Effect of Active Molluscicidal Component ofSpices on Different Enzyme Activities andBiogenic Amine Levels in the Nervous Tissue ofLymnaea acuminata

V. K. Singh, Sanjay Singh, Sushma Singh and D. K. Singh*Department of Zoology, DDU Gorakhpur University, Gorakhpur PIN-273 009, U.P., India

In vivo exposure ofLymnaea acuminatato thymol and [6]-gingerol (active molluscicidal components ofTrachyspermum ammiand Zingiber officinale, respectively) indicates that they significantly alter acetyl-cholinesterase, lactic dehydrogenase, succinic dehydrogenase and cyto-oxidase activity in the nervoustissue of snails.In vitro exposure showed that, except for acetylcholinesterase and lactic dehydrogenase,no significant changes were observed in cyto-oxidase and succinic dehydrogenase activity in the nervoustissue of L. acuminata. Sublethal exposure to thymol and [6]-gingerol reduced the levels of 5-hydroxy-tryptamine (5-HT) and dopamine (DA) in the nervous tissue ofL. acuminata. There was, however, no sig-nificant change in the level of 5-hydroxy indol acetic acid (5-HIAA). Thymol and [6]-gingerol thus affectsall the known neurotransmission mechanisms in the snail either separately or through a complex interac-tion between the different neurotransmitters. This may account for their toxicity to snails. Copyright#1999 John Wiley & Sons, Ltd.

Keywords:AChE; 5-HT; DA; snail; spices.

INTRODUCTION

We have previously observed that common spice plantssuch as Zingiber officinale, Trachyspermum ammi,Trigonella foenum-graecumand Allium cepa havemolluscicidal activity. The active molluscicidal compo-nent of Trachyspermum ammifruit and Zingiberofficinale rhizome, i.e. thymol and [6]-gingerol, werehighly toxic againstLymnaea acuminataand Indoplan-orbis exustus(Singh et al., 1997). In the present studyattention has been focused on the effect of thymol and[6]-gingerol on different enzymes and biogenic aminelevels in the nervous tissue of the snailLymnaeaacuminata. This snail is the vector of the liver flukesFasciola hepaticaandF. gigantica, which cause endemicfascioliasis in cattle and livestock in the northern part ofIndia (Singh and Agarwal, 1981; Agarwal and Singh,1988).

MATERIALS AND METHODS

Adult L. acuminata(2.6� 0.30 cm long) were collectedlocally and used as test animals. Snails were acclimatizedto laboratory conditions for 96 h and then treated with40% and 80% of 24 h LC50 of thymol (Sigma and Co.,USA) and [6]-gingerol (Tsumura Co., Japan). After 24 h

and 96 h exposure different enzymes, namely acetylcho-linesterase, lactic dehydrogenase, cytochrome oxidaseand succinic dehydrogenase activity, and after 96 hexposure biogenic amine levels were determined in thenervous tissue ofL. acuminata. For in vitro treatmentthymol and [6]-gingerol were dissolved in ether. Anappropriate volume containing 7.0, 9.0, 12.0mg thymoland 3.0, 5.0, 7.0mg [6]-gingerol were added to thecuvette. Ether was then allowed to evaporate. Thymoland [6]-gingerol were pre-incubated for 15 min at 25°Cwith the enzyme source and enzyme activity wasdetermined. The control contained ether only.

In withdrawal experiments the snails were transferredfor 72 h from 96 h exposure of 40% and 80% of 24 hLC50 of thymol and [6]-gingerol. Thereafter, enzymeactivity in the nervous tissue ofL. acuminata wasestimated.

Acetylcholinesterase.Acetylcholinesterase activity wasmeasured according to the method of Ellmanet al.(1961)as modified by Singhet al. (1982). The nervous tissue50 mg/mL ofL. acuminatataken from around the buccalmass was homogenized in 0.1M phosphate buffer pH 8.0for 5 min in an ice bath and centrifuged at 1000� g for30 min atÿ4°C. The supernatant was used as an enzymesource. 0.05 mL of the supernatant was pipetted to a10 mm pathlength cuvette. To this was added 0.1 mL(5� 10ÿ4) of freshly prepared acetyl thiocholine iodidesolution in distilled water, 0.05 mL of the chromogenicagent DTNB reagent, 1.45 mL of 0.5M phosphate bufferpH 8.0 was used. The change in optical density at 412 nmwas monitored for 3 min at 25°C. Enzyme activity wasexpressed asmmol ‘SH’ hydrolysed /min/mg protein.

PHYTOTHERAPY RESEARCHPhytother. Res.13, 649–654 (1999)

CCC 0951–418X/99/080649–06 $17.50Copyright# 1999 John Wiley & Sons, Ltd.

* Correspondence to: D. K. Singh, Department of Zoology, DDU GorakhpurUniversity, Gorakhpur PIN-273 009, U.P., India.Contract/grant sponsor: Indian Council of Agricultural Research, Governmentof India; Contract/grant number: ICAR 1-20/94.

Received 28 October 1998Accepted 19 January 1999

Lactic dehydrogenase.Lactic dehydrogenaseactivitywas measuredby the standard,modified method ofSingh and Agarwal (1991). 50mg of nervous tissuearoundthe buccalmasswashomogenizedin 1.0mL of0.1 M phosphatebuffer pH 7.5 for 5 min in an icebath and centrifuged at 10000� g for 30min atÿ4°C. The supernatantwasusedasan enzymesource:0.01mL was added to 0.5mL of pyruvate substrate(10mL of pH 7.5 pyruvate buffer, 0.75 M sodiumpyruvate),mixed with 0.01g NADH2 and incubatedat37°C (45 min). 0.5mL of 2, 4-dinitrophenylhydrazinesolution (0.2g of 2, 4-dinitrophenylhydrazine,8.5mLof conc HCl, distilled water to 1 L) was addedand themixture kept at room temperaturefor 20min. 5.0mLof 0.4 M NaOH was mixed and left for 30min atroom temperature.The optical density was measuredat 540nm and convertedto LDH units using standardcurves. Enzyme activity was expressedasmmol ofpyruvatereduced/min/mgprotein.

Cytochrome oxidase. The activity of cytochromeoxidase was measuredaccording to CoopersteinandLazarow (1951) as modified by Singh and Agarwal(1991).100mg of nervoustissuearoundthebuccalmasswas homogenizedin 1.0mL of 1/30 mol/L phosphatebuffer,pH 7.4,for 5 min in anicebathandcentrifugedat10000� g for 30min atÿ4°C. Supernatantswereusedas an enzyme source.Enzyme activity at 25°C wasmeasuredin a 10mm pathlengthcuvette. 3.0mL ofreducedcytochromeC solution(1.7� 10ÿ5 mol/L) wastakeninto the cuvetteand0.2mL of tissuehomogenatewas added.The reactantswere mixed by inverting thecuvetteseveraltimes, absorptionat 550nm was mon-itored for 3 min. A few grainsof potassiumferricyanidewere added(to oxidize cytochromeC completely)andthe extinction was redetermined.Enzymeactivity wasexpressedas the averageenzymeper min and per mgprotein.

Succinicdehydrogenase.Succinicdehydrogenaseactiv-ity wasdeterminedspectrophotometricallyby themethodof Arrigoni andSinger(1962)asmodifiedby SinghandAgarwal (1987). 50mg nervous tissue as before washomogenizedin 1.0mL of 0.5 M potassiumphosphatebuffer (pH 7.6)for 5 min in anicebathandcentrifugedat10000� g for 30min at ÿ4°C. The supernatantwaspreincubatedwith 50mmol succinateat 37°C for 7 min.This preincubationsamplewaskept on ice and0.05mLaliquotfrom thiswasusedfor enzymeassay.To a2.9mL‘cocktail’ containing 100mmol of phosphatebuffer,300mmol of KCN, 0.8mmol of CaCl2 and0.04mmol ofDCIP, 50mmol of succinate,0.05mL of 2% PMS and0.05mL of preincubatedenzymesourcewereadded.Thedecreasein absorbanceat 600nm was monitored for3 min. Enzyme activity was expressedasmmol dyereduced/min/mgprotein.

Estimation of biogenicamines.Biogenicamines(5-HT,DA and 5-HIAA) in the nervoustissueof treatedandcontrol snailsLymnaeaacuminataweremeasuredon anAminco Bowman spectrophotofluorometer. DA wasmeasuredaccording to the method of Maickel et al.(1968)and5-HT and5-HIAA by the methodof Curzonand Green(1970) as modified by Singh and Agarwal(1984).

25mgof nervoustissuewashomogenizedin 3.0mL of

acidifiedbutanolandcentrifugedat 2000� g for 10min.Supernatantswere shakenin cappedtubes for 10minwith 5.0mL n-heptaneand 1.0mL of 0.1 N HCl. Themixture wascentrifugedfor another5 min at 2000� g.Theaqueouslayerwasusedfor measurementof 5HT andDA. 5.0mL of the remaining organic layer wastransferredto cappedtubescontaining0.6mL of 0.5 MTris-buffer pH 7.0, shaken for another 10min andcentrifugedat 2000� g for 10min. The aqueouslayerwasremovedandusedfor estimationof 5 HIAA.

Estimation of 5-HT. The 0.2mL of aqueouslayerpipetted out previously for estimating 5-HT wastransferredto a tube containing 1.2mL OPT (0.004gopthaldehyde/100mL 10 N HCl). To this 0.02mL of 1%cysteinewasaddedandthe mixture washeatedat 60°Cfor 15min. It was cooled to room temperatureandfluorescencewas measuredat 360/470mm againsttheblank.

Estimation of 5HIAA. The Tris extract (0.4mL) fromthe organic layer was pipetted in a tube to which thefollowing chemicalswere addedsequentially0.05mL1% cysteine,1.0mL concentratedHCl, 0.05mL OPTin methanoland 0.05mL of sodiumperiodatesolution.The mixture was heated at 60°C for 15min andfluorescencewas measuredat 360/470mm againsttheblank.

Estimation of DA. 2.0mL of extractwas taken into atube containing 1.0mL of 1 M pH 8.0 potassiumhydrogen phosphatebuffer. The following solutionswerethenaddedsequentially—iodine0.05mL, alkalinesulphite 0.25mL and glacial acetic acid 0.1mL. Thetubeswereheatedin thedarkat60°C for 40min andthencooledto roomtemperature.Fluorescencewasmeasuredat 330/390mm againsttheblank.

The results are expressedas mean �SE of sixreplicates.Student’st-test,andproductmomentcorrela-tion coefficientwereappliedto locatesignificantchanges(SokalandRohlf, 1973).

RESULTS

In vivo 24h and96h exposureto 40% and80% of 24hLC50 of thymol and [6]-gingerol causeda significantchangein theacetylcholinesterase(AChE), cytochrome-oxidase(cyto-oxidase),succinic dehydrogenase(SDH)andlactic dehydrogenase(LDH) activity in the nervoustissueof L. acuminata. Significantreductionsin AChE(93.67%of control),LDH (87.24%of control)andcyto-oxidase(87.2%of control)wereobservedin thenervoustissueof L. acuminataexposedto 40% of 24h LC50 ofthymol (Table 1). Treatmentwith 40% of 24h LC50 of[6]-gingerol for 24h reducedthe AChE, LDH andcyto-oxidase activity to 91.13%, 85.64% and 82.37% ofcontrol, respectively.Therewasno significantchangeinSDH activity in the nervous tissue of L. acuminataexposedto 40% and 80% of 24h LC50 of thymol and40%of 24h LC50 of [6]-gingerol.A significantincreasein SDH activity in nervoustissuewasobservedonly at80% of 24h LC50 exposureof thymol and [6]-gingerolfor 96h (Table1).

The maximum inhibition of AChE, LDH and cyto-

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Table 1. In vivoeffectof 24h and 96h exposureto 40% and 80% of 24h LC50 to thymol (5.96and 11.92mg/L) and [6]-gingerol (2.89and 5.78mg/L) on acetylcholinesterase(AChE),lactic dehydrogenase(LDH), succinicdehydrogenase(SDH) and cytochrome oxidase(cyto-oxidase)activity in the nervous tissuseof L. acuminata

Enzyme activity/min/mg protein

24 h 96 h

Thymol 6-Gingerol Thymol 6-Gingerol

Enzyme Control 40% 80% 40% 80% 40% 80% 40% 80%

AChE 0.079� 0.001 0.074� 0.001a 0.064� 0.002a 0.072� 0.001a 0.069� 0.001a 0.065� 0.001a 0.059� 0.002a 0.067� 0.002a 0.063� 0.003a

(100) (93.67) (81.01) (91.13) (87.34) (82.87) (74.68) (84.81) (79.74)LDH 323.43� 7.89 282.18� 2.65a 224.82� 3.47a 277.01� 2.38a 212.87� 3.29a 232.50� 3.36a 208.16� 3.11a 222.66� 3.15a 170.67� 3.11a

(100) (87.24) (69.51) (85.64) (65.81) (71.88) (64.36) (68.84) (52.56)SDH 48.93� 1.69 50.24� 0.86 51.30� 1.29 49.50� 0.67 52.71� 0.59 53.66� 1.29 59.6� 1.54a 52.4� 1.59 58.2� 1.54a

(100) (102.67) (104.84) (101.16) (107.72) (109.66) (121.80) (107.09) (118.94)Cyto-oxidase 16.34� 0.73 14.25� 0.29a 10.65� 0.35a 13.46� 0.46a 9.67� 0.55a 11.51� 0.34a 8.25� 0.38a 10.25� 0.45a 7.55� 0.44a

(100) (87.20) (65.17) (82.37) (59.17) (70.44) (50.48) (62.72) (46.20)

Values are mean� SE of six replicates. Concentration (w/v) expressed as ®nal concentration in aquarium water. Values in parentheses indicate percent enzyme activity with control takenas 100%.Enzyme activity: acetylcholinesterase, mm `SH' hydrolysed/ min/ mg protein ; succinic dehydrogenase, mmol pyruvate reduced/ min/ mg protein; lactic dehydrogenase, mmol dye reduced/min/ mg protein; cytochrome oxidase, enzyme activity/ min/ mg protein. Arbitrary unit corresponding to the quantity of enzyme which catalyses an O2 uptake by the oxidation of reducedcytochrome.a Signi®cant (p< 0.05) when t-test was used for locating differences between experimental and control groups of animals.

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Table 2. Effect of 72h withdrawal from 96h exposureto 40% and 80% of 24h LC50 of thymol (5.96and 11.92mg/L) and [6]-gingerol (2.89and 5.78mg/L) on AChE, LDH, SDH and cyto-oxidase

Enzyme activity/min/mg protein

Thymol [6]-gingerol

Enzyme Control 40% 80% 40% 80%

Acetylcholinesterase 0.081� 0.002 0.080� 0.001a 0.075� 0.002a 0.078� 0.001a 0.076� 0.001a

(100) (98.76) (92.59) (96.29) (93.82)Lactic dehydrogenase 327.41� 6.65 310.55� 7.78a 282.65� 6.55a 265.35� 3.45a 257.25� 4.55a

(100) (94.85) (86.32) (81.04) (78.57)Succinic dehydrogenase 50.25� 1.75 49.30� 1.29a 51.45� 1.59a 52.34� 1.75 52.45� 1.85

(100) (98.10) (102.38) (104.15) (104.37)Cytochrome oxidase 16.27� 0.99 15.45� 0.85a 14.95� 0.47a 14.25� 0.39a 13.27� 0.36a

(100) (94.96) (91.88) (87.69) (81.66)

a Signi®cant (p< 0.05) when student's t-test was used for locating differences between enzyme activity in nervous tissue ofsnails exposed to 40% and 80% of 24 h LC50 for 96 h (Table 1) and 72 h withdrawn to freshwater (withdrawn group in abovetable).Other details as given in Table 1.

Table 3. Effect of in vitro exposure(15 min) to thymol on acetylcholinesterase,lactic dehydrogenase,succinicdehydrogenaseand cytochromeoxidaseactivity in the nervous tissueof L. acuminata

Enzyme activity/min/ mg protein at different concentration of thymol

Enzyme Control 7.0 mg (w/v)a 9.0 mg (w/v) 12.0 mg (w/v)

Acetylcholinesterase 0.078� 0.0009 0.071� 0.001bc 0.067� 0.002bc 0.062� 0.001bc

(100) (91.02) (85.89) (79.48)Lactic dehydrogenase 330.63� 9.65 295.43� 8.25bc 287.15� 7.15bc 273.25� 3.15bc

(100) (89.35) (86.84) (82.64)Succinic dehydrogenase 52.32� 1.65 51.25� 1.35 50.85� 1.90 51.35� 0.90

(100) (97.95) (97.19) (98.14)Cytochrome oxidase 17.25� 0.98 16.86� 0.82 16.91� 0.75 15.95� 1.10

(100) (97.73) (98.02) (92.46)

a Concentration of (w/v) of thymol has been given as a ®nal concentration in the incubation mixture present in the cuvette.b Signi®cant (p< 0.05) when student's t-test was applied between treated and control groups.c Signi®cant (p< 0.05) when analysis of variance was applied between different concentrations.

Table 4. Effect of in vitro (15 min) exposure to [6]-gingerol on acetylcholinesterase,lactic dehydrogenase,succinicdehydrogenaseand cytochromeoxidaseactivity in the nervous tissueof L. acuminata

Enzyme activity/min/ mg protein at different concentration of [6]-gingerol

Enzyme Control 3.0 mg (w/v) 5.0 mg (w/v) 7.0 mg (w/v)

Acetylcholinesterase 0.077� 0.002 0.069� 0.001bc 0.061� 0.002bc 0.057� 0.001bc

(100) (89.61) (79.22) (74)Lactic dehydrogenase 328.55� 8.25 289.25� 5.65bc 276.35� 4.25bc 262.15� 3.15bc

(100) (88) (84.11) (79.78)Succinic dehydrogenase 49.45� 0.95 48.86� 1.25 49.27� 0.89 50.13� 0.92

(100) (98.80) (99.63) (101.4)Cytochrome oxidase 15.25� 0.85 14.96� 0.67 15.24� 0.77 14.85� 1.15

(100) (98.09) (99.9) (97.37)

Other details as given in Table 3.

Table 5. Changesin the biogenicamineslevel in the nervoustissueof L. acuminataby 40% and 80% of 24h LC50 for 96h ofthymol and [6]-gingerol

Biogenic amines mg/g tissue

Biogenic amines ControlThymol [6]-gingerol

40% 80% 40% 80%

5-HT 19.45� 0.22 15.20� 0.87a 9.60� 0.67a 13.25� 1.12a 7.32� 0.88a

(100) (78.14) (49.35) (68.12) (37.63)DA 15.20� 0.44 11.00� 1.12a 6.40� 0.97a 10.8� 0.87a 5.15� 0.66a

(100) (72.36) (42.10) (71.05) (33.88)5-HIAA 0.92� 0.01 0.81� 0.11 0.80� 0.09 0.82� 0.12 0.81� 0.10

(100) (88.04) (86.95) (89.13) (88.04)

Values are mean �SE of six replicates.Doses (w/v) expressed as ®nal concentration in aquarium water.Values in parentheses indicate percent change with control. Values taken as 100%.a p< 0.05, signi®cantly different from controls when t-test was applied.

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oxidasewere observedin the nervoustissue of snailexposedto 80%of 24h LC50 of thymol and[6]-gingerolfor 96h. AChE, LDH and cyto-oxidaseactivity in thenervous tissue of L. acuminata were decreasedto74.68%,64.36%and 50.48%of the control after 96hexposureto 80%of 24h LC50 of thymol. Whereas,96hexposureto 80%of 24h LC50 of [6]-gingerolreducedtheAChE,LDH andcyto-oxidaseupto 79.74%,52.56%and46.20% of the control in the nervous tissue of L.acuminata(Table1).

Snails withdrawn to freshwaterfor 72h from 96hexposureto 40% and 80% of 24h LC50 of thymol and[6]-gingerol, showeda significant recovery in AChE,LDH, cyto-oxidaseactivity (Table 2). In snails with-drawnfrom 96h exposureto 80%of 24h LC50 of thymolAChE, LDH, cyto-oxidaseactivities recoveredup to92.59%,86.32%and91.88%of the control. RecoveredAChE, LDH, cyto-oxidaseactivity in snailswithdrawnfrom 96h exposureto 80%of 24h LC50 of [6]-gingerolwere93.82%,78.57%and81.66%of the control. After72h, total recoverywasobservedin theSDH activity inthe nervoustissueof L. acuminatawithdrawn to freshwater(Table2).

In vitro

In vitro pre-incubationof thymol causeda significantdose dependentdecreasein AChE and LDH activity(Table 3) in the nervous tissue of L. acuminata.Treatmentof 7.0mg (w/v) of thymol and [6]-gingerolreducedthe AChE activity to 91.02%and74.0%of thecontrol, respectively (Tables 3,4). Treatment with12.0mg (w/v) thymol and 7.0mg (w/v) [6]-gingerolreducedthe LDH activity to 82.64%and79.78%of thecontrol.Therewasno significantchangein theSDH andcyto-oxidaseactivity in in vitro treatmentwith thymoland[6]-gingerol (Tables3,4).

Table5 showsthat the endogenouslevel of 5-HT (5-hydroxytryptamine)in the nervoustissueof snailswas19.45� 0.22mg/g, that of DA (dopamine) 15.20�0.44mg/g and of 5-HIAA 0.92� 0.01mg/g tissue.Treatmentof snails with 40% and 80% of 24h LC50of thymol and[6]-gingerol for 96h causeda significant(p< 0.05)dosedependentdecreasein thelevelsof 5-HTand DA. A maximumreductionin 5-HT (37.63%)andDA (33.88%) level was observedafter treatmentwith80%of 24h LC50 of [6]-gingerol.In contrastto 5-HT andDA, treatmentwith thymol and [6]-gingerol did notsignificantlyalter the5-HIAA level in thenervoustissueof L. acuminata.

DISCUSSION

The resultsclearly demonstratethat in vivo treatmentwith 40% and 80% of 96h LC50 of [6]-gingerol andthymol, an active molluscicidal component of Z.officinale and T. ammi, causeda significant inhibitionof acetylcholinesterase,lactic dehydrogenase, cyto-oxidaseand an increasein succinic dehydrogenaseinthenervoustissueof L. acuminata. Thein vitro treatmentof [6]-gingerolandthymolcausedasignificantinhibitionof acetylcholinesteraseandlactic dehydrogenase. Succi-nic dehydrogenaseandcyto-oxidasewasnotsignificantlyaltered. The increasein succinic dehydrogenaseanddecreasein cyto-oxidaseactivity in in vivo treatmentmaybe due to interaction with other reactions. In vitroinhibition of acetylcholinesteraseandlactic dehydrogen-aseby [6]-gingerol and thymol indicatesthat they actdirectly on theseenzymes.

The presentstudy also demonstratesthat treatmentwith thymol and [6]-gingerol significantly reducedtheendogenouslevelsof 5-HT andDA after96h exposure.Itindicatesthat thymol and [6]-gingerol adverselyeffectbothcholinergicandmonoaminergicneuronsin thesnail.In mammalsit hasbeenshownthat thereis interactionbetweencholinergic and monoaminergicneurons.But-cher(1977)demonstratedthat in ratsACh facilitatestherelease of dopamine which subsequentlyacts as aneurotransmitter.Thesefindingsraisethepossibility thatcontinuedcholinergic stimulus causedby reduction inAChE activity might causeincreasedreleaseof mono-aminesin theneurons.Alternatively, it is possiblethat itmay affect the cholinergic tryptaminergicand dopami-nergic neuronsindependently.However,dataregardingthe interactionbetweencholinergic and other kinds ofneuronsin snailsare not available.Our dataalso showthat insteadof a reductionin 5-HT levelstherewerenochangesin the levelsof 5-HIAA. This is possibleif theincreasedreleaseof 5-HT andits subsequentbreakdownto 5-HIAA are accompaniedby an enhancedrate ofremovalof the later.

Withdrawal from in vivo treatmentclearly demon-stratesthat thereis a significantrecoveryin the AChE,LDH, SDH and cyto-oxidaseactivity. Reversibility intheir action thus would cause less damage to theenvironment.

Acknowledgements

The authors are thankful to the Indian Council of AgriculturalResearch,Governmentof India, for financialassistance.ICAR projectNo. 1-20/94pp.

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