Acute toxic effects of Medicinal Plant Jatropha gossypifolia on non ...

Wudpecker Journal <strong>of</strong> Agricultural Research Vol. 1(10), pp. 433 - 438, November 2012Available online at http://www.wudpeckerresearchjournals.org2012 Wudpecker Research JournalsISSN 2315-7259Full Length Research Paper<strong>Acute</strong> <strong>toxic</strong> <strong>effects</strong> <strong>of</strong> <strong>Medicinal</strong> <strong>Plant</strong> <strong>Jatropha</strong><strong>gossypifolia</strong> on non- target Fish and MicePratibha Singh * and Ajay SinghNatural Product Laboratory, Department <strong>of</strong> Zoology, D.D.U. Gorakhpur University, Gorakhpur- 273 001, (U.P.) IndiaAccepted 19 October 2012The medicinal properties attributed to <strong>Jatropha</strong> <strong>gossypifolia</strong> L. (Ratanjot) are extensive. The safety <strong>of</strong> J.<strong>gossypifolia</strong> is important in relation to its medicinal applications. This study was performed to elucidatethe possible <strong>toxic</strong> <strong>effects</strong> and mortality caused by crude latex and compound apigenin isolated fromleaves <strong>of</strong> J. <strong>gossypifolia</strong> on freshwater snakehead fish, Channa punctatus and Swiss albino mice Musmusculus. Toxicity <strong>of</strong> both the tested materials against Channa punctatus was time and dosedependent.But in case <strong>of</strong> Mus musculus, the <strong>toxic</strong>ity was only dose dependant and the LC doses wasmuch higher in comparison to fish Channa punctatus.Key words: <strong>Jatropha</strong> gosspifolia, Apigenin, Channa punctatus, Mus musculus, Piscicidal activity, Muricidalactivity.INTRODUCTIONThe use <strong>of</strong> plants and their products for curing andmanaging various ailments has been known to the worldsince time immemorial, and is gaining momentum thesedays due to <strong>toxic</strong> <strong>effects</strong> by synthetic drugs. Crudeextract <strong>of</strong> local plants are frequently being used bytraditional communities like tribal and rural population <strong>of</strong>India and elsewhere. This, and medicines manufacturedon principles <strong>of</strong> natural compounds even bypharmaceutical companies, may lead to a large-scaleexposure <strong>of</strong> humans to natural products (Awasthy et al.,2000). Natural products have always played a major rolein the development <strong>of</strong> organic chemistry. The variousstructural types <strong>of</strong> natural products contribute not only tonew finding or pose challenging synthetic problems butalso provide hope that they may become the basis fornew biologically active substances <strong>of</strong> commercialsignificance (Sukari et al., 1992). Though apparentlybelieved non-<strong>toxic</strong>, a clean chit <strong>of</strong>fered to these plantproducts requires strict scientific tests, besides clinicalones on different vital systems, moreso, because these* Corresponding Author E-mail: pratibha3gkp@rediffmail.comnatural products may contain a few harmful ingredients inthem as secondary metabolites (Nakamura andYamamoto, 1982) which may have perilous side <strong>effects</strong>including mutagenic potentials.It is, therefore, biological testing has played animportant role in <strong>toxic</strong>ity studies <strong>of</strong> crude latex and itscompound. There are numerous bioassay studies on plantextract. The first <strong>of</strong> such work involving some local plantswas reported as early as 1965 (Nakanishi et al., 1965).Thisstudy was conducted as part <strong>of</strong> the research to study thebiological activity <strong>of</strong> crude latex and compound apigeninisolated from leaves <strong>of</strong> local medicinal plant <strong>Jatropha</strong><strong>gossypifolia</strong> L. (Ratanjot). The report discusses thepiscicidal and muricidal activity <strong>of</strong> both the tested plantmaterial on fish and mice respectively.PLANTS<strong>Jatropha</strong> <strong>gossypifolia</strong> (Family Euphorbiaceae) wascollected locally from the Botanical garden <strong>of</strong> D.D.U.Gorakhpur University, Gorakhpur, (U.P.), India andidentified by Pr<strong>of</strong>. S.K. Singh, <strong>Plant</strong> Taxonomist,Department <strong>of</strong> Botany, D.D.U. Gorakhpur University,

Singh and Singh 434Gorakhpur, U.P. (India) where the boucher specimen wasdeposited.Uses in Traditional Medicine<strong>Jatropha</strong> <strong>gossypifolia</strong> is used in different countries inmany ways. The plant is antibiotic, insecticidal and usedfor toothache and as blood purifier (Balee, 1994). Itpossesses significant anticancer, hepatoprotective andpesticidal activity (Hartwell, 1969; Chatterjee et al., 1980;Panda et al., 2009; Panda et al., 2009). The roots, stems,leaves, seeds, and fruits <strong>of</strong> the plant have been widelyused in traditional folk medicine in many parts <strong>of</strong> differentcountries. The young stem <strong>of</strong> the plant is used astoothbrush as well as to clean the tongue in the treatmentthrush (Ogundare, 2007). The stem sap stops bleedingand itching <strong>of</strong> cuts and scratches (Labadie et al., 1989;Morton, 1968; Morton, 1980). The stem latex has beenshown to possess coagulant activity and its mechanism<strong>of</strong> action as haemostatic agent found to be byprecipitation <strong>of</strong> coagulant factors (Oduola et al., 2005).The leaves <strong>of</strong> J. <strong>gossypifolia</strong> are used for intermittentfevers, carbuncles, eczema, itches, and sores on thetongues <strong>of</strong> babies, swollen mammae, stomachache, andvenereal disease and as blood purifier (Balee, 1994). Theleaf decoction is used for bathing wounds (woundhealing) (Morton, 1968; Morton, 1981). In SouthernNigeria, the fresh leaf extract applied with crushed leaf byherbalists and local people used to stop bleeding fromskin and nose (Morton, 1981; Omoregbe et al., 1996).The leaf extract has been used as an anticoagulant forbiochemical and haematological analysis (Oduola et al.,2005).From leaves <strong>of</strong> <strong>Jatropha</strong> <strong>gossypifolia</strong>, threeflavonoids were isolated viz. apigenin, vitexin andisovitexin (Subramanian et al., 1971). The latex <strong>of</strong> J.<strong>gossypifolia</strong> yeilds the cyclic heptapeptide, cyclogossineA (Horsten et al., 1996) and cyclic octapeptidesCyclogossine B (Auvin-Guette et al., 1997).EXPERIMENTAL MATERIALSExperimental material 1The yellowish milky latex <strong>of</strong> <strong>Jatropha</strong> <strong>gossypifolia</strong> drained into glasstubes by cutting the stem apices. The latex was centrifuged at 1000rpm for 20 minutes to remove the resin. This resin free latexlyophilized at - 40°C, and the lyophilized powder stored for furtheruse. The wet weight <strong>of</strong> one ml latex <strong>of</strong> J. <strong>gossypifolia</strong> was 1.04 gmand dry weight (lyophilized at - 40°C) was 0.140 gm.Experimental material 2Extraction <strong>of</strong> Apigenin was done by washing the leaves <strong>of</strong> <strong>Jatropha</strong><strong>gossypifolia</strong> properly with water and dried in incubator at 37°C. Thedried leaves were then powdered. About 50 gm powdered leaf wassubjected to extraction through Soxhlet apparatus with about 250-300 ml ethyl alcohol for 72 hours at 20-40°C. After extraction, a littleamount <strong>of</strong> crude yellow powder was obtained. Dilute NaOHsolution was added to crude powder and then filtered. Again diluteHCl solution was added to filtrate and again filtered. Obtainedprecipitate was crystallized with Methanol, and it was Apigenin. Thepure compound isolated by this procedure was tested for their UVspectra, as well as their respective shifts (bathochromic andhypsochromic) that occur due to addition <strong>of</strong> specific agents:NaOMe, NaOAc, NaOAc + H 3BO 3, AlCl 3 + HCl. IR spectra werealso made. For the determination <strong>of</strong> Rf value in the TBA system(tertiary butanol acetic-water 6:2:2 v/v/v) Whatman 1chromatographic band was used.Apigenin is the main compound found in J. <strong>gossypifolia</strong> leaf andits molluscicidal (Singh and Agrawal, 1988) as well as piscicidalactivity (Singh and Singh, 2002) has already been known.Molecular weight: 270.24 g/molMolecular formulae: C 15H 10O 5Melting Point: 345-350 ° CApigeninThe spectral data <strong>of</strong> Apigenin were compared with the spectral data<strong>of</strong> (Dordevic et al., 2001).EXPERIMENTAL ANIMAL USEDExperimental animal 1The fish Channa punctatus (Bloch) (19.50 ± 2.50 cm in length,50.00 ± 4.00 gm in weight) were collected locally and acclimatizedin aquarium for 72 hours containing 100 L <strong>of</strong> de-chlorinated tapwater. The water in the aquarium was changed every 24 hour. Thebioassays were carried out at specific conditions, as recommendedby the American Public Health Association (APHA 1998). Themeasured values <strong>of</strong> pH, temperature, dissolved oxygen (DO) andhardness <strong>of</strong> water were 6.8- 7.0, 26°C, 7.2mg/l and 41mg/CaCO 3,respectively.Experimental animal 2: Four to six weeks old laboratory Swissalbino mice Mus musculus <strong>of</strong> body weight 37 ± 3 gm, male wereused for the study. The total five groups <strong>of</strong> experimental animals,having three replicates, each comprising five mice. All theexperimental animals were housed in stainless steel cages in aroom maintained at 25 ± 2°C with 12 hr day/night cycle. “Gulmuhar”(Hindustan Lever Limited, Mumbai, India) diet was the basal foodfor all the experimental animals. Drinking water was made availablead libitum. The animals used in the present study were maintainedin accordance with the guidelines <strong>of</strong> the Committee for the Purpose<strong>of</strong> Control and Supervision <strong>of</strong> Experiments on Animals (CPCSEA),

435 Wudpecker J. Agric. Res.India and approved by the Institute’s ethical committee.TOXICITY EXPERIMENTSToxicity Tests on Experimental animal 1Toxicity experiment was performed according to the method <strong>of</strong>(Singh and Agrawal, 1988). The fishes were exposed for 24h, 48h,72h and 96h at four different concentrations <strong>of</strong> both tested material<strong>of</strong> J. <strong>gossypifolia</strong>. Concentrations (w/v) <strong>of</strong> J. <strong>gossypifolia</strong> crude latexand apigenin used for <strong>toxic</strong>ity experiments were 10, 13, 16 and 19mgL -1 and 50, 55, 65, and 80 mgL -1 respectively. Three aquariawere set up for each dose and each aquarium contains ten fishes insix litre <strong>of</strong> de-chlorinated tap water. Control animals were kept insimilar condition without any treatment.Mortality was recorded every 24h throughout the 96h exposureperiod. Fishes were considered dead if they failed to respond tovigorous poking with glass rod. Dead fishes were removed fromaquaria as soon as possible in order to prevent their bodydecomposition.Toxicity Tests on Experimental animal 2: An initial test was doneto determine the approximate lethal and non-lethal doses <strong>of</strong> theextract according to method <strong>of</strong> (Turner, 1965). Five groups <strong>of</strong> fivemice, having three replicates <strong>of</strong> each, were used in theexperiments and treated with the aqueous latex extract andcompound apigenin <strong>of</strong> the plant at doses <strong>of</strong> 120, 150, 170, 200 mgextract /kg body weight and 1100, 1150, 1300, 1450 mg/kg bodyweight respectively. Single dose was administered to animalintraperitoneally (i.p.), by tuberculin syringe with needle no. 26. Thecontrol group was given an equal volume <strong>of</strong> water. The animalswere observed for 24 hr for signs <strong>of</strong> <strong>toxic</strong>ity including death and thenumber <strong>of</strong> dead mice was recorded and used in the calculation <strong>of</strong>the acute <strong>toxic</strong>ity value (LD 50).RESULTSThe results are reported in Table 1 and 2. Toxicityexperiments were performed by the method <strong>of</strong> (Singhand Agrawal, 1988) in case <strong>of</strong> fish and by the method <strong>of</strong>(Turner, 1965) in case <strong>of</strong> mice. Toxicity data obtainedfrom this study was computed through POLO PLUScomputer program version 2.0 <strong>of</strong> (Robertson et al.(2007). By using this program lethal concentration (LCvalues); upper and lower confidence limits and slopevalues were calculated through probit log analysismethod. Table 1 and Table 2 shows the effective doses(LC 10, 50, 90 values) <strong>of</strong> dried latex powder and compoundapigenin respectively, isolated from J. <strong>gossypifolia</strong> forperiod ranging from 24h to 96h against Channapunctatus and Mus musculus.From present study, it was clear that <strong>toxic</strong>ity <strong>of</strong> crudelatex and apigenin from J. <strong>gossypifolia</strong> was time anddose dependent in case <strong>of</strong> fish. There was a significantnegative correlation between LC 50 values and exposureperiods. Thus LC 50 <strong>of</strong> crude latex and apigenin decreasedfrom 22.334 mg/l (24h), 16.637 mg/l (48h), 12.614 mg/l (72h)and 10.490 mg/l (96h) and 76.485 mg/l(24h), 68.454mg/l (48h), 58.515 mg/l (72h) and 52.900 mg/l (96h)respectively. On administration <strong>of</strong> both tested materials,behavioral and physicals changes were noted i.e. theskin color <strong>of</strong> the fishes became light grey. Black spots onfins were found to lose their intensity and fishes startedscratching their nostril at the bottom <strong>of</strong> aquarium andfrequently came at the water surface for gasping air.Within 15-30 minutes, fishes try to escape from testaquaria. After 30 minutes, their movement was sloweddown, but they continue to swim near the water surface.Their after, fish shows irregular, erratic and sometimesjerky movement that was increase as exposure periodincreases.At higher doses after 10-12 hours, loss <strong>of</strong> bodyequilibrium and hemorrhage occurred, which manifesteditself as reddish color in head region and finally fisheswere died. In control group there were no such changesobserved, that means another factors other than the plantmoieties were responsible for alterations in behavior andmortality <strong>of</strong> fish.In case <strong>of</strong> mice, the median acute <strong>toxic</strong>ity value (LC 50 )<strong>of</strong> crude latex and apigenin determined to be 148.34mg/kg body weight within 95% confidence limits (127.2-165.2 mg/kg body weight) and 1218.16 mg/kg bodyweight within 95% confidence limits (1140.8-1294.4mg/kg body weight) respectively after 24h (Table 1 and2). No further mortality was observed after 24h exposureperiods (Table 1 and 2).On administration <strong>of</strong> the extract, no immediatebehavioral changes were noted. The mice moved and fednormally in both case <strong>of</strong> tested materials. After twentyminutes, piloerection was noticed and the animalsbecome restless, some trying to escape through theholes in the cages. The animals did not vomit, neitherwas there ptosis. The animals that received higher doseswent into convulsions and died in hyperextention.DISCUSSIONThe mortality <strong>of</strong> the fish and mice was chosen as themeasurable effect to determine the <strong>toxic</strong>ity <strong>of</strong> the crudelatex and apigenin. It is clear from the results that J.<strong>gossypifolia</strong> crude latex and compound apigenin is <strong>toxic</strong>against both freshwater fish Channa punctatus and Swissalbino mice Mus musculus, thus, the plant is piscicidal aswell as muricidal respectively.The plant J. <strong>gossypifolia</strong> may be used as a potentialsource <strong>of</strong> piscicides as a crude preparation <strong>of</strong> the latex aswell as compound apigenin has sufficient time dependentpiscicidal activity. As mentioned in result portion, the type<strong>of</strong> behavior responses indicate that both tested materialsare neuro<strong>toxic</strong>, which might be active at theneuromuscular system <strong>of</strong> fish C. punctatus. Animal

Singh and Singh 436Table 1. Toxicity (LC 10, 50, 90 values) <strong>of</strong> the crude latex <strong>of</strong> J. <strong>gossypifolia</strong> at different time intervals to the fishChanna punctatus and Swiss albino mice Mus musculus.Channa punctatusMus musculusExposure period Effective dose (mg/L) Slope valueEffective dose(mg/kg body weight)LC 10 = 11.28LC 10 = 101.272(6.46-13.26)(55.6-120.9)LC 50 = 22.33 4.322±1.404 LC 50 = 148.34024h(18.46-46.14)(127.2-165.2)LC 90 = 44.20LC 90 = 217.283(28.68-95.55)(187.4-351.2)LC 10 = 8.278(4.029-10.364)-LC 50 = 16.6348h(14.63-21.43) 4.228±1.196-LC 90 = 33.435(24.36-96.68)-LC 10 = 6.87(3.52-8.79)-LC 50 = 12.6172h(10.64-14.11) 4.861±1.192-LC 90 = 23.14(19.12-38.13)-LC 10 = 5.66(2.25-7.70)-LC 50 = 10.49096h(7.70-11.98) 4.785±1.249-LC 90 = 19.435(16.55-29.65)-Slope value7.731±2.269‣ -, values could not be calculated because no further mortality after 24 h.‣ There was no mortality in control groups‣ Mortality was determined at every 24h.‣ Regress coefficient showed that there was significant (P

437 Wudpecker J. Agric. Res.Table 2. Toxicity (LC 10, 50, 90 values) <strong>of</strong> the Apigenin extracted from leaves <strong>of</strong> J. <strong>gossypifolia</strong> at different time intervals to the fishChanna punctatus and Swiss albino mice Mus musculus.Channa punctatusMus musculusExposure period Effective dose (mg/L) Slope valueEffective dose(mg/kg body weight)LC 10 = 49.805LC 10 = 999.959(28.147-57.342)(782.179-1086.863)LC 50 = 76.485 6.879±2.38424h(67.505-117.594)LC 50 = 1218.165 (1140.895-1294.475)LC 90 = 117.455(90.318-432.273)LC 90 = 1483.986 (1370.040-1872.683)LC 10 = 45.554(26.272-52.956)-LC 50 = 68.45448h(61.362-85.383) 7.246±2.296-LC 90 = 102.865(83.475-236.361)-LC 10 = 38.344(16.148-46.847)-LC 50 = 58.51572h(49.074-66.295) 6.981±2.310-LC 90 = 89.298(74.882-186.850)-LC 10 = 36.677(15.732-44.790)-LC 50 = 52.90096h(41.456-58.492) 8.057±2.623-LC 90 = 76.300(66.946-124.651)-Slope value14.950±4.074‣ -, values could not be calculated because no further mortality after 24 h.‣ There was no mortality in control groups‣ Mortality was determined at every 24h.‣ Regress coefficient showed that there was significant (P

Singh and Singh 438water bodies or for medicinal purpose, without knowingtheir structural activity relationship with non-targetorganisms.Apigenin is less <strong>toxic</strong>ity than the crude latex. Higher<strong>toxic</strong>ity <strong>of</strong> crude latex may be due to the synergisticaction <strong>of</strong> several compounds present in latex powder(Horsten et al., 1996; Auvin-Guette et al., 1997). Dataemerged from this study will be helpful in thedevelopment <strong>of</strong> eco-friendly drugs and pharmaceuticalcompounds for other purposes.ACKNOWLEDGMENTSOne <strong>of</strong> the authors (Pratibha Singh) is thankful toUniversity Grants Commission, New Delhi, for the award<strong>of</strong> the Dr. D.S. Kothari Post Doctoral Fellowship Scheme(No. F.4-2/2006 (BSR)/ 13-214/2008 (BSR), dated 1 stOctober 2009) for financial assistance.REFERENCESAPHA/AWWA/WEF (1998). 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