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Partial characterisation of alkaline phosphatase in Fasciolopsis buski – an intestinal fluke treated with crude extract of Alpinia nigra (Zingiberaceae)

Background: Alkaline phosphatase is an important enzyme of helminth parasites that are found to be associated with absorption and/or digestion of food materials. Any interference to this enzyme could lead to paralysis and death of the parasites. In view of its functional significance, the present study was carried out to partially characterise the alkaline phosphatase of a fluke parasite, Fasciolopsis buski and to study the inhibitory effect of the extracts of Alpinia nigra, a medicinal plant of north-east India. Methods: Edible fresh shoots of Alpinia nigra were collected from Tripura, India and crude extract was extracted in ethanol. Live and mature F. buski was collected from the intestine of freshly slaughtered pig from the local abattoir and brought to the laboratory and processed for study. Various biochemical parameters like effect of pH, temperature, incubation, kinetic parameters and effects of A. nigra extract were studied on the alkaline phosphatase activity. A commercial anthelmintic drug praziquantel was taken as reference drug. Results: The optimal pH and temperature of the enzyme ranged between 9.0 to 10.0 and 35oC to 45oC, respectively. Alkaline phosphatase activity exhibited linear Arrhenius relationship with corresponding catalytic energy of activation (Ea) of 10.1 Kcal mole-1. The Km and Vmax values were 2.5 mM and 16.67 μM/min, respectively. The plant extract and the drug inhibited the enzyme activity in a dose-dependent manner with IC50 values of 1.10 mg/ml and 25 μM/ml for the plant extract and praziquantel, respectively. The kinetic parameters (Km and Vmax) were found to be altered both in the plant extract and drug treated parasite. Therefore, the alteration of the kinetic parameters by the crude extract of A. nigra and praziquantel treatment indicates a mixed type of alkaline phosphatase enzyme inhibition in F. buski. Conclusion: The altered enzyme activities, as observed under influence of the plant extract clearly indicate that the active principle of the plant acts as anthelminthic. However, to exploit the plant for commercial purpose, it is prerequisite to isolate and identify the active component responsible for anthelmintic activity.


Background
Alkaline phosphatase (AlkPase) is the enzyme that catalyses the hydrolysis of various phosphate esters and exhibit maximum activity in alkaline pH. The enzyme is found in the cells and extracellular fluids of a wide range of organisms including helminth parasites. Histochemical and biochemical studies have confirmed the presence of AlkPase in close associations with tegument, sub-tegument, somatic musculature, gut and cuticle of various helminth parasites, particularly in the adhesive organs like acetabulum and oral suckers [1][2][3][4][5]. Tegument is the outermost body covering of cestode and trematode parasites through which absorption and/or digestion of various food materials takes place [6][7]. Literatures have also shown that higher concentrations of AlkPase are found in the areas of intestine and sub-cuticular layers of the worm associated with proteins transport [8]. Thus, any interference or inhibition to this enzyme could lead to alteration of body physiology in helminth parasites.
Various commercial drugs (anthelmintic) and chemical compounds revealed to bring about changes in the activities of AlkPase in various soft-bodied helminth parasites like Echinococcus multilocularis, Raillietina echinobothrida, Hymenolepis diminuta, Schistosoma mansoni, Fasciolopsis buski and Fasciola hepatica [9][10][11][12]. In our earlier studies, in vitro exposure of F. buski to the alcoholic crude extract of A. nigra revealed alteration in the activities of different tegumental enzymes [13]. However, no literature is available on the altered kinetics of AlkPase in trematode parasites treated with anthelmintic phytochemicals. Therefore, in view of the functional significance, the present study was designed to characterise AlkPase in F. buski and analyse the in vitro effect of crude extract of Alpinia nigra on the activity of the enzyme.

Plant extract and test parasite
Collection of plant material and preparation of crude extract was carried out as described earlier [13]. Briefly, fresh edible shoots of Alpinia nigra were collected from

Quantification and characterisation of alkaline phosphatase
A 10% tissue homogenate was made in ice cold 100 mM sodium-glycine buffer (pH 9.5). The homogenate was centrifuged at 6,000 rpm for 30 min at 4ºC. Crude tissue supernatant obtained was used for enzyme studies. AlkPase activity was assayed following the method of Plummer [14]. The assay mixture consisted of 80 mM glycine-NaOH buffer (pH range 8-11), 1mM MgCl 2 , 2.5 mM p-nitrophenyl phosphate (pNPP), 0.05 ml of tissue supernatant (tissue protein = 0.10 mg/ml) and incubated for 30 min at 37±1ºC. The reaction was stopped by adding 2 ml of 0.2N NaOH at the end of incubation and absorbance measured at 410 nm in a double beam spectrophotometer (Carry 100, Varian). Influence of pH in the enzyme activities was studied by taking the pH range of 8.0 -11.0. The effect of incubation time and temperature was analysed within a range of 1 to 60 min and 5ºC to 70ºC, respectively. The activation energy of enzyme in relation to changes in temperature and rate of reaction has been calculated by Arrhenius equation following Njoku et al., [15] as follows: ln(k) = ln(A) -Ea/RT, Slope = -Ea/R and therefore, Ea = Slope x R; Where, k = Rate constant (enzyme activities), A = Pre-exponential factor, Ea = Activation energy, R = Gas constant (1.98 cal kelvin −1 mol −1 ) and T = Absolute temperature in kelvin.
The effect of crude extract of A. nigra and commercial drug praziquantel was determined by taking the concentration ranged from 0.1 to 5.0 mg/ml for the plant extract and 5 -200 µM/ml for the reference drug PZQ, respectively. To determine the kinetic parameters (K m and V max ), a nonlinear regression was employed at optimum pH 9.5 and temperature 37±1ºC, with substrate concentrations between 0.1 and 20 mM. The concentrations of the plant extract and the drug that inhibited 50% of enzyme activity (IC 50 ) was estimated following Kamal et al., [16] and Alhomida et al., [17]. The graphs were drawn by plotting % activity and % inhibition of alkaline phosphatase versus extracts of A. nigra (0.1-5.0 mg/ml) and PZQ (5-200 µM). The concentrations at the intersections of these two curves were taken as the IC 50 values. All the results are expressed as units/mg tissue protein [1 unit (U) = 1 µM p-nitrophenol min -1 mg protein -1 ].

Tissue protein and statistical calculation
The protein content of F. buski was measured following the standard method of Lowry et al., [18] using BSA (250 μg/ ml) as a standard protein. All experiments were carried out for four replicates (n = 4). All the calculations were carried out at Microsoft Excel and Origin software. Results were represented as the ±SEM (standard error of means). The probability analysis was done using Origin Pro8 software at 0.05% significance level.

Results and Discussion
The result indicates a good relationship between the AlkPase activity and the p-nitrophenyl phosphate (pNPP, the substrate) concentration. Increasing the substrate concentration from 0.5 to 10 mM, a linear increase in the enzyme activities (represented as units) in accordance with the normal pattern of enzymatic reactions has been observed (Figure 1a). Higher enzyme activity (velocity, V) has been observed in lower substrate concentrations ranging 0.5 to 4mM and thereafter, not much change has been observed, indicating the attainment of maximum velocity of the enzyme activity. When the data were plotted in a Lineweaver-Burk plot, the K m and V max were found to be 2.48 mM and 16.67U, respectively (Figure 1b, c). Lawton et al. , [19] studied the kinetic parameters of alkaline phosphatase in the membrane of hydatid cyst of Echinococcus granulosus and observed K m and V max values 0.24 ± 0.05 mM/L and 173 ± 21 nM/min/mg proteins for p-nitrophenylphosphate, respectively. Almost a similar kind of kinetic parameters has also been observed in other organism like Scrobicularia plana, where the enzyme attained half-maximum velocity at substrate concentration of 2.48 mM [20]. Whereas, in rabbit liver, a maximum activity of AlkPase (20 Umin -1 ) was observed at pNPP concentration of 0.5 mM [15]. The influences of pH, temperature and the incubation time on the AlkPase activity is depicted in figure 2. The affinity of an enzyme to bind to a specific substrate depends on the ionization states of the amino acid residues in the catalytic sites which are controlled by both the pH and the temperature. Variations of these factors modify the effective working efficiency of the enzymes by modifying the shape of the enzyme or denaturing its structure [21].
Increasing pH values has been found to have negative effect on the enzyme activities with lowest activity at pH 11.0. The enzyme activities have also been seen to be greatly controlled by temperature fluctuations. Highest activity of AlkPase at 9.41±0.13U was found to be at temperature range of 35ºC to 45ºC (Figure 2b) and decreased sharply with the increase of temperature above 40ºC. The Arrhenius plot (Figure 2d) of temperature-dependent enzyme activities revealed activation energy (Ea) of 10.1 Kcal mol -1 .
Activity of AlkPase increased with increasing incubation time up to 30 min and then decreased with the increase of incubation time (Figure 2). Probably, enzyme attains its maximum velocity within 30 min of incubation and then maintains the same speed and thereby less enzyme activity (units/mg protein) with the increase of incubation time. Effects of pH, various chemicals and anthelmintics on the AlkPase activities of Ascaridia galli, Centrorhynchus convi, Raillietina cesticillus and Cotylophoron cotylophorum has also been studied by Parshad and Guraya [22] and showed that the optimum pH for maximum activity of the enzyme are 9.1, 9.5, 8.7 and 8.4, respectively. In a similar kind of experiment Morales et al., [23] purified the AlkPase from common bean (Phaseolus vulgaris L.) roots and studied various kinetic parameters. In their experiment optimum pH and temperature for the enzyme assay was found to be within pH 8 and temperature of 37ºC, respectively. Similarly, the best pH range and incubation temperature was found to be 8.5 -10.0 and 30ºC -40ºC in crude tissue supernatant of Scrobicularia plana [20]. A significant (p < 0.05) difference in AlkPase activity has been observed between the control (11.64 ± 1.22U) and the extract of A. nigra treated tissue homogenate and also between control and drug (PZQ) treated homogenate (Figure 3). Lowest enzyme activities (4.51 ± 0.42U and 3.23 ± 0.26U) were observed when treated with highest doses of A. nigra (5 mg ml -1 ) and PZQ (200 µM ml -1 ), leading to almost 61% and 72% inhibition of enzyme activities, respectively (Figures 3a,3b). Two intersections between the % activity and % inhibition were taken as the A. nigra/drug concentrations where the enzyme activity showed 50% of reduction (IC 50 ). For both the A. nigra and PZQ, the IC 50 were found to be 1.10 mg and 25 µM respectively (Figures 3c,3d). Table 1 shows the comparative values of AlkPase activities of control and treated tissue. Significant difference (*labelled) of enzyme activities were observed in almost all the tissues treated with different concentrations of plant extract and commercial drug PZQ. At 0.5 mM pNPP concentration no such significant differences in the enzyme activities were noticed in both the doses of A. nigra treated supernatant, compared to control ones. Enzyme activities of both treated and control vs. pNPP concentrations (Figure 4a) showed hyperbolic curves, suggesting little interference with the substrate concentration. The enzyme activities when plotted in a Lineweaver-Burk plot, the kinetic parameters K m and V max showed little modifications (Figure 4b). Control tissue showed K m and V max values of 4.48 mM and 16.67 U; while the A. nigra treated tissue have 2.0 mM and 14.28 U for 0.5 mg ml -1 and  *Significance level at P < 0.05%. 2.0mM and 11.36 U for 1.0 mg ml -1 dose concentrations (Figure 1c). However, very little changes have been observed in y-intercept in PZQ treated tissue indicating competitive type of inhibition (Figure 4b).
A large number of drugs/chemicals have been seen to alter activities of AlkPase. According to Mahanty et al., [24], AlkPase secreation in the culture medium have been found to reduced when the Taenia solium Cysts was treated with PZQ and albendazole. Martins et al., [25] studied the effects of different drugs like levamisole, theophylline, quinidine, kaempferol, genistein, lidocaine and 3-isobutyl-1-methylxanthine on the activity of AlkPase and showed significant inhibitory effects by all the drugs on the enzyme activity. In a similar kind of experiment Parshad and Guraya [22] showed reduced activity of the enzyme in different helminth parasites treated with chemicals like MgSO 4 , CuSO 4 , FeCl 3 , KCN, NaF, sodium citrate, glycine and formaldehyde. Similarly, variable degrees of inhibition of the AlkPase activities were achieved following the addition of the anthelmintics like Bilevon, Mansonil, Vermex, Zanil, Distodin and Carbon tetrachloride. Like helminth parasites, AlkPase of rat heart has also been found to be strongly inhibited by levamisole, theophylline and aspirin [26].
A large number of plant-products/extracts have also been exploited in different parts of the world to see their anthelmintic properties. In vitro and in vivo studies of many medicinal plants like Flemingia vestita and its active component, genistein, Alpinia nigra, Potentilla fulgens, Acacia oxyphylla, Butea monosperma, Embelia ribes, Roltleria tinctoria etc. have been found to cause changes in both alkaline and acid phosphatase activities in helminths like R. echinobothrida and F. buski [12,13,[27][28][29][30].
Isatin, an indole derivative showed competitive type of AlkPase inhibition when the purified enzyme extract of Echinococcus multilocularis metacestodes was treated with it [31]. In a similar kind of experiment, Audin et al., [32] showed a promising inhibitory efficacy of acetylcarboxanilide and 2-methacrylanilides against E. multilocularis metacestodes in vitro. In vitro treatment of the cestode parasite Raillietina echinobothrida by Albendazole and crude extract of Acacia oxyphylla showed 40% -48% reduction in AlkPase activity [28]. Similarly, in our earlier studies, reduction in the alkaline phosphatase activity has been noticed when the fluke F. buski exposed to alcoholic extracts of different traditionally a b doi: 10.7243/2050-120X-2-5 used medicinal plants like Alpinia nigra, Potentilla fulgens and commercial drug praziquantel [12].

Conclusion
Plants parts or extract have been used since time immemorial to control helminth infections and are still in use as herbal remedies, particularly in developing countries. However, information on the mode of action of plantproducts is scant. In the present investigation we evaluated the activities of alkaline phosphatase, a vital tegumental enzyme of soft-bodied helminth parasite Fasciolopsis buski, under control and various stress conditions like, pH, temperature, plant extract (Alpinia nigra) and drug (praziquantel). The altered enzyme activities, as observed under influence of the plant extract clearly indicate that the active principle of the plant acts as anthelminthic. However, to exploit the plant for commercial purpose, it is prerequisite to isolate and identify the active component responsible for anthelmintic activity.