ASSESSMENT OF MOLLUSCICIDAL EFFECT OF SELECTED PLANT EXTRACTS ON PARTIALLY PURIFIED ACETYLCHOLINESTERASE FROM WHOLE BULINUS SNAILS (Bulinus africanus Krauss)

Abstract

Schistosomiasis is one of the most Neglected Tropical Diseases (NTDs) in terms of human suffering in the tropics and subtropics. It is caused by infectious trematode worms of the genus Schistosoma which penetrate a variety of small tropical freshwater snails of the Bulinus, Biomphalaria and Oncomelania species as intermediate hosts. This study was carried out to assess the molluscicidal effect of selected plant extracts on acetylcholinesterase purified from whole Bulinus africanus. The leaves of Bryophyllum pinnatum, Acalypha fimbriata and Mallotus oppositifollus were shredded by blender and then pulverized to powder, weighed and suspended separately in absolute ethanol for three (3) days. These mixtures were sieved to collect their respective ethanol extracts which were then subjected to vacuum evaporation in a rotary evaporator at 70o C. The Bulinus africanus snails were maintained in an aquarium containing 5 L de-chlorinated water and of standard aeration and temperature (20oC) for acclimatization to laboratory condition. They were fed fresh lettuce leaves prior to being used for experiments. Crude enzyme was prepared by homogenizing and centrifuging whole snails to obtain clarified supernatant, the supernatant was stored in the freezer and used as the crude enzyme solution. Crude enzyme solution was purified using DEAE-Sephacel and Sephadex G-100. The acetylcholinesterase (AChE) activity was measured and quantified by the Ellman method. Bryophyllum pinnatum showed an LC50 of 3.2% (32 mg/ml), Acalypha fimbriata showed an LC50 of 4.5% (45 mg/ml) while Mallotus oppositifolus showed an LC50 of 4.1% (41 mg/ml). Copper sulfate and bayluscide yielded LC50 at 1.9 μg/ml and 1.0 μg/ml concentrations respectively. The purified enzyme was highly active over a pH range of 7.0 to 9.0 with optimum activity observed at pH 7.0. It was found to be active at temperature range of 40 oC and 60 oC with optimum temperature at 50 oC. The purified AChE showed different IC50 values for the plant extracts and chemical inhibitors. Bryophyllum pinnatum required 84 μg/ml to inhibit 50% of AChE activity while Acalypha fimbriata and Mallotus oppositifollus required 91 μg/ml and >100 μg/ml respectively. The chemical inhibitors, copper sulfate and bayluscide showed IC50s of 0.8 μg/ml and 0.08 μg/ml respectively. The catalytic activity of AChE towards the plant extracts using acetylcholine iodide (AChI) as substrate showed that the kinetic parameters Ki, Vmax and Km were 13.50, 83.33 μmol/min/ml and 4.00 mg/ml respectively for Bryophyllum pinnatum, 27.50, 108.70 μmol/min/ml and 6.67 mg/ml respectively for Acalypha fimbriata and 20.50, 166.67 μmol/min/ml and 11.11 mg/ml respectively for Mallotus oppositifollus. Bayluscide showed Ki, Vmax and Km values of 0.02, 142.86 μmol/min/ml and 5.56 mg/ml respectively. Bryophyllum pinnatum, Mallotus oppositifollus and Bayluscide caused mixed inhibition while Acalypha fimbriata caused a non-competitive inhibition. These findings showed that AChE is present in Bulinus africanus and the catalytic activity of AChE towards the selected plants can be explored in novel molluscicidal formulations in a bid to eradicate schistosomiasis especially in the tropics and sub-tropics