Interference Potential of Tannins and Chlorophylls in Zebrafish Phenotypic-Based Assays.

Natural products are prolific producers of diverse chemical scaffolds, which have yielded several clinically useful drugs. However, the complex features of natural products present challenges for identifying bioactive molecules using high-throughput screens. For most assays, measured endpoints are either colorimetric or luminescence based. Thus, the presence of the major metabolites, tannins, and chlorophylls, in natural products could potentially interfere with these measurements to give either false-positive or false-negative hits. In this context, zebrafish phenotypic assays provide an alternative approach to bioprospect naturally occurring bioactive compounds. Whether tannins and/or chlorophylls interfere in zebrafish phenotypic assays, is unclear. In this study, we evaluated the interference potential of tannins and chlorophylls against efficacy of known small-molecule inhibitors that are known to cause phenotypic abnormalities in developing zebrafish embryos. First, we fractionated tannin-enriched fraction (TEF) and chlorophyll-enriched fraction (CEF) from Camellia sinensis and cotreated them with PD0325901 [mitogen-activated protein kinase-kinase (MEK) inhibitor] and sunitinib malate (SM; anti-[lymph]angiogenic drug). While TEF and CEF did not interfere with phenotypic or molecular endpoints of PD0325901, TEF at 100 µg/mL partially masked the antiangiogenic effect of SM. On the other hand, CEF (100 µg/mL) was toxic when treated up to 6 dpf. Furthermore, CEF at 100 µg/mL potentially enhanced the activity of γ-secretase inhibitors, resulting in toxicity of treated embryos. Our study provides evidence that the presence of tannin and/or chlorophyll in natural products do interfere with zebrafish phenotype assays used for identifying potential hits. However, this may be target/assay dependent and thus requiring additional optimization steps to assess interference potential of tannins and chlorophylls before performing any screening assay.

A tannin-blocking agent does not modify the preference of sheep towards tannin-containing plants.

Sheep have been suggested to use their senses to perceive plant properties and associate their intake with consequences after ingestion. However, sheep with browsing experience do not seem to select against tannin-rich browsing materials in cafeteria trials. Thus, the objective of the present study was to evaluate the relationship between the chemical composition, selectivity index (SI), preference and intake rate (IR) of tannin-containing forage trees offered to sheep in cafeteria experiments. Four trees were selected for their condensed tannin content and their varying biological activities. Havardia albicans (high biological activity), Leucaena leucocephala (medium biological activity), Acacia gaumeri (low biological activity) and Brosimum alicastrum (very low biological activity) were used in this study. Ten hair sheep (23.7kg±1.43LW) with eight months of browsing experience in native vegetation were used in this study. Polyethylene glycol (PEG 3600MW) was administered to five sheep during all experiments. In experiment 1, fresh foliage from all trees was offered ad libitum for 4h. In experiment 2, B. alicastrum was withdrawn and the preference was determined again. The forage preference in experiment 1 was A. gaumeri (14.77gDM/kgLW)>B. alicastrum (11.77gDM/kgLW)>H. albicans (3.71gDM/kgLW)=L. leucocephala (1.87gDM/kgLW) (P<0.05). The preference in experiment 2 was A. gaumeri>H. albicans=L. leucocephala. PEG administration had no effect on the preference or IR. The intake rate seemed to have been affected by the plant density. Moreover, fiber compounds were found to be better predictors of DM intake than polyphenolic compounds at levels typically found in the evaluated forages. It was concluded that tannins and PEG did not modify the preferences of sheep in cafeteria trials. Thus, tannins are not involved in the preference regulation of animals with browsing experience.

Polyphenol-rich beverages enhance zinc uptake and metallothionein expression in Caco-2 cells.

The effect of red wine (RW), red grape juice (RGJ), green tea (GT), and representative polyphenols on Caco-2 cell (65)Zn uptake was explored. RW, RGJ, and GT enhanced the uptake of zinc from rice matrix. Fractionation of RW revealed that enhancing activity of zinc uptake was exclusively resided in the polyphenol fraction. Among the polyphenols tested, only tannic acid and quercitin stimulated the uptake of zinc while others did not influence the uptake. In tune with these results, only tannic acid and quercitin competed with zinquin (a zinc selective fluorophore) for zinc in vitro. Although all the polyphenols tested appear to enhance the expression of metallothionein (MT), the induction was higher with tannic acid, quercitin, and RW extract. Furthermore, phytic acid abrogated the tannic acid-induced MT expression. These results suggest that polyphenol-rich beverages, tannic acid, and quercitin bind and stimulate the zinc uptake and MT expression in Caco-2 cells.

Value addition to finger millet (Eleusine coracana) by germination and fermentation with Monascus purpureus.

The present investigation has been carried out to highlight the importance of germination and fermentation of finger millet with Monascus purpureus. Finger millet was subjected to (i) germination, (ii) to fermentation with M. purpureus, and (iii) germination followed by fermentation with M. purpureus. The results of this experiment suggest that the germinated (72 h) finger millet fermented (10 days) with M. purpureus showed reduction in phytic acid and tannin contents by 88.8% and 90.1%, respectively, with an increase of 61.5% HCl-extractable minerals, reducing sugars and soluble proteins thereby supporting the production of antihypercholesterolemic metabolite, statin.

In vitro larval migration and kinetics of exsheathment of Haemonchus contortus larvae exposed to four tropical tanniniferous plant extracts.

As for some temperate forage, some tropical tanniniferous plants (TTP) from browsing might represent an alternative to chemical anthelmintic. The anthelmintic effect of four TTP (Acacia pennatula, Lysiloma latisiliquum, Piscidia piscipula, Leucaena leucocephala) on Haemonchus contortus was measured using two in vitro assays. First, the effects of increasing concentrations of lyophilized extracts (150, 300, 600, 1200 microg/ml PBS) were tested on H. contortus larvae (L(3)) using the larval migration inhibition (LMI) test. An inhibitor of tannin, polyvinyl polypyrrolidone (PVPP), was used to verify whether tannins were responsible for the AH effect. Secondly, the effects of extracts on larval exsheathment were examined. Larvae (L(3)) were in contact with extracts (1200 microg/ml) for 3h, and then were exposed to an artificial exsheathment procedure with observations of the process at 10 min intervals. A general lineal model (GLM) test was used to determine the dose effect in the LMI test and the difference of the percentage of exsheathed larvae between the control and the treatment groups. A Kruskal Wallis test was used to determine the effect of PVPP on LMI results. The LMI test showed a dose-dependent anthelmintic effect for A. pennatula, L. latisiliquum and L. leucocephala (P<0.01), which disappeared after PVPP addition, confirming the role of tannins. No effect was found for P. piscipula on H. contortus in the LMI test. However, all four plant extracts interfered with the process of L(3) exsheathment which might be involved as a mechanism of action of tannins on H. contortus larvae. A. pennatula, L. latisiliquum and L. leucocephala could be used as an anthelmintic for the control of H. contortus after confirmation based on in vivo studies.

Salivary proteins as a defense against dietary tannins.

Tannins, a diverse group of water-soluble phenolics with high affinity to proteins, are widely distributed in various parts of plants, and have negative effects in herbivores after ingestion. Some mammalian species are thought to counteract tannins by secreting tannin-binding salivary proteins (TBSPs). Several types of TBSPs are found in the saliva of laboratory animals, livestock, and wildlife. Among them, proline-rich proteins (PRPs) and histatins are effective precipitators of tannins. It is widely accepted that, at the least, PRPs act as a first line of defense against tannins. Many observations support this idea: in vitro affinity of PRPs to tannins is far higher than that of other proteins such as bovine serum albumin; complexes formed between PRPs and tannins are stable even under the conditions in the stomach and intestine; and PRP production is induced by ingesting tannins. It is believed that species that usually ingest tannins as part of their natural diets produce high levels of PRPs, whereas species not exposed to tannins produce little or no PRPs. This hypothesis is generally supported, although studies on TBSPs in wildlife are limited. This work stresses the importance of gathering basic information on such items as the characteristics of unidentified TBSPs, and seasonal and geographical variations in PRP production.

Prevention of hydrolysable tannin toxicity in goats fed Clidemia hirta by calcium hydroxide supplementation.

Although plants containing hydrolysable tannins can be hepatotoxic, such poisoning has not been reported in Indonesia despite the presence of these plants. In order to determine the hepatotoxic potential of Indonesian plants, goats were intoxicated experimentally with the Indonesian plant Climedia hirta (harendong), which contained 19% hydrolysable tannin. The prophylactic effect of Ca(OH)2 supplementation on the disease was also examined. Two groups of goats were fed for 28 days with grain-based pellets containing 50% harendong leaf or 50% harendong leaf + 8% Ca(OH)2. Two control groups were fed similar pellets containing 50% of the non-toxic elephant grass (Pennisetum purpureum) with and without 8% Ca(OH)2. Serum enzymes indicative of liver damage were monitored during the experiment and histopathological examination of selected tissues was done at the conclusion of the experiment. In goats given unsupplemented harendong pellets there was a significant increase in aspartate aminotransferase and glutamate dehydrogenase from 50.2 and 20.6 U l-1 to 219.6 and 63.3 U l-1, respectively. These changes were associated with moderate to severe nuclear plemorphism, vacuolation and megalocytosis of hepatocytes and deposits of brown pigment in the Kupffer cells. There was also nephrosis of the renal convoluted tubules and collecting ducts, abomasitis and enteritis. Biochemical and histological changes were reduced significantly in the harendong + Ca(OH)2 group and virtually absent from control groups. It is concluded that hydrolysable tannins in harendong leaf are hepato- and nephrotoxic and associated with gastroenteritis, but that poisoning may be ameliorated by Ca(OH)2 supplementation.

Protection by plasma proteins against condensed tannin-mediated platelet activation.

Condensed tannins isolated from cotton bracts are potent platelet agonists. They promote the secretion of 5-hydroxytryptamine from washed bovine platelets with an EC50 of 35 micrograms/ml. However, when platelet-rich plasma was used in lieu of washed platelets, the potency of tannin was decreased over 50-fold. Reconstitution experiments demonstrated that some component in the plasma was responsible for the diminished potency of tannin. Fractionation of platelet-poor plasma suggested that albumin may be the inhibitory plasma component. Confirmation of this hypothesis was obtained from the finding that purified serum albumin at physiologic concentrations inhibited the tannin-mediated release of platelet 5-HT by more than 70%. These data suggest that any activation of platelets which may occur in vivo by the tannins present in inhaled cotton dust would require higher tannin concentrations than would have been predicted from our previous studies using washed platelets.