P. J. Whitfield

In vitro screening for anthelmintic and antitumour activity of ethnomedicinal plants from Thailand.

AIM OF STUDY This study screened for anthelmintic and/or antitumour bioactive compounds from Thai indigenous plants and evaluated effectiveness against three different worm species and two cancer cell lines. MATERIALS AND METHODS Methylene chloride and methanol extracts of 32 plant species were screened for in vitro anthelmintic activity against three species of worms, the nematode Caenorhabditis elegans, the digeneans Paramphistomum epiclitum and Schistosoma mansoni (cercariae). Cytotoxicity of the extracts was evaluated against two cancer cell lines: human amelanotic melanoma (C32) and human cervical carcinoma (HeLa) by the SRB assay. Anthelmintic and anticancer activities were evaluated by the inhibiting concentration at 50% death (IC(50)) and the selectivity index (SI) relative to human fibroblasts. RESULTS AND CONCLUSIONS None of the extracts were active against Paramphistomum epiclitum. Plumbagin, a pure compound from Plumbago indica, had the strongest activity against Caenorhabditis elegans. The methylene chloride extract of Piper chaba fruits had the strongest activity against schistosome cercariae. Strong cytotoxicity was shown by the methylene chloride extract of Michelia champaca bark and the methanol extract of Curcuma longa rhizome against C32 and HeLa, respectively. These extracts had higher SI (>100) than positive controls in relation to either the worms or the cell lines. The methanol extract of Bouea burmanica had a slightly lower activity towards C32 cells than did Michelia champaca but had a much higher SI (>27,000). ETHNOPHARMACOLOGICAL RELEVANCE The plant species screened in this research was recorded by several indigenous medicinal practitioners as antiparasitic, anticancer and/or related activities to the human major organ system.

Experimental studies of infection dynamics: infection of the definitive host by the cercariae of Transversotrema patialense

The number of cercariae of Transversotrema patialense which attach to the fish host Brachydanio rerio, during a fixed exposure period, is shown to be directly proportional to cercarial density within an experimental infection arena. The distribution of successful infections/host is shown to change from a random pattern to an over-dispersed form as cercarial exposure density or duration of host exposure to infection increases. A stochastic simulation model is used to demonstrate that small differences in host susceptibility to infection, within a population of hosts, can generate patterns of dispersion in parasite numbers/host similar to those observed in the experimental studies. Differences in host behaviour, during the period of exposure to infection, are thought to generate variability in host susceptibility to cercarial infection.

Parasite utilization of a host community: the distribution and occurrence of metacercarial cysts of Echinoparyphium recurvatum (Digenea: Echinostomatidae) in seven species of mollusc at Harting Pond, Sussex

The occurrence, in September 1979, of Echinoparyphium recurvatum metacercarial cysts in a community of molluscan hosts at Harting Pond, West Sussex, was investigated. The parasite exhibited broad host specificity with all 7 species comprising the molluscan community being infected. There were marked differences, however, in the extent to which each host species was utilized and using the mean number of parasites/ individual mollusc as a parameter of infection the mollusc species may be ranked in descending order of utilization as follows: Sphaerium corneum, Lymnaea peregra, Valvata piscinalis , planorbids, Pisidium subtruncatum and Potamopyrgus jenkinsi . Combination of basic infection data with population estimates for each mollusc species in the study area showed that the bivalve P. subtruncatum and the prosobranch V. piscinalis were the most important hosts because they contained approximately 90% of the total number of cysts. In both of these hosts the cyst population was over-dispersed and the degree of over-dispersion increased with host size. The pattern of second intermediate host utilization is discussed in relation to the likely flow of E. recurvatum between the first intermediate host ( L. peregra ) and wildfowl definitive hosts at Harting.

ANTISCHISTOSOMAL BIOACTIVITY OF ISOFLAVONOIDS FROM MILLETTIA THONNINGII (LEGUMINOSAE)

Extracts of the seeds of the leguminous tree Millettia thonningii are molluscicidal and schistosomicidal and contain a mixture of isoflavonoids and 3-phenylcoumarins. Robustic acid and a pure mixture of alpinumisoflavone and dimethylalpinumisoflavone, as characterized by nuclear magnetic resonance, were isolated from a dichloromethane extract. The bioactivity of these compounds against Schistosoma mansoni miracidia, cercariae, and adult worms was tested in vitro. Both robustic acid and the alpinumisoflavone mixture killed cercariae and adult schistosomes in vitro. At sublethal concentrations of the isoflavonoids, reductions in miracidial motility were measured using a video-imaging technique. The inhibition of mitochondrial electron transport at site I by the isolated isoflavonoids was also investigated. The results suggest that robustic acid and at least 1 alpinumisoflavone compound from the dichloromethane extract of the seeds of M. thonningii are responsible for some of the observed bioactivity of this extract against schistosomes.

The infection of human skin by schistosome cercariae : studies using Franz cells

Franz cells (2-chambered, air/fluid phase static diffusion devices, previously used for the study of drugs across viable human skin) are utilized for the first time to investigate the process of infection of human skin by Schistosoma mansoni cercariae. Skin obtained from cosmetic surgery sources was used in the Franz cells to describe the temporal dynamics of the early interaction of cercariae with skin. At 38 degrees C, about 50% of cercariae applied in water to the epidermal surface of the skin were irreversibly attached within 1 min and after 5 min about 85%, were similarly irrecoverable. The technique also provides the means of following the early penetration path of cercariae by histological methods. Franz cell results on the dynamics of attachment/early penetration have been compared with those obtained using artificial skin equivalents and non-human mammalian skin models in the context of the physical and chemical differences between these systems and viable human skin. It is concluded that Franz cells provide a convenient system for directly investigating the early phases of S. mansoni cercariae interaction with human skin.

The egg sorting function of the uterine bell of Polymorphus minutus (Acanthocephala)

An adult female Polymorphus minutus releases only mature eggs into the intestine of its final host. These eggs come from the pool of eggs in the body cavity of the worm which contains only about 30% of mature eggs, the rest being immature. An analysis of the age structures of the egg populations in the body cavity and the uterus shows that an assortment of mature and immature eggs has taken place as the eggs pass from the body cavity to the uterus. The uterus contains only mature eggs and these are the eggs which are about to be released. The only pathway whereby eggs can enter the uterus from the body cavity is through the uterine bell. This suggests that it is the uterine bell which is able to select mature eggs from the mixture of eggs in the body cavity. A uterine bell in vitro engages in precisely patterned muscular activity which propels eggs through its branching lumen. In one part of the bell (the grooves between the median wall cells and the lappets of the uterine duct cells), the patterned muscular activity passes mature eggs into the uterus and immature eggs back into the body cavity to complete their development. The greater length of mature eggs seems to be the character which enables them to be ‘recognized‘ by the uterine bell.

Delayed tail loss during the invasion of human skin by schistosome cercariae.

Schistosomiasis is initiated when cercarial larvae invade human skin. Contrary to long-held assumptions, most cercariae of Schistosoma mansoni do not shed their propulsive tails as they penetrate. Scanning electron microscopy studies and infection experiments with entire human skin and differentiated, stratum corneum-like, human keratinocyte cultures, have shown that most cercarial tails enter the skin along with their bodies. We propose that this behaviour is an adaptive trait linked with concomitant immunity.

Dimethicone barrier cream prevents infection of human skin by schistosome cercariae: evidence from Franz cell studies.

One approach to the prevention of schistosomiasis is the use of topical formulations to inhibit cercarial penetration of skin. A number of formulations containing either cercaricidal ingredients or components designed to inhibit penetration have been studied, but with variable results. Such studies have rarely considered the persistence of inhibitory effects through time, and to date, there have been no systematic investigations of barrier formulations. The aim of this study was to use Franz cells to investigate the effect of such barrier creams on the penetration of S. mansoni cercariae into human skin. The results show that a single application of a barrier cream based on dimethicone offers a high level of protection against penetration that is sustained for at least 48 hr.

A comparison of topical formulations for the prevention of human schistosomiasis

Recently, a dimeticone formulation has been shown to be effective at preventing Schistosoma cercariae infecting skin, while DEET (N,N‐diethyl‐m‐toluamide), a highly effective insecticide, has been shown to have activity against cercariae. Seven formulations, 3 containing DEET, were prepared and applied to excised human skin in Franz cells for 1 h. Schistosoma cercariae were applied for 30 min at 1 and 24 h, and the number that penetrated the skin calculated (n = 9). DEET could not be incorporated into the dimeticone formulation, yet it remained the most effective at preventing cercarial penetration, both 1 and 24 h after application. The ointments that contained DEET did prevent penetration but their mode of action was due to the toxicity of DEET against the cercariae. The persistence of the protection afforded by the dimeticone formulation after washing suggests that the formulation may be interacting with the stratum corneum to prevent cercarial recognition of skin.

The use of video-imaging to assesss the sub-lethal impact of plant secondary compounds on Schistosoma mansoni miracidia

The study describes methods developed for using video-imaging technology to record and measure the velocity of Schistosoma mansoni miracidia. The efficacy of the classical bioassay procedure (a qualitative behavioural assay) was compared with that of the new quantitative protocol, for assessing the sub-lethal impact of a larvicidal dichloromethane extract of the seeds of Millettia thonningii on miracidia. The new technique confirmed the efficacy of the classical bioassay for rapid determination of the lethal and sub-lethal impact of larvicides but also provided quantitative information on sub-lethal impacts on miracidial velocity and shape.