Chemical Stability and Bioactivity of tanshinone I, tanshinone IIA, cryptotanshinone and dihydrotanshinone in in vitro test systems.

Danshen Si Wu is a Traditional Chinese Medicine used for menopausal complains. Beside tanshinone IIA (Tan IIA), Danshen also contains tanshinone I (Tan I), cryptotanshinone (CT) and dihydrotanshinone (DT). The aim of this study was to compare the biological activity of these tanshinones and to determine their cytotoxicity and genotoxicity. Purities and stabilities of the substances were analyzed by LC-DAD and LC-MS analyses. DT and CT concentrations decreased rapidly in dimethylsulfoxide and were converted to Tan I and Tan IIA, respectively. In aqueous solution concentration of all tanshinones decreased after 24 h. Tan I and Tan IIA showed dose-dependent bioactivity mediated by ERα and ERß. No cytotoxic and genotoxic effects for Tan I and Tan IIA were detected. In a yeast transactivation assay Tan I and Tan IIA showed antiandrogenic activity. A significant anabolic activity in C2C12 cells could be detected for Tan I and Tan IIA. In conclusion our data provide evidence that Tan I and Tan IIA are the most relevant bioactive tanshinones in Danshen. Our finding that all tanshinones display a certain instability in aqueous solutions is relevant when discussing their potential therapeutic benefits in humans.

Bioactive Phenolic Compounds from Lingonberry (Vaccinium vitis-idaea L.): Extraction, Chemical Characterization, Fractionation and Cellular Antioxidant Activity.

Lingonberries contain high contents of bioactive compounds such as chlorogenic acids and anthocyanins. In addition to radical scavenging and antioxidant activities, these compounds can protect cells from DNA damage. For this reason, lingonberries might be well suited for nutraceuticals or natural biomedicines. To assess these applications, the present study characterized and identified the most effective extract, only consisting of anthocyanins, copigments or a mixture of both, obtained from a lingonberry juice concentrate. An extract was generated by using a XAD-7 column followed by fractionation into anthocyanins and copigments using adsorptive membrane chromatography. After identification of main polyphenols by HPLC-photodiode array-electrospray ionization-tandem mass spectrometry, free radical scavenging activity was analyzed by electron spin resonance spectroscopy using 2,2-diphenyl-1-picrylhydrazyl and galvinoxyl radicals. Furthermore, cyclic voltammetry analyses and the Trolox equivalent antioxidant capacity (TEAC) assay were applied. Finally, the reactive oxygen species (ROS) reducing effects of the lingonberry extract and its fractions were evaluated in HepG2 cells. While the combination of anthocyanins and copigments possessed the highest antioxidant activities, all samples (XAD-7 extract, anthocyanin and copigment fraction) protected cells from oxidative stress. Thus, synergistic effects between phenolic compounds may be responsible for the high antioxidant potential of lingonberries, enabling their use as nutraceuticals.

Synthesis and in vitro characterization of the genotoxic, mutagenic and cell-transforming potential of nitrosylated heme.

Data from epidemiological studies suggest that consumption of red and processed meat is a factor contributing to colorectal carcinogenesis. Red meat contains high amounts of heme, which in turn can be converted to its nitrosylated form, NO-heme, when adding nitrite-containing curing salt to meat. NO-heme might contribute to colorectal cancer formation by causing gene mutations and could thereby be responsible for the association of (processed) red meat consumption with intestinal cancer. Up to now, neither in vitro nor in vivo studies characterizing the mutagenic and cell transforming potential of NO-heme have been published due to the fact that the pure compound is not readily available. Therefore, in the present study, an already existing synthesis protocol was modified to yield, for the first time, purified NO-heme. Thereafter, newly synthesized NO-heme was chemically characterized and used in various in vitro approaches at dietary concentrations to determine whether it can lead to DNA damage and malignant cell transformation. While NO-heme led to a significant dose-dependent increase in the number of DNA strand breaks in the comet assay and was mutagenic in the HPRT assay, this compound tested negative in the Ames test and failed to induce malignant cell transformation in the BALB/c 3T3 cell transformation assay. Interestingly, the non-nitrosylated heme control showed similar effects, but was additionally able to induce malignant transformation in BALB/c 3T3 murine fibroblasts. Taken together, these results suggest that it is the heme molecule rather than the NO moiety which is involved in driving red meat-associated carcinogenesis.

Molecular identification of potential Th1/Th2 responses-modulating bacterial genes using suppression subtractive DNA hybridization.

BACKGROUND AND OBJECTIVES: We characterized the immunomodulating potential of a number of lactobacilli isolated from an African fermented food by co-incubation with peripheral blood mononuclear cells (PBMCs). Two strains with different immune modulating properties were genetically compared by suppression subtractive hybridization (SSH). METHODS: From 48 Lactobacillus strains isolated from Kimere, African fermented pearl millet dough, 10 were selected based on their bile salt tolerance. Their effects on secretion by PBMCs of the T-helper cells Th1- and Th2-cytokines IFN-γ and IL-4, respectively, in the presence or absence of staphylococcal enterotoxin A (SEA) were assessed. To study the genetic basis of different immune-modulating properties, a subtracted cDNA library for L. fermentum strains K1-Lb1 (Th1 inducer) and K8-Lb1 (Th1 and Th2 suppressor) was constructed using SSH. Finally, adhesion of these strains to hydrocarbons (relative hydrophobicity) and to human HT-29 colonic epithelial cell line was assessed. RESULTS: Two strains, K1-Lb1 and K4-Lb6, induced basal IFN-γ secretion. Four strains, K1-Lb6, K6-Lb2, K7-Lb1, and K8-Lb1 diminished INF-γ secretion by SEA-stimulated PBMCs. All strains, except K1-Lb1, K2-Lb4, and K9-Lb3, inhibited SEA-stimulated IL-4 secretion. Comparing the genomes of K1-Lb1 and K8-Lb1 by SSH indicated that K1-Lb1 is able to synthetize polysaccharides, for the synthesis of which K1-Lb8 appears to lack enzymes. A difference in the hydrophobicity properties of the surfaces of both strains indicated that this has impact on their surface. CONCLUSION: The K1-Lb1-specific sequences encoding putative glycosyltransferases and enzymes for polysaccharides synthesis may account for the observed differences in immunomodulation and surface properties between the two strains and for mediating potential probiotic effects.

Characterization of Intestinal Lactobacillus reuteri Strains as Potential Probiotics.

This study was conducted to evaluate the probiotic properties of Lactobacillus reuteri isolated from human infant feces (less than 3 months). Out of thirty-two representative L. reuteri strains isolated from the infant human feces, nine isolates (i.e. LR5, LR6, LR9, LR11, LR19, LR20, LR25, LR26 and LR34) showed survival in acid, bile and simulated stomach-duodenum passage conditions, indicating their high tolerance to gastric juice, duodenal juice and bile environments. The nine isolates did not show strong hydrophobic properties because the percentages of adhesion to the apolar solvent, n-hexadecane, did not exceed 40%, showing that their surfaces were rather hydrophilic. Functionality of these nine probiotic isolates was supported by their antagonistic activity and their ability to deconjugate bile salts. The safety of the nine indigenous L. reuteri isolates was supported by the absence of transferable antibiotic resistance determinants, DNase activity, gelatinase activity and hemolysis. The results obtained so far suggest that the nine strains are resistant to low pH, bile salts and duodenum juice, so they could survive when passing through the upper part of the gastrointestinal tract and fulfill their potential probiotic action in the host organism. According to these results, the L. reuteri strains isolated from human infant feces possess interesting probiotic properties that make them potentially good candidates for probiotics.

Functional properties of lactic acid bacteria isolated from ethnic fermented vegetables of the Himalayas.

A total of 94 strains of Lactic acid bacteria (LAB), previously isolated from ethnic fermented vegetables and tender bamboo shoots of the Himalayas, were screened for functional properties such as acidification capacity, enzymatic activities, degradation of antinutritive factors and oligosaccharides, production of biogenic amines, hydrophobicity and adherence to mucus secreting HT29 MTX cells. Strong acidification and coagulation activities of LAB strains were recorded. Most of the LAB strains showed antimicrobial activities against the used indicator strains; however, only Lb. plantarum IB2 (BFE 948) isolated from inziangsang, a fermented leafy vegetable product, produced a bacteriocin against Staphylococcus aureus S1. LAB strains showed enzymatic activities and also degraded oligosaccharides. Almost all the strains of LAB were non-producers of biogenic amines except few strains. Some strains of Lb. plantarum showed more than 70% hydrophobicity. Adherence to the mucus secreting HT29 MTX cells was also shown by seven strains indicating their probiotic nature.

Functional characteristics of Lactobacillus spp. from traditional Maasai fermented milk products in Kenya.

In this study functional characteristics of 23 representative Lactobacillus strains isolated from the Maasai traditional fermented milk 'Kule naoto' were determined. The Lb. acidophilus group strains showed resistance to gastric juice and bile. In addition, some Lb. acidophilus strains expressed bile salt hydrolase activity, and had ability to assimilate cholesterol in vitro. In-vitro adhesion to HT29 MTX cells of up to 70% was recorded. Lb. fermentum strains showed almost 100% survival under simulated stomach acidic conditions and physiological salt concentrations of bile salts, hydrophobicity values were over 80%. Most strains of the Lb. casei and Lb. acidophilus groups showed aggregation abilities of above 50%. Many strains expressed a protective effect against N-methyl-N'-nitro-N-nitrosoguanidine induced DNA damage according to the 'comet assay' and none was virulent. The antibiotic minimum inhibitory concentration of selected strains was established. According to these results, the Lactobacillus spp associated with 'Kule naoto', contain potentially probiotic (functional) strains.

Functional properties of Lactobacillus plantarum strains isolated from Maasai traditional fermented milk products in Kenya.

Lactobacillus plantarum was the major species among the lactic acid bacterial strains isolated from traditional fermented milk of the Maasai in Kenya. Selected strains were characterized for their functional properties using in vitro standard procedures. All strains expressed acid tolerance at pH 2.0 after 2-h exposure of values that ranged from 1% to 100%, while bile tolerance of acid-stressed cells at 0.3% oxgal varied from 30% to 80%. In vitro adhesion to the mucus-secreting cell line HT 29 MTX and binding capacity to extracellular protein matrices was demonstrated for several strains. The four strains tested in a simulated stomach duodenum passage survived with recovery rates ranging from 17% to 100%. Strains were intrinsically resistant to several antibiotics tested. From these in vitro studies, a number of Lb. plantarum strains isolated from the Maasai traditional fermented milk showed probiotic potential. The strains are good candidates for multifunctional starter culture development.