ASIAN J. EXP. BIOL. SCI. VOL 4 (1) 2013: 28-35

ASIAN J. EXP. BIOL. SCI. VOL 4 (1) 2013: 28-35 Sciety f Applied Sciences Effective Dse (ED) and Quality Cntrl Studies f the Crude Ethanlic Extract (CEE) Mixture f Makabuhay, Caimit and Makahiya (MCM) as Dewrmer fr Gats Against Haemnchus cntrtus Tmas J. Fernandez, Jr., Harvie P. Prtugaliza, Flres B. Braga, Erlinda A. Vasquez, Andresit 1 2 3 4 5 6 7 D. Acabal, Billy P. Divina & Winefred B. Pedere ORIGINAL ARTICLE 1,2,3,7 Cllege f Veterinary Medicine, Visayas State University, Visca, Baybay City, 6521-A Leyte, Philippines 4 Philippine Rt Crp Research and Training Center, Visayas State University 5 Department f Pure and Applied Chemistry, Visayas State University 6 Cllege f Veterinary Medicine, University f the Philippines, Ls Baňs, 4031 Laguna, Philippines Received 06-09-2012 Revised 02-01-2013 Accepted 08-01-2013 ABSTRACT The study was cnducted with the fllwing bjectives: 1) T determine the effective dse (ED) f CEE mixture f makabuhay (Tinspra rumphii), caimit (Chrysphyllum cainit) and makahiya (Mimsa pudica) in gats experimentally infected with Haemnchus cntrtus, and 2) T find ut the effect f temperatures and duratin f strage n the quality f CEE mixture. Results shwed that the ED f granulated CEE mixture with the green and white precipitates was 6 ml/kg BW. While that f CEE withut the green precipitate but with white precipitate was 3.5 ml/kg BW. Althugh the FECR f gats treated with 3.5 ml/kg BW f CEE mixture was lwer than thse grups treated with cmmercial dewrmers, this was nt statistically significant. The AM and GM %FECR f treated grups were lwer than grups treated with cmmercial dewrmers. Based n Kassai (1999) categry f percent efficacy f an anthelmintic, the CEE mixture was nly mderately effective at 3.5 ml/kg BW. Hwever, based n Riek and Keith (1957) percent efficacy, the dsage level f 3.5 ml/kg BW CEE mixture was highly effective. Keywrds Chrysphyllum cainit, FECR, Haemnchus cntrtus, Mimsa pudica, MCM, Tinspra rumphii INTRODUCTION In terms f cnstraints that hamper prductivity and full develpment f gat subsectr, high mrtality rate at weaning, develpment f anthelmintic resistance, and endparasitism were identified as the tp three cncerns that shuld be addressed [18]. One f these endparasites that is f cncern t gat raisers due t its pathlgic effects is Haemnchus cntrtus. Haemnchus spp. is an imprtant genus f Family Strngylidae that lcalizes in the abmasum f gats. It pssesses a buccal cavity with a slender tth r lancet. It is a bldsucker parasitic nematde [22]. There are tw species f Haemnchus that are f ecnmic imprtance amng gats and sheep in the lcality. These are Haemnchus cntrtus and Haemnchus placei [20]. Bth species are imprtant ecnmically in terms f stunted grwth, infertility, drp in milk prductin and/r death f gats [9]. The develpmental cycle f Haemnchus spp cnsists f eggs, larvae (L1, L2, L3 and L4), and adult [16]. These stages are srt f linked tgether that, if ne f the stages is severed, develpment f the next stage des nt prceed [9]. Thus, if gats, infected with H. cntrtus, are treated with apprpriate anthelmintics, ne f the links that is cutff is the adult wrm. In the absence f adult parasites, disseminatin f viable eggs int the envirnment is stpped; hence develpment f the parasite cannt perpetuate [5]. T address this parasitic infectin due t H. cntrtus is the use f anthelmintic. There are tw kinds f anthelmintic, the synthetic and herbal anthelmintics. These anthelmintics have their advantages and disadvantages. The main advantages f synthetic drug are their high efficacy and prven safety if used accrding t the manufacturer's recmmendatin. Hwever, lng term usage f synthetic anthelmintic wuld result t develpment f drug resistance by parasites thereby decreasing its efficacy [10, 12 & 24]. Als, synthetic dewrmers are als quite expensive and nt always readily available. Further, drug residue in meat and milk, 28 ASIAN J. EXP. BIOL. SCI. VOL 4 (1) 2013

Effective Dse f Makabuhay, Caimit and Makahiya As Dewrmer Fr Gats Against Haemnchus Cntrtus... T.J.Fernandez et al. and ptential harmful effects in the envirnment have been a cncern [21 & 25]. On the ther hand, herbal dewrmer, althugh less effective than synthetic drug, des nt have the disadvantages f cmmercial dewrmers. Hence, it is imperative t tap alternative anthelmintic, and ne f these alternatives is herbal dewrmer such as makabuhay ( Tinspra rumphii), caimit ( Chrysphyllum cainit) and makahiya ( Mimsa pudica). Previus study had shwn that the crude aqueus and crude ethanlic extract (CEE) f each plant part f makabuhay, caimit and makahiya (MCM) were effective against adult and infective larvae (L3s) f Haemnchus cntrtus [6], respectively. When the CEEs f MCM were cmbined, using varying rati cmbinatins, and assayed fr their larvicidal activity, it was fund ut that, regardless f the rati cmbinatins assayed, the CEE mixture f MCM killed a significant number f L3s. Results f this in vitr study were the basis fr the Mean Lethal Dse (LD 50), Effective Dse (ED), and quality cntrl studies f CEE mixture using a rati f 1:2:1 (1 part Makabuhay: 2 parts Caimit: 1 part Makahiya). An ideal anthelmintic shuld kill all stages f parasites by its cyttxic actin, eliminate dead parasites by its purgative actin, and heal injury in the mucsa brught abut by the sucking ability f strngyle wrm by its astringent effect. These cyttxic, purgative, and healing prperties are facilitated by flavnids and alkalids (cyttxic actin), anthraquinnes (purgative actin), and tannins (astringent actin) [11]. Phytchemical studies f makabuhay, caimit and makahiya shwed that each plant cntains biactive cmpunds such as tannins, alkalids, anthraquinnes, and flavnids [1]. Hwever, the cncentratins f these biactive cmpunds in each plant vary such that C. cainit has high cncentratin f flavnids and tannins [1] T. rumphii has high triterpenes [1], and M. pudica has high alkalids, sapnin, flavnids and triterpenes [7] (Table 1). By cmbining the CEEs f each plant part, the in viv synergistic actins f these cmpunds wuld be enhanced. Objectives 1. T determine the effective dse (ED) f CEE mixture in gats experimentally infected with Haemnchus cntrtus after treatment with CEE mixture. 2. T find ut the effect f temperatures and duratin f strage f CEE mixture n its larvicidal and adulticidal activities against L3s and adults f Haemnchus cntrtus, respectively. Table 1. Phytchemical prfile by varius tests f Crude Ethanlic Extracts (CEEs) f Makabuhay, Caimit and Makahiya Phytchemicals Detectin Test Reactin Detectin Makabuhay Alkalid Mayer s +/- Trace, slightly turbid Sapnins Frth - - Flavnid Bate-Smith & Metcalf +/- Trace, light red Tannins Ferric Chlride - - Triterpenes Lieberman & Buchard + Reddish Caimit Alkalid Mayer s - - Sapnins Frth +/- Trace, slightly frthy Flavnids Bate-Smith & Metcalf + Magenta red Tannins Ferric Chlride + Blue black Triterpenes Liebermann & Buchard - - Makahiya Alkalid Mayer s + Turbid Sapnins Frth + Frthy Flavnid Bate-Smith & Metcalf + Reddish Tannins Ferric Chlride - - Triterpenes Lieberman & Buchard + Reddish MATERIALSAND METHODS Effective Dse (ED) Study Native gats f varying sexes and weights were each experimentally infected rally with 500 L3s f Haemnchus cntrtus. The L3s were derived frm cultured eggs f macerated adult female Haemnchus. Experimentally infected gats were examined fecally, using the Whitlck methd, 28 days after infectin, t get the pre-treatment eggs per gram (EPG) feces; and thereafter 3 times a week, fr 3 cnsecutive weeks, t get the pst-treatment EPG. The indicatrs fr the efficacy f the CEE mixture f MCM were the fecal egg cunt reductin and arithmetic (AM) and gemetric means (GM) percent fecal egg cunt reductin (%FECR) [2 & 3]. ASIAN J. EXP. BIOL. SCI. VOL 4 (1) 2013 29

Effective Dse f Makabuhay, Caimit and Makahiya As Dewrmer Fr Gats Against Haemnchus Cntrtus... T.J.Fernandez et al. TheAM and GM % FECRs were btained: % FECR [2] = [(AM r GM EPG pre-treatment AM r GM EPG pst-treatment) (AM r GM EPG cunts pretreatment)] X 100 Where:AM= arithmetic mean GM= gemetric mean % FECR [3] = [(1-AM r GM f T2/AM r GM f T1) x (AM r GM f C1/AM r GM f C2)] X 100 Where: T = treated EPGs 1 = pre-treatment EPG C = cntrl EPG 2 = pst-treatment EPG Quality Cntrl Study The CEEs mixtures f MCM were stred in three strage temperatures (26 C-28 C, 4 C, and 37 C). Their physical characteristics (dr, clr and cnsistency), micrbial cunt, and larvicidal and adulticidal activities were mnitred mnthly. Experimental Design Effective Dse (ED) Study. The experiments were laid ut in a cmpletely randmized design (CRD). There were 6 treatment grups, with 6 gats in each treatment grup, and each gat served as a replicate. T0 served as the untreated cntrl, T1 was treated with albendazle, T2 was treated 1% ivermectin, and T3, T4 and T5 were treated with 6 ml/kg BW, 3.5 ml/kg BW r 1.5 ml/kg BW f CEE mixture. Quality Cntrl Study. Fr in vitr study, the experiment was laid ut in cmpletely randmized design (CRD). There were 6 treatment grups, replicated 3 times with at least 30 L3s but nt mre than 40 L3s. T0, which served as the cntrl grup, was treated with tap water; T1, which served as the reference cntrl grup, was treated with fresh CEE mixture; and T2, T3 and T4 were treated with 3.5 ml/kg BW CEE mixture that was kept in the fridge (4 C), ambient (26 C-28 C) and ven (37 C), respectively. Fr in viv study, there were 4 treatment grups, with 4 gats in each treatment grup, and each gat served as a replicate. T1 was treated with 3.5 ml/kg BW CEE mixture stred in the fridge (4 C), T2 was treated with 3.5 ml/kg BW CEE mixture stred in ambient (26 C-28 C), and T3 was treated with 3.5 ml/kg BW CEE mixture stred in the ven (37 C). StatisticalAnalysis The data n % mrtality f L3s and FECR were analyzed by Analysis f variance (ANOVA). Multiple pairwise cmparisn test was als dne. The significant differences amng the treatment grups were analyzed by Tukey's Hnestly Significant difference (HSD). The efficacy f CEE mixture based n its larvicidal activity and % FECRs was categrized accrding t either Kasai classificatin f anthelmintic efficacy: <80% efficacy, the drug is said t be ineffective; 80%-89% efficacy, the drug is said t be mderately effective (still acceptable); 90-98% efficacy, the drug is said t be effective; and >98% efficacy, the drug is said t be highly effective [15]; r Riek and Keith classificatin f anthelmintic efficacy: <70% efficacy, the drug is said t be ineffective; 70-80% efficacy, the drug is said t be effective; and >81% efficacy, the drug is said t be highly effective [19]. RESULTSAND DISCUSSION ED Study Frm a series f preliminary studies, the purpse f which was t establish the effective dse (ED) f CEE mixture, an actual study n the ED f granulated CEE mixture f MCM was cnducted using tw dsage levels lwer than the LD50 f 41.0 g/kg BW [8]. Results shwed that the eggs per gram (EPG) feces f all treatment grups befre dsing with CEE mixture, including thse in the cntrl grup (TO), were within the high categry (>500 EPG) (Fig. 1), and this was nt statistically significant. After treatment with CEE mixture, there was a reductin f the ttal EPG in all treatment grups at weeks 2, 3 and 4, except in grup treated with 1.5 g/kg BW (T3) f CEE mixture at weeks 2 and 3 pst-treatment in which the FECR was fluctuating (Fig. 1). 30 ASIAN J. EXP. BIOL. SCI. VOL 4 (1) 2013

Effective Dse f Makabuhay, Caimit and Makahiya As Dewrmer Fr Gats Against Haemnchus Cntrtus... T.J.Fernandez et al. 6500 t 6000 n u 5500 C 5000 G 4500 P 4000 E n 3500 a e 3000 M2500 2000 1500 1000 500 0 Pre-Tx n= 6 T0= Untreated Cntrl T1= 11.25%Albendazle T2= 1% Ivermectin T3= 1.5g/kg BWCEE MCM T4= 6g/kg BWCEE MCM T5= 41g/kg BWCEE MCM Week2 Pst-Tx Week3 Pst-Tx Week4 Pst-Tx Figure 1. Mean EPG cunt f gats treated with granulated CEE mixture f MCM with green and white precipitate Cmparisn f the mean fecal egg cunt reductin (FECR) between TO (Untreated Cntrl) and T3-T4 at weeks 2, 3 and 4 pst-treatment shwed a statistically significant difference (p<0.01). The fecal egg cunt reductin culd be attributed t the biactive cmpunds cntained in CEE mixture. On the ther hand, cmparisn f the mean FECR between grups treated with 11.25% Albendazle (T2) r 1% Ivermectin (T3) and grups treated with 1.5 g/kg BW (T3), 6 g/kg BW (T4) and 41 g/kg BW (T5) CEE mixture at weeks 2, 3 and 4 fecalysis was nt statistically significant. This bservatin shws that the anthelmintic activity f CEE mixture f MCM is as gd as the cmmercial dewrmers. Althugh the arithmetic (AM) and gemetric (GM) means %FECR f grups treated with varying dsage levels f CEE mixture was higher than the untreated cntrl (TO) at weeks 2, 3 and 4 fecalysis, these were lwer than grups treated with the cmmercial dewrmers. Based n Kassai categry f anthelmintic efficacy [15], with GM % FECR f Cles et al. [2], at 6 ml/kg BW, the CEE mixture was nly effective regardless f weeks pst-treatment fecalysis. On the ther hand, based n GM % FECR f Dash et al. [3], at 6 ml/kg BW, the CEE mixture was mderately effective at week 2 and effective at weeks 3 and 4 pst-treatment. Hwever, based n the percent efficacy f Riek and Keith categry f anthelmintic efficacy, at secnd week pst-treatment the efficacy f 6 mg/kg BW was nly effective, hwever at weeks 3 and 4 this was highly effective [19]. The AM % FECR f Cles et al. measures the anthelmintic activity f a particular treatment grup with reference t its pre-treatment EPG (self), while the GM %FECR measures the anthelmintic activity f treatment grups with reference t their pre-treatment EPG [2]. On the ther hand, the AM % FECR f Dash et al. measures the anthelmintic activity f a particular treatment grup in relatin t its untreated cntrl grup (T0), while the GM % FECR measures the anthelmintic activity f treatment grups in relatin t their untreated cntrl grup (TO) [3]. Frm the results f granulated frm f CEE mixture, the dsage level that reduced the FECR f 6 g/kg BW was quite high. Thus, it was necessary t reduce the dsage level f CEE mixture while increasing its %FECR t make the herbal dewrmer attractive t lcal gat racers. Hence, tw experiments were cnducted. One f these experiments was the remval f the green precipitate frm the CEE f each plant. When the CEEs f each plant part were kept vernight in the fridge (4 C), a green precipitate frmed at the bttm f the test tubes. Based n phytchemical prfiling by thin layer chrmatgraphy (TLC), this green precipitate is a chlrphyll [7]. A study in which the green precipitates frm each CEE f the three plants were remved was cnducted. Tw dsage levels (3.5 ml/kg BW and 6 ml/kg BW) f CEE mixture were assayed. Results shwed that, regardless f the week fecalysis after treatment, there was a reductin f fecal egg cunt in all treatment grups. The weekly FECR after treatment f grups treated with 3.5 ml/kg BW and 6 ml/kg BW was higher than the FECR in untreated grups (T0) (Fig. 2). ASIAN J. EXP. BIOL. SCI. VOL 4 (1) 2013 31

Effective Dse f Makabuhay, Caimit and Makahiya As Dewrmer Fr Gats Against Haemnchus Cntrtus... T.J.Fernandez et al. Again, this result culd be due the biactive cmpunds in the CEE mixture. Althugh the FECR f grups treated with CEE was lwer than grups treated with cmmercial dewrmers at weeks 1, 2 and 3, this was nt statistically significant (p<0.01). Based n Dash et al. AM and GM %FECR, grups treated with 3.5mL/kg BW was abve 80% and 90%, respectively. This % FECR efficacy was nly mderately effective and effective, respectively based n Kassai categry f anthelmintic efficacy [15], but highly effective based n Riek and Keith categry f anthelmintic efficacy [19]. Fllwing mixing the CEE f makabuhay and caimit, a white precipitate was prduced. It was thught that the precipitate cntained cmpunds that might suppress the actin f biactive cmpnents in CEE mixture having anthelmintic activity by cnjugatin r binding [13]. Thus, an experiment was cnducted in which the green and white precipitates were remved. Results shwed that the FECR f 1.5 ml/kg BW was nt as gd as the cmmercial dewrmers, but was better than the untreated cntrl, regardless f the number f weeks pst-treatment EPG (Fig. 3). Statistically, the numerical difference in FECR between TO untreated cntrl) and T3 (1.5 g/kg BW) was significant (p< 0.01) at weeks 2 and 3 pst-treatment and between T3 (1.5 g/kg BW) and grups treated with the cmmercial dewrmer. This shws that dsage level f CEE mixture f 1.5 ml/kg BW culd be ptentially used against Haemnchus infectin in gats. T0= Untreated Cntrl T1= 11.25% Albendazle T2= 1% Ivermectin T3= 1.5 ml/kg BW CEE MCM t 12000 n u 11000 C10000 G 9000 P E 8000 n 7000 a e 6000 M 5000 4000 3000 2000 1000 0 Pre-Tx n= 6 Week 1 Pst-Tx Week 2 Pst-Tx Week 3 Pst-Tx Figure 3. Mean EPG cunt f gats treated with CEE mixture f MCM withut green and white precipitate Fllwing per s administratin f a drug, the highest cncentratin (threshld) f a drug usually ccurs at tw weeks [4]. Thus the FECR tw weeks after treatment culd be attributed t residual effect f a dewrmer in tissues f animals and/r the threshld f a drug in the circulatin. On the ther hand, the FECR at three weeks after treatment culd be attributed t the drug depsited in tissues befre its threshld is reached in the circulatin [13]. Quality Cntrl Study Results n the effect f mnthly strage at varying strage temperatures f CEE mixture against L3s f Haemnchus cntrtus shwed that, after ne mnth strage, T3 (26 C-28 C) had the highest percent mrtality f 81.98%, fllwed 32 ASIAN J. EXP. BIOL. SCI. VOL 4 (1) 2013

Effective Dse f Makabuhay, Caimit and Makahiya As Dewrmer Fr Gats Against Haemnchus Cntrtus... T.J.Fernandez et al. by T2 (4 C) f 76.03%, and T4 (37 C) f 67.5%. Hwever, this percent mrtality was lwer than that f grup treated with fresh CEE mixture (T1) (Fig. 4). These numerical differences in the number f L3s that died fllwing expsure with CEE mixture withut the green and white precipitates were statistically different (p< 0.01) when cmpared with T0 (Tap Water). This shws that the CEE mixture cntains biactive cmpunds that are lethal t L3s. On the ther hand, there was a statistically significant difference (p< 0.01) n the mean mrtality f L3s between T1 (Fresh CEE) and T2-T4. This shws that the larvicidal activity f CEE mixture kept in varying strage temperatures fr ne mnth was nt as gd as the fresh CEE mixture Figure 4. %Mrtality f L3s Expsed with CEE Mixture f MCM stred fr ne mnth and Tw Mnths at varying strage temperature Results f the in viv study n CEE stred in different strage temperatures shwed that, after ne mnth strage, T1 (4 C) had the highest mean EPG reductin, fllwed by T3 (37 C), and T2 (26 C-28 C) at weeks 1, 2 and 3 fecalysis pst-treatment (Fig. 5). Cmparisn f the mean FECR between T0 (untreated Cntrl) and T1 (4 C), T2 (26 C-28 C) and T3 (37 C) shwed a statistically significant differences (p< 0.01). This bservatin shws that, regardless f strage temperatures, keeping the CCE mixture fr ne mnth wuld nt affect its adulticidal activity against Haemnchus cntrtus. After tw mnths strage f CEE mixture, the percent mrtality was drastically reduced t 21.9% (T2-4 C), 6.6% each fr T3 (26 C-28 C) and T4 (37 C) (Fig. 5). Cmparisn f the mean mrtality f L3s between grups treated with CEE mixture stred at varying strage temperatures and T0 (Tap Water) is nt statistically different, but statistically different (p< 0.01) when cmpared with T1 (Fresh CEE Mixture). This shws that, regardless f strage temperatures, keeping the CEE mixture fr tw mnths had lst its larvicidal activity. The same bservatin was nted in the previus study that CEE mixture kept in the fridge (4 C) had high larvicidal activity when this was used within ne week f strage [6]. The reductin in the larvicidal activity f CEE mixture after ne mnth strage culd prbably be attributed t either unstable state f biactive cmpunds in CEE mixture r bacterial cntaminants. After 2 mnths strage, the FECR f grups treated with CEE stred at varying strage temperatures was higher than T0 (Untreated Cntrl) (Fig. 5), and this was statistically significant. Hwever, based n the AM and GM %FECR f Cles et al. [2] and Dash et al. [3], the CEE mixture was ineffective. The micrbial cntaminatins, which were ccci and rds that have fermented the CEE mixture, as indicated by change in clr, dr and cnsistency might have affected the efficacy f CEE mixture after 1 mnth strage. Figure 5. Mean EPG cunt f gats treated with 3.5 ml/kg BW CEE mixture f MCM withut green and white precipitate fr 1 mnth and 2 mnths at varying strage temperature ASIAN J. EXP. BIOL. SCI. VOL 4 (1) 2013 33

Effective Dse f Makabuhay, Caimit and Makahiya As Dewrmer Fr Gats Against Haemnchus Cntrtus... T.J.Fernandez et al. There was n attempt t prfile the cmpunds f the white precipitate in the present study. Hwever, whether r nt the anthelmintic activities f biactive cmpunds cntain in CEE mixture might have been suppressed by cnjugatin f free radicals in the white precipitate f CEE mixture culd nt be explained in the present study. Suffice it t say that the biactive cmpunds present in CEE mixture are fat sluble that are extracted by ethanl. These cmpunds culd be alkalids, tannins, anthraquinnes, flavnids and triterpenes [1, & 7] (Table 1). Hwever, as t what specific cmpnent frm these cmpunds is having a lethal effect against adult and larvae f H. cntrtus is smething that shuld be lked int in the future study. Tannins have been shwn by varius authrs [14, 17 & 21] t have an anthelmintic activity. It has been demnstrated that tannins adversely affected the integrity f the cuticle f larvae and adult nematdes (14 & 21]. On the ther hand, in the study n the anthelmintic activity f tannins against Haemnchus cntrtus and Trichstrngylus clumbrifrmis, it was shwn that tannins have a direct effect n the fecundity f the nematdes [17]. Likewise, tannins stimulated the lcal immune respnse f treated cattle, thereby increasing the inflammatry cells f hst in the fundus f abmasum. These inflammatry cells are capable f neutralizing either stages f the nematde. Thus, tannins in apprpriate amunt culd be used as a dewrmer withut an adverse effect n the animals being treated. At dsage level f 3.5 ml/kg BW, withut the green and white precipitates, the CEE mixture culd be used as a dewrmer fr gats against H. cntrtus. In the absence f a field trial study, at this dsage level, the CEE mixture culd be used t cntrl the Haemnchus infectin in a farm prvided that this shuld be cmplimented with gd management practices. SUMMARYAND CONCLUSION The research study has tw cmpnents: These are 1) Establishment f Effective Dse (ED) f CEE Mixture in Gats Experimentally Infected with Haemnchus cntrtus, and 2) Effect f Strage Temperature and Duratin n the Quality f CEE Mixture. Each f these cmpnents had specific bjectives. The CEE mixture was prepared as befre [8]. The ED f CEE mixture was mnitred in gats experimentally infected with 500 L3s f H. cntrtus. Abut 28 days after infectin, the gats were examined fecally (Whitlck methd) t get the pre-treatment EPG, and after treatment with apprpriate dsage level f CEE mixture, t get the pst-treatment EPG. The ED f CEE mixture was indicated by a significant fecal egg cunt reductin. The AM and GM % FECR were cmputed based n the frmula f Cles et al. [2] and Dash et al. [3]. The CEE mixtures were kept in varying strage temperatures. Each mnth fr tw mnths the stred CEEs were mnitred fr their larvicidal and adulticidal activity against H. cntrtus, bacterial cntaminants, and physical characteristics such as clr, dr and cnsistency base n Likert scale frm 1-5, with 1 crrespnding t riginal clr, dr and cnsistency, and 5 the deterirated quality. Results f the ED study shwed that the dsage level f CEE mixture that gave a high %FECR culd be reduced frm 6 ml/kg BW t 3.5 ml/kg BW r ptentially t 1.5 ml/kg BW with the remval f the green precipitate frm each plant CEE and white precipitate frm CEE mixture. The anthelmintic activity f 3.5 g/kg BW f CEE mixture against H. cntrtus infectin in experimentally infected gats was better than the untreated cntrl, and is as gd as the cmmercial dewrmers. Strage f CEE mixture fr ne mnth in ambient temperature did nt adversely affect its physical characteristics, and larvicidal and anthelmintic activities as indicated by a significant number f L3s that died and the high FECR, respectively. Hwever, after tw mnths strage, the larvicidal and anthelmintic activities f CEE were drastically reduced brught abut prbably by deteriratin f CEE mixture due prbably t bacterial cntaminants Based n the results f the study, the fllwing cnclusins culd be derived: 1. The granulated CEE mixture was effective at dsage level f 6 g/kg BW. 2. With the remval f green precipitate, the effective dse f CEE was reduced t 3.5 ml/kg BW. 3. With the remval f green and white precipitates, the dsage level f CEE mixture culd be reduced t 1.5 ml/kg BW. 4. By TLC, the green precipitate cntains abundantly with chlrphyll. 5. Strage f CEE mixture in the fridge (4 C) fr 1 mnth did nt affect its physical characteristics, and the efficacy against L3 and adult f H. cntrtus. 6. Strage f CEE mixture beynd 1 mnth renders it less effective against H. cntrtus. 7. The larvicidal and adulticidal activities f CEE mixture decreased after 1 mnth strage. IMPLICATIONSAND RECOMMEDATIONS CEE mixture f MCM culd be used as a dewrmer fr gats against H. cntrtus at dsage level f 3.5 ml/kg BW, prvided this shuld be kept within 1 mnth at 26 C-28 C (ambient temperature) r 4 C (fridge). Hwever, the efficacy has t be evaluated by field trial study and establishment f dewrming regimen, with cnsideratin f hypbisis f L3. There is a need t reduce the dsage level f CEE mixture f MCM t make it attractive t gat raisers. Reducing the 34 ASIAN J. EXP. BIOL. SCI. VOL 4 (1) 2013

Effective Dse f Makabuhay, Caimit and Makahiya As Dewrmer Fr Gats Against Haemnchus Cntrtus... T.J.Fernandez et al. dsage level and increasing the efficacy culd be dne thrugh islatin f biactive cmpnents. If the biactive cmpnents are islated, these can be cmbined t cme up with apprpriate frm f the herbal dewrmer. ACKNOWLEDGMENT The research study was supprted by a grant frm the Philippine Cuncil fr Agriculture, Aquatic and Natural Resurces Research and Develpment (PCAARRD). Likewise, the Visayas State University had cntributed 20% f the ttal grant fr the research study. REFERENCES [1]. Bautista,R.M.C. (2009). Phytchemical Study f Larvicidal Extracts frm Mimsa pudica Linn. Chrysphyllum cainit Linn. and Tinspra rumphii Berl. Unpublished Undergraduate Thesis Wrk, Cllege f Arts and Science, Visayas State Cllege f Agriculture, Visca, Baybay, Leyte. [2]. 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Haemnchus cntrtus: parasite prblem n. 1 frm trpics-plar circle, prblems and prspects fr cntrl based n epidemilgy. Trpical Bimedicine, 22(2): 131-137. Crrespnding Authr : T.J. Fernandez, Jr., Cllege f Veterinary Medicine, Visayas State University, Visca, Baybay City, 6521-A Leyte, Philippines (phne: +63 053 563 7071; fax: +63 053 563 7148; e -mail: tmasfernandez18@yah.cm). ASIAN J. EXP. BIOL. SCI. VOL 4 (1) 2013 35