Phenolics and condensed tannins of forage plants from Botswana and their biological significance : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry at Massey University, Turitea campus, Palmerston North, New Zealand
The main objectives of this study were to isolate and characterise phenolics and CT from Botswanan forage plants, and to investigate their potential anti-parasitic and immunostimulatory properties. Viscum rotundifolium, Viscum verrucosum, Tapinanthus oleifolius, Grewia flava and Ipomoea sinensis forage leaves and small stems were harvested over two summers (February 2009 and 2010), extracted twice with acetone:water (7:3) and subsequently defatted with dichloromethane. Each crude extract (6 g) was loaded onto a Sephadex LH-20 column and eluted with aqueous methanol (1:1) to yield four fractions, and subsequently eluted with acetone:water (7:3) to yield three fractions. Phytochemical screening of the fractions for the presence of CT and phenolics was conducted by a reverse phase high performance liquid chromatography coupled to a photodiode array (RP-HPLC-PDA) detector at 280 nm. The butanol-HCl colorimetric assay revealed that the total CT concentrations from the forage plants ranged from 0.2 to 12.7 (g/100g dry matter). These results indicated that significant amounts of CT were present in V. verrucosum, T. oleifolius and G. flava.
The potential impact of each purified CT fraction was evaluated for anti-parasitic effects using a larval development assay (LDA). Three different species of gastrointestinal nematodes (Haemonchus contortus, Trichostrongylus colubriformis and Teladorsagia circumcincta) from sheep were tested with the fractions at 100 and 500 μg/mL. CT fractions from V. rotundifolium and I. sinensis samples, collected in 2009 and 2010, did not inhibit larval development (L1 and L2) to the infective L3 stage. CT isolated from V. verrucosum and T. oleifolius which were collected in 2009 completely inhibited the development of all parasite species at both concentrations. Also, complete inhibition of larval development of all tested parasite species was obtained in CT fractions from G. flava collected in 2009, with the lowest inhibitory activity at 62.5 μg/mL. These findings suggest that CT extracts have anti-parasitic effects in vitro, which may be translated into reduction of the effects of parasitism in ruminants in vivo.
The potential impact of the extracts on priming of γδ T cells from the peripheral blood of lambs, calves and kids at 5 and 10 μg/mL was also evaluated in vitro. Condensed tannins (CT) from G. flava significantly primed γδ T cells in kids by up to 64.75% at 10 μg/mL, which was statistically significant relative to the negative control at 22.66% (p=0.004). CT from T. oleifolius also induced priming of γδ T cells in kids, while fractions from V. rotundifolium and V. verrucosum induced minimal priming of γδ T cells. These findings suggest that CT from a selected Botswanan forage plants can stimulate the immune system in vivo in selected ruminant species.
The anti-parasitic and immunostimulatory effects could be influenced, among other things, by the chemical structure and concentration of CT in the forage sample. The 13C-NMR and thiolysis results revealed that CT from V. verrucosum, T. oleifolius and G. flava were procyanidin (PC) and cis dominant. Further purification of the thiolysis adducts of CT from these plants led to the isolation of (-)-epicatechin which was found to be the dominant compound in the extension units of the CT polymer. The final stage of the research was the re-chromatography of methanolic fractions containing low molecular weight phenolics. The low molecular weight phenolics from the methanolic fractions were successfully purified and isolated. The characterisation of flavonoids by NMR and LC-ESI-MS/MS showed that quercetin was predominant in the purified fractions with attached sugars such as rhamnose, glucose and apiose.