Rooibos (Aspalathus linearis) beyond the farm gate: From herbal tea to potential phytopharmaceutical

Aspalathus linearis (Burm.f.) Dahlg. (Fabaceae, Tribe Crotalarieae), an endemic South African fynbos species, is cultivated to produce the well-known herbal tea, rooibos. It is currently sold in more than 37 countries with Germany, the Netherlands, the United Kingdom, Japan and the United States of America representing 86% of the export market in 2010. Its caffeine-free and comparatively low tannin status, combined with its potential health-promoting properties, most notably antioxidant activity, contributes to its popularity. First marketed in 1904 in its fermented (oxidised) form, green rooibos is a new product recently on the market. The utilisation of rooibos has also moved beyond a herbal tea to intermediate value-added products such as extracts for the beverage, food, nutraceutical and cosmetic markets. Its potential as a phytopharmaceutical, shown in recent scientific studies, has not yet been exploited. This review focuses on past and current research aimed at enhancing the value of rooibos herbal tea as a specialised, niche product and expanding its value-adding potential against the background of its traditional use and the current market. The focus falls specifically on aspects such as composition, processing, quality and rooibos as food and potential medicine.     

Secondary metabolite profile and phytotoxic activity of genetically distinct forms of Colletotrichum gloeosporioides from yam (Dioscorea spp.)

Highly virulent, slow-growing grey (SGG); moderately virulent, fast-growing salmon (FGS); and avirulent/weakly virulent, fast-growing grey (FGG) forms of Colletotrichum gloeosporioides have been described from yam (Dioscorea spp.), but little is known about their chemodiversity or the role of toxins in their pathogenesis. Secondary metabolite profiles in high performance tlc (hptlc) showed that the pathogenic SGG and FGS forms have a chemotype (A or B) that is distinct from the non-pathogenic FGG form (chemotype C). Crude extracts of 35-d-old Czapek-Dox yeast broth cultures of FGS and SGG isolates caused tissue necrosis on treated yam leaves but not those of FGG isolates. Extracts from uninoculated broth cultures showed no phytotoxic activity. Toxicity of the culture filtrate was not host specific and toxic substances were thermostable. Dioscorea genotypes with varying levels of resistance to anthracnose differed in their sensitivity to crude toxin extract of FGS (Cg33) and SGG (Cg25) isolates, indicating that these extracts may be useful in evaluating host resistance to anthracnose in vitro. Analysis of two toxin fractions unique to the pathogenic FGS and SGG forms using hlpc, mass spectrometry and nuclear magnetic resonance suggested the presence of a low molecular weight amide peptide. However, possibly due to low yield and the presence of impurities, the chemical structure of the compound(s) could not be fully elucidated.     

Fate of resting cysts of Alexandrium spp. ingested by Perinereis nuntia (Polychaeta) and Theola fragilis (Mollusca)

The ingestion of resting cysts of Alexandrium spp. by Perinereis nuntia (Polychaeta) and Theola fragilis (Mollusca) was experimentally examined in the laboratory. P. nuntia and T. fragilis were cultured in bottom sediment containing a high density of Alexandrium cysts under dark conditions. Moreover, to evaluate the degree and consequence of being ingested, the density of cysts in the control sediment (no macrobenthic organisms) and the germination capability of the cysts in the faecal pellets of the two species of macrobenthos were examined.Cysts in the culture sediment were found to be ingested by both P. nuntia and T. fragilis. No difference in the density of cysts between the sediments cultured with and without P. nuntia was observed. However, the density of cysts in the sediments with T. fragilis decreased by 24% compared to the density in the control sediment. It is possible that most of the cysts ingested were digested by T. fragilis. The rate of Alexandrium cyst digestion by this species is estimated 594 cysts/individual/day. It is estimated that 91% of the cysts ingested by T. fragilis were partially or totally digested and only 9% were excreted in a viable state during the experiment. Thus, T. fragilis has a stronger affect on the abundance of Alexandrium cysts compared with P. nuntia.No significant difference was observed between the germination success of the cysts from faecal pellets of P. nuntia and T. fragilis compared to the cysts in the control sediment. If, however, the necessary light for the cysts to germinate is cut off by being enclosed within the faecal pellet, the germination rate of cysts from the faecal pellets may be suppressed.     

The complex effects of the invasive polychaetes Marenzelleria spp. on benthic nutrient dynamics

The effects of the polychaetes Marenzelleria sp. (Polychaeta, Spionidae), nonindigenous, rapidly increasing species in the Baltic Sea, on benthic nutrient fluxes, denitrification and sediment pore water nutrient concentration were studied in laboratory experiments using a flow-through setup with muddy sediment from coastal regions of the Gulf of Finland. In addition, different forms of sediment phosphorus (P), separated by chemical fractionation, were studied in three sediment layers. At a population density corresponding to about half the highest measured in the northern Baltic Sea, Marenzelleria sp. increased the fluxes of P and ammonium to the water column. No effect could be recorded for denitrification. Since the previously dominant species of the area, Monoporeia affinis, can enhance denitrification and lower the amount of dissolved P in the pore water, the replacement of M. affinis with Marenzelleria spp. may lead to increased P flux to the water column and decreased denitrification, further increasing the ammonium flux to the water column. However, sediment reworking by Marenzelleria spp. also oxidizes the surface sediment in the long run, improving its ability to retain P and support nitrification. Therefore, the impact of Marenzelleria spp. on sediment nutrient release may not be as drastic as the initial reactions seen in our experiments suggest.     

Identification of Nosema bombi Fantham and Porter 1914 (Microsporidia) in Bombus impatiens and Bombus sandersoni from Great Smoky Mountains National Park (USA)

Ninety three bumble bees belonging to the genus Bombus, subgenus Pyrobombus (three Bombus vagans, seven Bombus bimaculatus, 17 B. sandersoni and 68 B. impatiens) from Great Smoky Mountains National Park were examined for microsporidia. Light microscopy of calcoflour and trichrome-stained smears, and PCR revealed infection with N. bombi in one specimen each of B. sandersoni and B. impatiens. Sizes and shapes of spores in both N. bombi isolates were similar to those described for European isolates of the microsporidium. A region of the rRNA gene from the B. impatiens isolate (1689 bp, accession GQ254295) aligned with homologous sequences from eight European isolates, with only three variable sites. Sequence variability of this region between novel isolates and the European ones was the same as among European isolates.     

Vulnerability of newly settled plaice (Pleuronectes platessa L.) to predation: effects of habitat structure and predator functional response

Plaice (Pleuronectes platessa) nursery grounds on the Swedish west coast have been subject to increasing cover of annual green macroalgae during recent years, with growth of algae starting at the time of plaice settlement in April to May. A laboratory experiment was performed to investigate how the vulnerability to predation of metamorphosing plaice was affected by the presence of filamentous algae. Predation by shrimps (Crangon crangon) on settling plaice larvae was higher on sand than among algae, whereas predation by crabs (Carcinus maenas) was unaffected by habitat type, suggesting a lower overall mortality of plaice in the vegetated habitat. When predators and prey were presented with a combination of the two habitats, predation by shrimps was as high as that in the sand treatment alone, whereas predation by crabs was lower than that in the two treatments with one habitat. Based on these results, an additional experiment was performed, investigating the functional response of shrimps to six densities of juvenile plaice in a sand habitat with alternative prey present. The proportional mortality of juvenile plaice (12-16 mm total length (TL)) was density-dependent and was best described by a type III (sigmoid) functional response of the predatory shrimps. The results suggested that the combined predation pressure from shrimps and crabs was lower among algae than on sand, but settling plaice and predatory shrimps chose the sand habitat. Plaice densities in the sigmoid part of the obtained functional response curve represented normal to high field densities of plaice on the Swedish west coast, suggesting that shrimp predation could have a stabilising effect on plaice recruitment. The formation of macroalgae mats could therefore lead to a concentration of plaice juveniles in the remaining sand habitat and increased mortality through density-dependent predation by shrimps.     

Analysis of somatic and salivary gland antigens of third stage larvae of Rhinoestrus spp. (Diptera, Oestridae)

Larvae of Rhinoestrus spp. (Diptera, Oestridae) infect nasal and sinus cavities of horses, causing a nasal myiasis characterized by severe respiratory distress. Presently, the diagnosis of horse nasal botfly relies on the observation of clinical signs, on the post mortem retrieval of larvae or on molecular assays performed using pharyngeal swabs. The present study was carried out to characterize larval somatic proteins and salivary glands of Rhinoestrus spp. in a preliminary assessment towards the immunodiagnosis of equine rhinoestrosis. Out of the 212 necropsied horses 13 were positive for the presence of Rhinoestrus spp. larvae. The analysis of the sera from the infected animals by Western blotting assay showed the presence of a specific host humoral immune response against Rhinoestrus spp. larvae and proved that the salivary glands are the major immunogens in horse nasal botflies.     

Nucleic acid aptamers for capture and detection of Listeria spp

The purpose of this study was to identify biotinylated single-stranded (ss) DNA aptamers with binding specificity to Listeria and use these for capture and subsequent qPCR detection of the organism. For aptamer selection, SELEX (systematic evolution of ligands by exponential enrichment) was applied to a biotin-labeled ssDNA combinatorial library. After multiple rounds of selection and counter-selection, aptamers separated, sequenced, and characterized by flow cytometry showed binding affinities to L. monocytogenes of 18–23%. Although selected for using L. monocytogenes, these aptamers showed similar binding affinity for other members of the Listeria genus and low binding affinity for non-Listeria species. One aptamer, Lbi-17, was chosen for development of a prototype capture and detection assay. When Lbi-17 was conjugated to magnetic beads and used in a combined aptamer magnetic capture (AMC)-qPCR assay, the pathogen could be detected at concentrations <60 CFU/500 μl buffer in the presence of a heterogeneous cocktail of non-Listeria bacterial cells, with a capture efficiency of 26–77%. Parallel experiments using immunomagnetic separation (IMS)-qPCR produced the same detection limit but lower capture efficiency (16–21%). Increasing assay volume to 10 and 50 ml resulted in reduced capture efficiency and higher limits of detection, at 2.7 and 4.8 log₁₀ CFU L. monocytogenes per sample, respectively, for the AMC-qPCR assay. Biotinylated ssDNA aptamers are promising ligands for food-borne pathogen concentration prior to detection using molecular methods.     

Distribution and Seasonal Abundance of Trematode Parasites (Trematoda: Allocreadiidae: <Emphasis Type="Italic">Crepidostomum</Emphasis> spp.) in burrowing-mayfly nymphs (Ephemeroptera: Ephemeridae: <Emphasis Type="Italic">Hexagenia</Emphasis> spp.) from Connecting Rivers of the Laurentian Great Lakes

Burrowing-mayfly nymphs such as Hexagenia spp. have been used extensively in North America and Europe as a biomonitoring tool to indicate mesotrophic water quality, yet infestation by associated parasites has not been well documented. We performed laboratory analysis of archived samples of Hexagenia spp. nymphs collected in 1985 and 1986 to provide base-line data on the distribution (1985) and seasonal infestation (1986) of the trematode parasite Crepidostomum spp. in Hexagenia spp. nymphs in connecting rivers between Lakes Superior and Erie of the Laurentian Great Lakes. In May and June 1985, frequency of occurrence of metacercariae was widely distributed throughout the connecting rivers (63% of 203 stations with nymphs), except in areas where nymph densities were relatively low (i.e.,≤69 nymphs/m²). Distribution was probably underestimated in the present study because of low probability (mean = 31%, range = 0–57%) of detecting infestation in a small number of collected nymphs ( ≤10) at nymph densities ≤69/m². In 1986, seasonal infestation between April and October occurred in 3.3% (627) of 18696 nymphs. Overall prevalence, mean intensity, and mean abundance of parasites at one station in the St. Marys River indicate parasite transmission occurred between June and September. This period of transmission is dependent on the life-cycle of the parasite. In addition, the life-cycle of Hexagenia spp. determines which annual cohort of nymphs is infested and therefore, the duration of infestation. Although, no impacts of infestation on Hexagenia spp. nymphs were observed in the present study, infestation intensities were high enough (≥25 metacercariae per nymph) at one station in the St. Marys River to potentially cause tissue damage in a high proportion (53%) of infested nymphs.     

The impact of infauna (Nereis diversicolor and Saduria entomon) on the redistribution and biomass of macroalgae on marine soft bottoms

Mass occurrence of macroalgae is a phenomenon attributed to eutrophication, and can lead to drastic changes in the benthic communities on soft bottoms. While the negative effects of macroalgal blooms on the macrozoobenthos have been studied extensively, the effects of the infauna on the macroalgal material have not previously been studied in the northern Baltic Sea. The impact of the infaunal species Nereis diversicolor and Saduria entomon on the burial and biomass of Enteromorpha spp., Cladophora glomerata and Fucus vesiculosus, was assessed through a series of microcosm experiments. Results show that S. entomon did not significantly affect the biomass of the algae, nor actively relocate them. N. diversicolor redistributed the filamentous green algae into the sediment, down to 4 cm at most, and decreased the biomass of the filamentous algae by 140-360%. Furthermore, the loss of biomass promoted in presence of polychaetes proved to be a density dependent process. The effect on the perennial macroalgal species, F. vesiculosus, was less clear, as no redistribution or significant change in macroalgal biomass was observed. Our findings show that infauna can contribute to a loss in macroalgal biomass through feeding and burrowing activities leading to the redistribution and incorporation of the detritus into bioturbated sediment.     

A comparative study on the antimutagenic properties of aqueous extracts of Aspalathus linearis (rooibos), different Cyclopia spp. (honeybush) and Camellia sinensis teas

Antimutagenic activity of aqueous extracts of the South African herbal teas, Aspalathus linearis (rooibos) and Cyclopia spp. (honeybush) was compared with that of Camellia sinensis (black, oolong and green) teas in the Salmonella mutagenicity assay using aflatoxin B₁ (AFB₁) and 2-acetylaminofluorene (2-AAF) as mutagens. The present study presents the first investigation on antimutagenic properties of C. subternata, C. genistoides and C. sessiliflora. The herbal teas demonstrated protection against both mutagens in the presence of metabolic activation, with the exception of “unfermented” (green/unoxidised) C. genistoides against 2-AAF, which either protected or enhanced mutagenesis depending on the concentration. Antimutagenic activity of “fermented” (oxidised) rooibos was significantly (P < 0.05) less than that of Camellia sinensis teas against AFB₁, while for 2-AAF it was less (P < 0.05) than that of black tea and similar (P > 0.05) to that of oolong and green teas. Antimutagenic activity of unfermented C. intermedia and C. subternata exhibited a similar protection as fermented rooibos against AFB₁. Against 2-AAF, fermented rooibos exhibited similar protective properties than unfermented C. intermedia and C. sessiliflora. Unfermented rooibos was less effective than the C. sinensis teas and fermented rooibos, but had similar (P > 0.05) antimutagenicity to that of fermented C. sessiliflora against AFB₁ and fermented C. subternata against 2-AAF. Fermented C. intermedia and C. genistoides exhibited the lowest protective effect against 2-AAF, while fermented C. intermedia exhibited the lowest protection when utilising AFB₁ as mutagen. Aspalathin and mangiferin, major polyphenols in rooibos and Cyclopia spp., respectively, exhibited weak to moderate protective effects when compared to the major green tea catechin, (−)epigallocatechin gallate (EGCG). Antimutagenic activity of selected herbal tea phenolic compounds indicated that they contribute towards (i) observed antimutagenic activity of the aqueous extracts against both mutagens and (ii) enhancement of the mutagenicity of 2-AAF by unfermented C. genistoides. Antimutagenic activity of the South African herbal teas was mutagen-specific, affected by fermentation and plant material, presumably due to changes and variation in phenolic composition.     

Campylobacter spp. detection in the 21st century: A review of the recent achievements in biosensor development

Campylobacter spp. are an important cause of acute bacterial diseases in humans worldwide. Many bacterial species in the Campylobacter genus are considered harmful and may cause several infectious diseases. Currently, there are no commercial biosensors available to detect Campylobacter spp. in food matrices, and little to no testing has been done in research laboratories with actual food matrices. Biosensors potentially provide a powerful means to detect Campylobacter spp. with the advantages of high sensitivity (low limits of detection with a high signal to noise ratio), high specificity (able to selectively detect the target among several similar targets), real time sensing, and in-site monitoring. This review summarizes the latest research in biosensing technologies for detection of Campylobacter spp. based on a variety of transducers and recognition elements. Finally, a comparison is made among all recently reported biosensors for the detection of Campylobacter spp.     

Impact of fiddler crabs (Uca spp.) on rates and pathways of benthic mineralization in deposited mangrove shrimp pond waste

The impact of fiddler crabs (Uca spp.) on benthic mineralization rates and pathways in deposits of shrimp pond waste (SPW) with planted mangrove trees (Rhizophora apiculata) were determined in the Ranong mangrove forest, Thailand. Sediment metabolism, measured as CO₂ flux, increased by 2- to 3-fold when either fiddler crabs or mangrove trees were present compared to control plots. Sulfate reduction rates (SRR) were always high and partitioning of various electron acceptors to total carbon oxidation revealed that sulfate reduction contributed by >90%, with iron reduction being important only near the sediment-water interface. However, significant iron reduction appeared down to 7 cm when bioturbation and plant roots were combined in the easily oxidizable mangrove sediment (MS), indicating that infaunal activity and plant roots were able to alter the substratum. Microprofiles around individual Uca burrows showed 46% lower SRRs in a 15-mm-thick oxidized layer around the burrows compared to the surrounding sediment. The burrow wall environment appears to be a zone of intense reoxidation of reduced compounds as indicated by low pools of reduced sulfide compounds and a high Fe(III) content. Despite the decreased SRRs near the burrow, and the introduction of Fe(III) to deeper sediment layers, fiddler crabs and mangrove trees have only limited impact on rates and partitioning of anaerobic carbon mineralization in the SPW. This lack of response was attributed to the relative small volume of sediment affected by crab activities.     

Genetic evidence for three discrete taxa of Melampsora (Pucciniales) affecting willows (Salix spp.) in New York State

Rust fungi in the genus Melampsora (Pucciniales) are the most important pathogens of shrub willows (Salix spp.) cultivated for biomass in New York State and temperate regions worldwide. The taxonomy and species identification of these fungi historically have been problematic as they are morphologically indistinguishable on willow and often have complex life histories. Melampsora of Salix in North America, therefore, have been circumscribed to the collective species Melampsora epitea Thüm. and further delineated to formae speciales by aecial host. Ribosomal DNA (rDNA) data was obtained from 75 collections/isolates of Melampsora in NY State affecting either native and cultivated Salix spp. or suspected alternate hosts. Maximum likelihood (ML), maximum parsimony (MP), and Bayesian (BI) analyses were conducted on three data partitions (individual and concatenated): complete internal transcribed spacer (ITS) and partial large subunit (LSU) rDNA sequences for all collections. Analyses of the ITS and concatenated ITS-LSU sequences revealed that Melampsora on native and cultivated willows in NY State consisted of three phylogenetically delineable taxa (phylotaxa); monophyly for each phylotaxon was strongly supported by ML, MP, and BI credibility values. Phylotaxa were also delimited phylogenetically by aecial host: Alpine currant (Ribes alpinum), eastern larch (Larix laricina), or balsam fir (Abies balsamea).     

The use of a rapid assay to detect the neuraminidase production in oral Porphyromonas spp. isolated from dogs and humans

Neuraminidase was produced by 32.1% and 28.5% of Porphyromonas from dogs with and without periodontitis, respectively; and by 31.8% of bacteria from humans. The presence of neuraminidase in Porphyromonas spp. suggests that this enzyme can be involved with the pathogenesis of the periodontal disease, and the use of this assay to detect the neuraminidase production in oral Porphyromonas species is suggested.     

Chloroplast DNA analysis in Tunisian fig cultivars (Ficus carica L.): Sequence variations of the trnL-trnF intergenic spacer

We investigated the nucleotide variation of a non-coding, chloroplast DNA (cpDNA) region to infer relationships among Tunisian fig cultivars. In this study, we examine the level of genetic diversity and its distribution using sequences of the trnL and trnF genes intergenic spacer. The non-coding region displays 28 substitution sites. Insertions and deletions involving 6 sites were found. By using the Kimura-2 method, nucleotide sequences have been aligned using the MEGA program to calculate pairwise divergence of trnL-trnF spacer sequences between cultivars. The size of this non-coding region varied from 430 to 464 bases. The relatively high A + T values (63.7–64.4%) of trnL-trnF intergenic spacer in Ficus carica may explain the high proportion of the identified transversions (t_i/t_v = 0.9). These results suggest the occurrence of nucleotide diversity with a large variation level of chloroplast non-coding region. The analysed data illustrate a considerable level of variability in the genetic pool of the local germplasm. In fact, relationships inferred from the cpDNA analysis suggest several clades, which do not show geographical correspondence. Fourteen haplotypes were detected among 20 individuals examined, yielding a haplotype diversity of 0.983 and a high level of nucleotide diversity (0.0100). The observed variation pattern of plastid DNA provides evidence that the fig germplasm has been undergoing rapid expansion. Neutrality tests rejected the neutrality assumption in the total sample. The cytoplasm variability indicates a narrow genetic base in the cultivated common fig. Despite the high level value of the apparent diversity, we may conclude that fig chloroplast genome provides a new conceptual and practical opportunity to evaluate genetic diversity and to identify local cultivars, making it a valuable source to include into potential breeding programs.