Photoactivated Chlorophyllin and Acetylcholinesterase / Cytochrome Oxidase Activity in Fasciola gigantica cercaria Larvae

Objective: The effect of chlorophyllin (CHL), CHL + freeze dried cow urine (FCU) and FCU on acetylcholinesterase (AChE) and cytochrome oxidase activity in the nervous tissue of the host snail Lymnaea acuminata, and parasitic cercaria larvae of Fasciola gigantica were studied. Methods: Whole cercaria larvae and snail nervous tissue was homogenized and supernatant was used as an enzyme source. Enzyme activity was measured in 10-mm path length cuvette using incubation mixtures and change in optical density was recorded. Results: Sublethal 4 h LC50 40% and 80% of these formulations in sunlight and laboratory conditions caused no significant change in AChE activity in the nervous tissue of host snail. It was noted that sublethal in vivo 8 h exposure to 40% and 80% of 4 h LC50 of single and binary combination of CHL+FCU, CHL and FCU significantly altered the AChE and cytochrome oxidase activity in the cercaria larvae. Binary combinations of CHL+FCU in sunlight were more effective in inhibiting both the enzymes. Conclusion: The present study reported that enzymes AChE and cytochrome oxidase were inhibited by chlorophyllin formulations and thus causing the larval death inside the snail body without affecting the snail itself which is an important bioindicator of aquatic ecosytem.


INTRODUCTION
Fasciolosis is one of the most devastating food borne zoonosis caused by parasitic trematode Fasciola.2][3][4][5] The parasite is transmitted by ingestion of metacercaria of Fasciola species found on the leaves of aquatic plants in contaminated fresh water.Fasciola gigantica caused endemic fasciolosis in cattle population of northern part of India. 6,7and their intermediate hosts are Lymnaea acuminata and Indoplanorbis exustus.Scientists working on fasciolosis control programmed have advocated that control of the host snail population is one of the possible approaches to control fasciolosis. 8,9Environmental changes on the planet earth have compelled us to use sustainable and non-hazardous methods of parasite control.In aquatic ecosystem of ponds and lakes host snails and Fasciola larvae encounter with each other.Snails are the important component of fresh water ecosystem.1][12] If these larvae will be destroyed by plant larvicides without killing the infected snails, the rate of Fasciola infection can be reduced.In the last three decades, we are using plant derived larvicides against host snails for effective control of fasciolosis. 7,13,14In this connection, we have recently observed that chlorophyllin (CHL) a chlorophyll product possesses potential larvicidal activity against F. gigantica larvae in different wavelengths of visible light. 7,14The mechanism of action of chlorophyllin in cercarial death is still unknown.9][20][21] The aim of the present study is to evaluate the effects of sublethal exposure of CHL and FCU formulations on acetylcholinesterase (AChE) and cytochrome oxidase activity in host snails as well as in cercaria larvae of F. gigantica.

Test materials Preparation of chlorophyllin
Chlorophyllin was prepared by the method. 22hlorophyll was isolated from spinach using 100% ethanol (for about 2 h at 55°C).To avoid transformation of chlorophyll into pheophytin by the acidic content of the cell vacuoles, 1 mg CaCO 3 /g plant material was added as a buffer.The extract was subsequently filtered, and petroleum benzene was added.After shaking the mixture, chlorophyll moved into lipophilic benzene phase.The two phases were separated using a separatory funnel and about 1 mL methanolic KOH was added to 50 mL of benzene phase.On agitation, the chlorophyll met the methanolic KOH and transformed to water soluble chlorophyllin (cleavage of the ester bond between the chlorophyllin and the phytol tail by saponification).The phytol tail is responsible for the lipophilic property of chlorophyll.Chlorophyll is found as chlorophyllin in the KOH phase.

Collection of cow urine
Geer breed cow urine was collected in sterilized bottles, from green grass grazing 3-5 years old healthy cows.Different formulations of cow urine were tested against the larvae of F. gigantica.

Preparation of freeze dried cow urine (FCU)
Geer cow urine was kept in sun light (8h/day)/ laboratory conditions for 15 days.After 15 days cow urine was freeze dried in lyophilizer.Binary combinations (1:1) of chlorophyllin with FCU kept for 15 days in sunlight (8h/day) and laboratory conditions were prepared at the time of treatment.

Animals
Adult L. acuminata, each measuring 2.6 ± 0.4 cm in length were collected locally from Ramgarh Lake and low lying submerged areas of Gorakhpur district of Uttar Pradesh, India.Cercariae shedding infected snails were separated from uninfected snails by the method of Largue et al. 23 and Sunita et al. 24

In vivo treatment
The snails could acclimatize for 24 h in laboratory conditions and then treated with sublethal concentrations 40% and 80% 4 h LC 50 of binary combinations (1:1) of chlorophyllin formulations (CHL, CHL+FCU and FCU) (Table 1).Six batches were set up for each concentration.Control group (for each batch) contained only dechlorinated tap water without any treatment.All the treatments were made in the dechlorinated water in laboratory conditions.Thereafter, enzymes AChE and cytochrome oxidase activity were determined.Effect of toxicant on host (snails L. acuminata) and parasite (F.gigantica larvae) were observed simultaneously.The treated animals were removed from the aquaria after 4 h and rinsed with water.After 4 h of treatment snails were dissected for procurement of cercariae and nervous tissues of snails.About 15 to 20 snails were dissected, which yielded about 70-100 mg nervous tissues.In each concentration 100 to 150 infected snails were used.Nearly 4000 to 6000 cercaria larvae were procured in dissection of a single infected snail.In control group, infected snails were not treated with cercaricides.Both cercariae as well as nervous tissues of infected snails (positive control) were used in the same manner as treated group for AChE and cytochrome oxidase activity measurement.Protein levels were estimated according to the method of Lowry et al. 25

AChE activity
AChE activity was estimated in treated as well as control group of animals by the method of Ellman et al. 26 as modified by Singh et al. 27 Cercaria larvae as well as brains of the infected snails were taken for the measurement of acetylcholinesterase (AChE) and cytochrome oxidase activity.The brains of snails were removed separately and placed on ice cubes.Afterwards, they were placed on filter paper to remove the adherent water and not to be weighty.Snail nervous tissue (50 mg) was homogenized in 1.0 mL of 0.1M phosphate buffer, pH 8.0, for 5 min in ice bath, and then centrifuged at 1,000×g for 30 min at 4ºC.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 0.1M phosphate buffer (pH 8.0), 0.1 mL of chromogenic agent DTNB (5,5-dithio-bis-2-nitrobenzoic acid) and 0.02 mL of freshly prepared ATChI (acetylthiocholine iodide) solution in distilled water.The change in optical density at 412 nm was recorded for 3 min after every 3-min interval at 25ºC.Enzyme activity has been expressed μ mole 'SH' hydrolysed/ min/ mg protein.

Homogenization of F. gigantica cercaria larvae
Fresh Cercaria (1×10 6 ) larvae were centrifuged at 4 ºC for 2 min at 800×g.Then 1.0 mL 0.1M phosphate buffer pH 8.0 containing a cocktail of protease inhibitors (10 mM EGTA, 40 μg/mL leupeptin, 20 μg/mL pepstatin, 1.0 mg/mL bacitracin, 20 units/mL aprotinin and 1 mM benzamidine) was added (10 mL), followed by sonication at 50% duty cycle/output control 4 in a bath Ultrasonics sonicator, (Plainview, NY, U.S.A).Sonication was performed for 3×3 min at 4 ºC.Each homogenate was used as enzyme source for AChE activity measurement.Protein levels were estimated according to the method of Lowry et al. 25 Cytochrome oxidase Activity of cytochrome oxidase was measured according to Cooperstein and Lazarow. 28As modified by Singh and Agarwal. 29Homogenate obtained from fraction of cercaria larvae (1×10 6 ) of F. gigantica in 1.0 mL of 1/30 mol/L phosphate buffer, pH-7.4 centrifuged at 10,000×g for 30 min at 4 ºC.Supernatants were used an enzyme source.Enzyme activity at 25ºC was measured in a 10-mm path length cuvette.Reduced cytochrome solution (3-mL) (1.7×10 -5 mol/L) was taken into cuvette and 0.2 mL of cercaria larvae homogenate was added.The reactants were mixed by inverting the cuvette several times and absorption at 550 nm was monitored for 3 min.A few grains of potassium ferricyanide were added (to oxidize cytochrome c completely) and the extinction was redetermined.Enzyme activity has been expressed as the average enzyme activity/min/mg protein (arbitrary unit).

Acetylcholinesterase
Fractionated homogenate obtained from fractionation of cercaria larvae (1×10 6 ) of 0.1M phosphate buffer pH 8.0 centrifuged at 1000×g for 30 min at 4ºC.The supernatant was used as an enzyme source 0.1 mL (5×10 -4 M) of enzyme source, 2.9 mL of 0.1M phosphate buffer (pH 8.0 mL) 0.1 mL of chromogenic agent DTNB (5, 5-dithio-bis-2-nitrobenzoic acid) and 0.02 mL of freshly prepared ATChI (acetylthiocholine iodide) solution in distilled water was added in 10 mm path length cuvette.The change in optical density at 412 nm was monitored for 3 min interval at 25ºC.Enzyme activity has been expressed μ mole 'SH' hydrolysed/ min/ mg protein.

Statistical analysis
Concentration mortality data for each group of cercaria larvae of F. gigantica were analyzed using the probit analysis program, POLO-PC (LeOra Software) Robertson et al. 30 Rank correlation coefficient was applied to observe significant variation in between treated and control group AChE and cytochrome oxidase activity. 31

RESULTS
In control groups, AChE activity in the nervous tissues of L. acuminata in sunlight and in laboratory conditions was 0.91-0.92μ mole 'SH' hydrolyzed/min/mg protein (Table 2).No significant change in AChE activity in sunlight and laboratory conditions was noted in control as well as treated (40 % and 80 % of 4 h LC 50 of CHL, CHL+FCU and FCU) snails group (Table 2).In untreated cercaria larva AChE activity in sunlight and laboratory conditions was 1.01-1.3μmole `SH` hydrolyzed/min/mg protein (Table 3).
Pharmacognosy Journal, Vol 10, Issue 4, Jul-Aug, 2018  Treatment with 80 % and 40 % of 4 h, LC 50 of CHL+FCU, CHL and FCU for 4 h caused significant (P<0.05)inhibition in the AChE activity in the cercaria larvae (Table 3).Maximum inhibition upto 8.9 % and 3.9 % of control in AChE activity was observed in cercaria larvae found in the snails exposed to 40 % and 80 % of 4 h LC 50 of CHL+FCU in sunlight (Table 3).Significant (P < 0.05) variation in AChE activity in cercaria larvae was noted in sunlight and laboratory conditions exposure of chlorophyllin.There was a significant (P < 0.05) rank correlation in between the treated concentration of larvicides and corresponding changes in the AChE activity in cercaria larvae (Table 3).In untreated (control) cercariae, cytochrome oxidase activity was 13.12-12.41/ min / mg protein (Table 4).Significant (P < 0.05) decrease in cytochrome oxidase activity was noted in the whole cercaria larvae inside the host snails, exposed to 40 % and 80 % of 4 h LC 50 of CHL+FCU, CHL and FCU.Maximum inhibition upto 6.17 % and 5.25 % of control in cytochrome oxidase activity was noted in the cercariae inside snails exposed to 40 % and 80 % of 4 h LC 50 of CHL+FCU (Table 4).There was a significant rank correlation in between the cytochrome oxidase activity and corresponding concentrations of the CHL, CHL+FCU and FCU (Table -4).

DISCUSSION
It is evident from the results section that in in vivo sub-lethal treatment of binary combinations of CHL+FCU, CHL and FCU caused no significant change in AChE activity in the nervous tissue of snails L. acuminata.While significant inhibition of AChE activity was noticed in treated cercaria larvae in sunlight and laboratory conditions.It indicates that concentration toxic to cercaria larvae is safe against host snail L. acuminata.Presence of AChE is reported in several developmental stages of the parasite, i.e. in cercaria, schistosomula and adult worms. 32,33Fried et al. 34 reported that AChE is an active enzyme in cercaria larvae of Echinostoma revolutam (Trematoda).AChE is widely used as a biomarker in various toxicological studies. 35,36Seasonal variation in AChE activity in the nervous tissue of the clam Tapes philippinarum. 35and snail Cronia contracta were noted in different Goa cost of India. 36It is reported that acetylcholinesterase and cytochrome oxidases are very important enzymes in activation of cercaria. 37The enzyme AChE plays an important role in animals exposed to various natural/unnatural stimulants and in nerve impulse transmission in both vertebrate and invertebrates. 38Effect of sublethal concentrations of different larvicides formulations of CHL and FCU are potent inhibitors of acetylcholinesterase activity of cercaria larvae.Cytochrome oxidase is an important enzyme of electron transport system, responsible for active movement of cercaria larvae. 37,39A classical electron transport chain is reported in larval stages of trematode but is generally absent in adults. 39Cercarian tails have more cytochrome oxidase activity to meet the energy demands for movement. 40Earlier it has been reported that phyto cercaricides citral, ferulic acid, umbelliferone, azadirachtin and allicin caused significant inhibition of acetylcholinesterase (AChE) and cytochrome oxidase activity in the treated cercaria larvae of F. gigantica. 41umar et al. 42 reported that chlorophyllin is a potent inhibitor of cytochrome P450 enzymes involved in bioactivation of several environmental carcinogens because of radical scavenging mechanism and suppress metabolic activation.There is a significant (p<0.05)decrease in the cytochrome oxidase activity in the treated cercaria.It indicates that inhibition of AChE and cytochrome oxidase may be the cause of cercarial death.Present study, clearly indicates that we can reduce the incidence of fasciolosis without killing the snails with different formulations of chlorophyllin in sunlight.Among all the formulations CHL+FCU in sunlight are more effective cercaricides as it caused highest inhibition of AChE and cytochrome oxidase in cercaria larvae.

CONCLUSION
It seems that inhibition of AChE and cytochrome oxidase by chlorophyllin formulations in cercaria larvae of F. gigantica inside the snail body is a major cause of larval death.Thus, the larvicidal activity of these natural compounds may be helpful in solving the problem of fasciolosis by killing the larvae inside the host.So, the present study is one of the promising methods to control fasciolosis without killing the snails.

Table 3 : AChE activity (µ mole SH hydrolyzed/min/mg protein) in the cercaria larva of F. gigantica exposed to 40% and 80% of 4h LC 50 of active larvicides chlorophyllin formulations CHL, CHL+FCU and FCU in sunlight and laboratory condition. Larvicides 4 h LC 50 Sunlight Laboratory
Values are mean ± SE of six replicates.Values in paranthesis indicate % enzyme activity with untreated control taken as 100%.(*) significant (P<0.05),when rank correlation coefficient in between sublethal LC 50 and corresponding AChE activity in the whole cercaria larvae of F. gigantica in treated group.