. The metabolite, tectorigenin, had more potent hypoglycemic activity as well as in vitro cytotoxic activity against tumor cell lines than 6"-O-xylosyltectoridin and tectoridin. These results suggest that 6"-O-xylosyltectoridin and tectoridin are prodrugs which can be transformed to the active agents by human intestinal bacteria.

Metabolism of puerarin and daidzin by human intestinal bacteria and their relation to in vitro cytotoxicity.

When puerarin or daidzin were incubated for 24 h with human intestinal bacteria, two metabolites, daidzein and calycosin, were produced from them, respectively. The metabolic time course of puerarin was as follows: at an early time, puerarin was converted to daidzin, and then calycosin. The metabolic time course of daidzin by human intestinal bacteria was also similar to that of puerarin. The in vitro cytotoxicities of these metabolites, calycosin and daidzein, were superior to those of puerarin and daidzein.