Enzyme Inhibition by Molluscicidal Components of Myristica fragrans Houtt. in the Nervous Tissue of Snail Lymnaea acuminata

This study was designed to investigate the effects of molluscicidal components of Myristica fragrans Houtt. (Myristicaceae) on certain enzymes in the nervous tissue of freshwater snail Lymnaea acuminata Lamarck (Lymnaeidae). In vivo and in vitro treatments of trimyristin and myristicin (active molluscicidal components of Myristica fragrans Houtt.) significantly inhibited the acetylcholinesterase (AChE), acid and alkaline phosphatase (ACP/ALP) activities in the nervous tissue of Lymnaea acuminata. The inhibition kinetics of these enzymes indicates that both the trimyristin and myristicin caused competitive noncompetitive inhibition of AChE. Trimyristin caused uncompetitive and competitive/noncompetitive inhibitions of ACP and ALP, respectively whereas the myristicin caused competitive and uncompetitive inhibition of ACP and ALP, respectively. Thus results from the present study suggest that inhibition of AChE, ACP, and ALP by trimyristin and myristicin in the snail Lymnaea acuminata may be the cause of the molluscicidal activity of Myristica fragrans.


Introduction
The snail Lymnaea acuminata Lamarck (Lymnaeidae) is the vector of liver flukes, Fasciola gigantica Cobbold (Fascioliodae) and Fasciola hepatica Linnaeus (Fascioliodae) which are responsible for endemic fascioliasis in cattle population of northern India [1,2]. One way to reduce the risk of fascioliasis is to delink the life cycle of the flukes by killing the vector snail [3,4]. Recently, it has been reported that Myristica fragrans (Myristicaceae) seed (nutmeg) and aril (mace) have potent molluscicidal activity against Lymnaea acuminata [5]. The active moieties responsible for the molluscicidal activity are trimyristin and myristicin [5]. The mechanism by which these active components cause snail death is not known. The present study is an extension of our previous study aimed at elucidating the effect of the active moieties on the different enzymes namely, acetylcholinesterase (AChE), acid phosphatase (ACP), and alkaline phosphatase (ALP) in the nervous tissue of snail Lymnaea acuminata.

Test Material.
Trimyristin (1,2,3-tritetradecanoylglycerol ) catalog no. T 5141, and myristicin (4-methoxy-6-(2-pro-penyl)-1,3-benzodioxole), catalog no. M 9411, were purchased from Sigma Chemical Co., USA.   [5]. Control animals were kept in an equal volume of dechlorinated water under similar conditions without any treatment. After 24 and 96 hours of treatment snails were removed from the aquaria and rinsed with water. Nervous tissue, present around the buccal mass of the snail, was taken out for the measurement of AChE, ACP, and ALP activities in the treated and control groups of snails.
In vitro experiments were performed by dissolving the molluscicides in ether and an appropriate volume containing 3, 5, 7, and 9 μg of trimyristin and 0.3, 0.5, 0.7, and 1.0 μg of myristicin (0.04, 0.07, 0.09, 0.1, and 0.15 mM of trimyristin and 0.01, 0.02, 0.03, 0.05, and 0.09 mM of myristicin) was added to 10 mm path length cuvette separately. Ether was then allowed to evaporate. Molluscicides were preincubated for 15 minutes at 25 • C with an enzyme source and then enzyme activity was determined. The control cuvette contained ether only. The Michaelis-Menten constant (K m ) and maximum velocity (V max ) of different enzyme inhibitions were calculated by nonlinear regression. Lineweaver-Burk plots for the hydrolysis of different concentrations of substrate by the treated (0.09 mM trimyristin and 0.03 mM myristicin) and untreated enzymes were plotted to observe mode of enzyme inhibition [6]. IC 50 values of myristicin and trimyristin were calculated in between the negative log concentration of inhibitor versus relative activity between inhibited and uninhibited enzymes.

Enzyme Assay
2.3.1. Acetylcholinesterase. Acetylcholinesterase (AChE) activity was measured by the method of Ellman et al. [7] as modified by Singh and Agarwal [8]. 50 mg of nervous tissue of Lymnaea acuminata taken around the buccal mass was homogenized in 1.0 mL of 0.1 M phosphate buffer pH 8.0 for 5 minutes in an ice bath and centrifuged at 1000 g for 30 minutes at 4 • C. The supernatant was used as an enzyme source. Enzyme activity was measured in a 10 mm path length cuvette using an incubation mixture consisting of 0.1 mL of enzyme source, 2.9 mL of 0.1 M buffer pH 8, 0.1 mL of chromogenic agent DTNB (5, 5 -dithio-bis-2nitrobenzoic acid), and 0.02 mL of freshly prepared ATChI (aetylthiocholine iodide) solution in distilled water. The change in optical density at 412 nm was recorded for 3 minutes after every 30 s interval at 25 • C. Enzyme activity has been expressed as μmole "SH" hydrolyzed min

Acid
Phosphatase. Acid phosphatase (ACP) activity in the nervous tissue of Lymnaea acuminata was measured by the method of Bergmeyer [9] as modified by Singh and Agarwal [10]. Tissue homogenate (2%, w/v) was prepared in ice cold 0.9% NaCl and centrifuged at 5000 g for 15 minutes at 4 • C. The supernatant was used as an enzyme source. 0.2 mL of enzyme source was added to 1.0 mL of acid buffer substrate (0.41 g citric acid, 1.125 g sodium citrate, and 165 mg 4-nitrophenyl phosphate sodium salt to 100 mL of double distilled water) preincubated at 37 • C for 10 minutes. The incubation mixture was mixed thoroughly and incubated for 30 minutes at 37 • C. 4 mL of 0.1 N NaOH was then added to the incubation mixture. The yellow colour, developed due to the formation of 4-nitrophenol, was determined colorimetrically at 420 nm. Standard curves were drawn with different concentrations of 4-nitrophenol. The ACP activity has been expressed as μmole substrate hydrolyzed 30 min −1 mg −1 protein. For the determination of kinetic constants of acid phosphatase, in vitro inhibition of the enzyme was carried out at different concentrations

Protein.
Protein estimation was carried out by the method of Lowry et al. [11] using bovine serum as a standard.

Statistical Analysis.
Each experiment was replicated at least six times and results were expressed as mean ±SE of six replicates. Student's t-test was applied between control and treated groups to locate significant (P < .05) variations [12].      (Table 1). In vivo treatment of 40 and 80% of 24 and 96 h LC 50 of trimyristin and myristicin caused significant (P < .05) inhibition in ALP activity in the nervous tissue of Lymnaea acuminata. Maximum inhibition of ALP activity (52% of control) was observed in snails exposed to 80% of 96 h LC 50 of myristicin (Table 1).  (Table 2). IC 50 values of trimyristin/myristicin against AChE, ACP, and ALP were 0.11, 0.16 and 0.18 / 0.03, 0.07 and 0.6 mM, respectively ( Table 2).

Discussion
In vivo and in vitro sublethal treatments of trimyristin and myristicin caused a significant inhibition of AChE, ACP and ALP activities in the nervous tissue of Lymnaea acuminata. In in vitro condition, the activity of the enzyme is in the presence of drug. Extent of enzyme inhibition in in vivo and in vitro conditions is the almost same. In in vivo condition titer of the drug at action site may be low, yet it inhibited the enzyme up to the same extent as in in vitro condition. IC 50 values of trimyricitin and myristicin clearly indicate that both are more potent inhibitors of AChE than ACP and ALP. AChE inhibition results in accumulation of acetylcholine at the nerve synapses, so that the postsynaptic membrane is in a state of permanent stimulation producing paralysis, ataxia, general lack of coordination in neuromuscular system, and eventual death [13]. It has been reported that n-hexane extract of M. fragrans seeds significantly inhibited AChE activity in brain of Swiss albino mice [14] and in in vitro, hydroalcoholic extracts of M. fragrans inhibited 50% of AChE activity at concentration of 100-150 μg/mL using AChE obtained from bovine erythrocytes [15].
The kinetic study clearly indicates that the inhibition of AChE by trimyristin and myristicin is competitive noncompetitive, as K m and V max values of uninhibited and inhibited enzymes were different and slopes of inhibited and uninhibited AChE were also changed; both were not parallel to each other. Inhibitions of ACP by trimyristin and ALP, by myristicin are uncompetitive. It is evident from the Lineweaver-Burk plots that the slopes of trimyristin inhibited ACP, myristicin inhibited ALP and uninhibited ACP/ALP were not changed; both were parallel to each other, whereas the intercepts of inhibited and uninhibited ACP/ALP were changed. The K m and V max , of uninhibited and inhibited enzymes were different. Inhibition of ACP by myristicin is competitive, as the K m values of the uninhibited and 6 Enzyme Research inhibited myristicin enzymes were different and V max of both were same, as evident from same intercept (1/V max ) on the Y axis of Lineweaver-Burk plots. Inhibition of ALP by trimyristin is also competitive noncompetitive.
Inhibition of AChE, ACP, and ALP by trimyristin and myristicin indicate, different types of inhibition kinetics. It seems that the molluscicidal components of Myristica fragrans kill the snails by inhibiting these enzymes in different ways.