Sub-Acute Toxicity and Biochemical Effects of Extracts of Anaphe Venata Larvae in Mice

African Journal of Biomedical Research, Vol. 8 (2005): 89 - 93 ISSN 1119 – 5096 © Ibadan Biomedical Communications Group Available online at http://www.bioline.org.br/md Full length Research Article Sub-Acute Toxicity and Biochemical Effects of extracts of Anaphe venata larvae in mice E.O.Iwalewa1i, O.A Onayade2, O.O Oyedapo3, O.M Daniyan1 Departments of Pharmacology1, Pharmacognosy2 and Biochemistry3, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria. Received: February, 2005 Accepted: May, 2005 ABSTRACT Ataxia syndrome which is characterized by sudden onset of severe muscular tremor and gait ataxia has been shown to be associated with the consumption of the larvae of Anaphe venata in South Western part of Nigeria. In this report, the sub -acute toxicity and biochemical effects of polar and non- polar extracts of Anaphe venata larvae were investigated in mice. The sub-acute toxicity study showed an increase in the behavioural components as tremor, jerking and stretching, after the administration of the extracts for 7 days, an indication of Ataxia syndrome. Also, no significant difference in these components occurred between polar and non-polar extracts, an indication of similarity in the chemical composition and level of toxicity. There was a corresponding increase in enzymatic activities coupled with increase in weight of essential organs investigated, but not significantly different from the controls. However, the involvement of receptors and neurotransmitters, in the action of the extracts to cause ataxia syndrome require further investigation. Keywords: Anaphe venata, larvae, toxicity, Free radicals, In vitro, Cissus quadrangularis , CCl4, Antioxidant activity INTRODUCTION therefore necessary to evaluate the sub-acute toxicity and biochemical effects of the polar and Studies have shown that in many developing non -polar extracts of the larvae of Anaphe countries of the world, insects are a good source venata on certain serum biochemistry of mice. of quality proteins, fats and minerals (Ene 1963 and Defoliart 1989, 1992) as such it mass MATERIALS AND METHODS rearing has even been advocated. Anaphe venata larvae (African Silkworm) Sample Collection: The larvae of Anaphe found commonly in Western part of Nigeria venata were obtained as commercial sample during the period of July-September each year, from a local market in Ile Ife, Nigeria and have been shown to be of high protein content authenticated by comparison with known sample (Umoh et al. 1980, Ashiru 1988) Other known with similar vernacular names at the National constituents of the larvae include amino acids, History Museum of the Obafemi Awolowo fats and sterols (Onayade and Adamolekun, University, Ile Ife. 1996). However, a seasonal ataxia syndrome Sample Preparation: Anaphe venata larvae associated with the consumption of these larvae (1kg) were parboiled in water to which ashes has been reported (Adamolekun 1993a). This have been added. They were dried and roasted syndrome is characterized by sudden onset of in hot white sand to remove their numerous severe muscular tremor and gait ataxia, and it tough and long tufts of hair. The larvae were occurrence coincides with the period of it wide then cooked in salt water followed by sun dried availability in the market. for preservation. Based on its wide acceptability and it high seasonal availability, coupled with high Extraction Procedure: The extraction consumption rate, in Western Nigeria, it is procedure followed the earlier described method 90 African Journal of Biomedical Research 2005 (Vol. 8) /Iwalewa, Onayade, Oyedapo & Daniyan of Onayade and Adamolekun (1996). It involved intraperitoneally at varying doses of 200, 800, the crushing of dried larvae to powder followed 3200 and 12,800mg/kg body weights for both by maceration in absolute ethanol for 48 hours in polar and non-polar extracts. All the groups were the dark. The resulting alcoholic extract was observed continuously for the first 24 hours and then partitioned in ethyl acetate to give the daily for the next 15 days. At the peak of aqueous (polar) and ethyl acetate (non-polar) convulsive episode which shortly precedes fractions. The fractions were concentrated in death in any of the groups, the set of mice in the vacuo at room temperature and freeze dried. group were quickly sacrificed blood samples collected into an anticoagulant tube containing Animals: Adult Swiss Albino mice weighing 3.8%w/v sodium citrate followed by between 18-24g were obtained from the animal centrifugation at 5000rpm for 10 minutes. The house of the Department of Pharmacology, plasma samples were stored at -20oC until Obafemi Awolowo University, Ile Ife, Nigeria. analysed. Also, essential organs- lung, spleen, They were fed with standard diets (Pfizer mouse brain, heart, kidney and liver- were aseptically cubes) with regular supply of water ad libitum. removed, weighed and kept in the deep freezer. They were kept within a lighted environment on Meanwhile, all the animals that survived were 12 hours light-dark cycle maintained at room sacrificed on the 15th day and blood as well as temperature. the essential organs were collected and treated as earlier described. Finally, the plasma samples Sub-Acute Toxicity Testing: Adult Swiss were analysis for Total protein (Gornorlet et al. Albino mice (10) were used in this study. The 1949), Aspertate / Alanine aminotranferase animals were randomly divided into two groups (Retman and Frontal 1957), and Alkaline of 5 mice each for polar and non-polar extracts Phosphatase (Saini and Von-Etten, 1979, administration. A sub-lethal dose of 500mg/kg Oyedapo, 1996). body weight for polar and 750mg/kg body weight for non-polar were used. Intraperitoneal Statistical Analysis: The variations in the administrations of both extracts were performed observed behavioural components among the once daily consistently for seven days at 24 mice in each group (polar and non-polar) were hours interval. The animals were observed for tested using Kruskal Wallis Analysis of Variance behavioural display, which include freezing, (ANOVA). The Wilcoxin sign ranked test for forward walking, rearing, grooming, cycling, paired observations was used to test the sniffing, hyperactivity, tremor, licking, respiratory hypothesis of no difference between the effects distress, stretching, jerking and raising of tail, for produced before and after extracts 15 minutes before the administration, 5 minutes administration. Mann-Witney U test was used to for drug stability and then for the next 40 test the difference in the effects produced by minutes. The animals were kept under close polar and non-polar extracts on mice. Analysis of observation and treatment for the 15 days. The multiple biochemical treatments effects and observed behavioural components were scored pathophysiological changes on essential organs on a rating scale of 0-4 depending on the were conducted using one way analysis of intensities or frequencies of the observe variance (ANOVA). All analysis was performed response as shown in Table 1: using PRIMER statistical software. Table 1: Ratings of observed behavioural components in mice RESULTS AND DISCUSSIONS Score Behavioural frequencies 0 Absent The behavioural effects elicited by the polar and non -polar extracts of Anaphe venata larvae in 1 Mild intensity, present 1-4 times. response to seven days sub-lethal dosage administration (Fig.1) showed no significant 2 Moderate intensity, present 5-8 times. difference among the treatment groups. This implied that the mice gave similar responses to 3 High intensity, present 9-12 times. the effects of extracts at the tested doses. However, when compared with the observed 4 Severe, present for a prolong period behavioural responses before the administration i.e. > 12 times of the extracts (Table 2) a significant difference were observed. Such behavioural activities like Biochemical study: Adult Swiss Albino mice tremor, jerking and stretching were (27) of both sexes were used. They were conspicuously present, which were consistent randomly divided into nine groups of 3 mice with our earlier preliminary findings (Onayade et. each. Group 1 serves as control while the other al. 2004). Maricker and Paltabiraman (1988) eight groups were divided into two of four groups have shown that heat treatment of foods each for polar and non-polar extract increases the nutritional values of protein and administration. The extracts were administered partial inactivation of protease inhibitors. 90 Toxicity of Anaphe venata larvae in mice Polar Extracts 4 Non Polar Extract 3.5 3 2.5 2 Score 1.5 1 0.5 0 Cycling Sniffing Tremor Licking Jerking Freezing Rearing Grooming Stretching Hyperactivity Tail Raising Forwardwalking Resp. Distress Fig. 1 Summary of effects of polar and non-polar extracts of Anaphe venata larvae on mice behaviour. Polar: H=2.047 with df =4 and p=0.727, Non -Polar: H=1.477 with df =4 and p=0.831; Observed p>a=0.05 in both extracts, therefore there is no significant difference in the observed activity among the mice used. Table 2: Behavioural components exhibited by mice after i.p administration of polar and non-polar Extracts of Anaphe venata larvae. Components Polar Average Ratings Non-Polar Average Polar Vs Non-Polar Ratings A1 B1 A2 B2 B1 B2 Freezing 0.2 3 0.2 3.2 3 3.2 Forwardwalking 3 1.8 3 2.2 1.8 2.2 Rearing 3.8 2 3.4 2.4 2 2.4 Grooming 1.8 3.2 2.4 3 3.2 3 Cycling 0.2 2 0.8 2.4 2 2.4 Sniffing 3.6 2.2 3 2.8 2.2 2.8 Hyperactivity 2.2 1 2.4 1.2 1 1.2 Tremor 0 0.4 0.2 1.8 0.4 1.8 Licking 0.6 3 1.4 3 3 3 Resp. Distress 0 1.2 0 0.6 1.2 0.6 Stretching 0 2.2 0 3 2.2 3 Jerking 0 1.2 0 2 1.2 2 Tail Raising 0 0 0 0.4 0 0.4 A1/A2 and B1/B2 signify rating scores before and after extract administration.

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