ORIGINAL PAPER
Bioassay-guided isolation and identification of active compounds from Fructus Arctii against Dactylogyrus intermedius (Monogenea)in goldfish (Carassius auratus )
Gao-xue Wang &Jing Han &Ting-ting Feng &Fu-yuan Li &Bin Zhu
Received:2October 2009/Accepted:8October 2009/Published online:27October 2009#Springer-Verlag 2009
Abstract Dactylogyrus intermedius is a significant mono-genean parasite on the gills of cyprinid fishes and can cau rious problem in fish aquaculture.In the prent study,bioassay-guided fractionation was employed to identify the active compounds from Fructus Arctii against D.interme-dius.Five solvents (petroleum ether,chloroform,ethyl acetate,ethanol,and water)were applied for the extraction of Fructus Arctii.Among them,only the chloroform extract exhibited promising anthelmintic efficacy and therefore,subjected to the further isolation and purification using various chromatographic techniques.Two compounds showing potent activity were obtained and identified by spectral data (infrared,nuclear magnetic resonance,and mass spectrometry)as:arctigenin (1)and arctiin (2).They were found to be significantly effective against D.intermedius with median effective
concentration (EC 50)values of 0.62and 3.55mg L −1,respectively.Arctigenin exhibited higher activity as compared with the positive control mebendazole with an EC 50value of 1.25mg L −1.The 48-h acute toxicity tests (LC 50)of arctigenin and arctiin were found to be 8.47and 14.14mg L −1for goldfish,respectively.The results provided evidence that the studied plant extract,as well as the isolated compounds,might be potential sources of new antiparasitic drug for the control of Dactylogyrus.
Introduction
Fish aquaculture in China is an important economic activity that has been growing in the last few years.The main drawbacks for the extensive commercial production of the freshwater fish are associated with dias including bacterial,viral,and parasitic infections.The ecoparasites monogenean,Dactylogyrus ,usually parasites on the gills of cyprinid fishes and caus a rious problem in fish culture (Buchmann et al.1995;Dove and Ernst 1998).This parasite has a direct life cycle without intermediate host.They relea eggs into the water column that hatch prior to attaching to the gills of a fish host and developing into free-swimming larvae.The larvae are then carried to a new host by water currents and their own ciliated movement (Klinger and Floyd 2002).This parasite is favored by high densities and dias signs include inflamed gills,excessive mucous cretions,a
nd accelerated respiration (Reed et al.2009).Mixed infections with other parasites and condary bacterial infections are common (Woo et al.2002),resulting in a rious damage to the host such as lowered growth performance,loss of appetite,and high mortalities.
To cope with the parasitism and deleterious conquences,various effective parasiticides have been developed in the past relying on such as praziquantel,toltrazuril,and mebendazole (Schmahl and Mehlhorn 1985;Schmahl et al.1988;Treves-Brown 1999).However,the frequent u of the chemical parasiticides has had limited efficacy in reducing mono-genean infestations and is often accompanied by rious drawbacks,including the development of drug-resistant parasites,environmental contamination,and even toxicity to host (Goven et al.1980;Marshall 1999).Other chemicals,such as trichlorfon and formalin,are also far from being satisfactory (Goven et al.1980;Klinger and Floyd 2002).For the reasons,an effective therapy has to be improved,
G.-x.Wang (*):J.Han :T.-t.Feng :B.Zhu College of Animal Science and Technology,Northwest A&F University,Yangling 712100,China
e-mail:wanggaoxue@nwsuaf.edu
F.-y.Li
College of Veterinary Medicine,Northwest A&F University,Yangling 712100,China
Parasitol Res (2009)106:247–255DOI 10.1007/s00436-009-1659-7
and new drugs are needed.Current rearch efforts focud on developing alternative drug formulations including medical plants.The methanol extracts of Piper guineen eds(Piperaceae)were effective to monogenean parasites (Ekanem et al.2004).Two compounds,osthol and isopim-pinellin,from Frutus cnidii have been reported to have the anthelmintic efficacy against Dactylogyrus intermedius (Wang et al.2008).
初级经济法基础Fructus Arctii(Niubangzi in Chine),as the dried fruits of Arctium lappa L.,a well-known herb belonging to the Composita family,has been widely ud for dispelling pathogenic wind-heat,promoting eruption,relieving sore throat,removing toxic substances,and subduing swelling (Lei1995).The dominant active constituents of the herb are the lignan compounds which have been shown to have antioxidant,anti-inflammatory,and antimicrobial effects (Cho et al.2002;Ayres and Loike1990).Arctigenin,a reprentative dibenzylbutyrolactone lignan from Fructus Arctii was found to have an inducible nitric oxide syntha inhibitory activity(Park et al.2005).On the other hand, arctiin,another important lignan,showed anticancer proper-ties in animals models(Matsuzaki et al.2008).However, there is no report available in controlling the monogenean parasite D.intermedius.
Therefore,the aim of the prent study was to investigate the ability of Fructus Arctii extracts against D.intermedius and subquently,to isolate the possible active constituent (s)through bioassay-guided fractionation techniques and identified by spectral analysis.
Materials and methods
Infected fish preparation
Healthy goldfish(mean weight3.7±0.3g)were obtained from Shaan’xi Fisheries Rearch Institute in China and maintained in a180-l glass aquarium with an oxygen content higher than85%saturation at23±1°C.Fish were acclimatized under laboratory conditions for7days and cohabitated with the ones infested with D.intermedius which were cultured in our laboratory.The infected fish were prepared following the method described in our previous work(Wang et al.2008).Briefly,this procedure consisted of the collection of eggs,hatching of the eggs, and reinfection of D.intermedius.After3weeks of postinfection,five fish were randomly lected,killed by spinal verance,and checked for the prence and intensity of parasite under a light microscope(Olympus BX41, Tokyo,Japan)at10×4magnification prior to the experi-ment.Fish were chon for the tests when the infection rate was100%,and a moderate gill infestation level(40–50 parasites per fish)was obtained.Plant material
Fructus Arctii(dried fruits of A.lappa L.)was collected from Tsinling Mountains(altitude from800to1,000m), Shaan’xi province of China,in September2006.The plant was identified by Prof.X.L.He(Northwest A&F University, Shaanxi,China),and a voucher specimen have been deposited in the College of Life science,Northwest A&F University,China.Mature fruits were air-dried under sunlight for a week and finally oven-dried at45°C for 48h.Dried fruits were crushed and reduced to fine powder with a strainer(30–40mesh).The powdered sample was freeze-dried at−45°C to ensure a complete removal of water.
Selection of extraction solvent
Five powdered samples,each weighing20.0g,were respectively extracted with petroleum ether,chloroform, ethyl acetate,ethanol,and water for2h,and the process was repeated for three times.The ratio of sample to solvent was1:10(m/v).Each extract was subquently filtered,and the five filtrates were concentrated under reduced pressure in a vacuum rotary evaporator until a constant yield was obtained.The crude extracts were dissolved in40ml of dimethyl sulfoxide(DMSO)to obtain0.5gml−1stocking solutions which were ud for the preparations of the desired concentrations for each extract.And the control groups containing no plant extract were t up under the same conditions as the test groups.To discard the possible effects of DMSO on the par
asites,another control contain-ing the corresponding percentage of DMSO was also included.Among them,only the chloroform extract exhibited the most significant activity as compared with other four extracts.
Extraction and isolation of bioactive compounds
Bad on the previous trials,chloroform was ud for the extract of Fructus Arctii in order to obtain the most effective constituents.The powdered dry sample(3.1kg) was extracted with chloroform(10L×3times)overnight and then filtered through Whatman no.1filter paper (Whatman,Maidstone,England).The chloroform filtrates were combined and evaporated to dryness under reduced pressure at50°C yielding237.4g of chloroform extract. Part of chloroform extract(200.0g)was put through a silica gel column(silica gel:100–200mesh)and eluted with a solvent mixture of petroleum ether/ethyl acetate(200: 1–0:1,v/v)and finally eluted with methanol affording339 fractions(300ml each).Thin layer chromatography analysis was performed on silica gel using the same solvent system as the mobile pha,and compounds were visualized under ultraviolet(UV)light(254and365nm)or by spraying the
plates with ethanol–sulfuric acid reagent(Wagner et al. 1984).The fractions were then pooled in fiv
e new fractions as follows:(Fr.A:1–64,Fr.B:65–170,Fr.C: 171–205,Fr.D:206–271,Fr.E:272–302,Fr.F:303–337, Fr.G:338–339).Among the,only the Fr.A and Fr.C showed significant anthelmintic efficacy as compared with other fractions and were lected for the further isolation.
Rechromatography of the Fr.A(12.5g)was conducted on silica gel column(silica gel:200–300mesh)and eluted with a mixture solvent of chloroform/methanol(100:1–5:1,v/v)and finally eluted with methanol affording six major subfractions (Sfr.A1:1–2,Sfr.A2:3–8,Sfr.A3:9–19,Sfr.A4:20–28,Sfr. A5:29–30,Sfr.A6:31–32).Subfraction eluted with chloroform/methanol(50:1,v/v;Sfr.A2)revealed the highest efficacy as compared with other subfractions.
The active Fr.C(68.0g)was subjected to open column chromatography on normal pha silica gel(silica gel:200–300mesh)and eluted with a solvent mixture of ethyl acetate/methanol(50:1–2:1,v/v)and finally eluted with methanol yielding six subfractions(Sfr.C1:1–56,Sfr.C2: 57–80,Sfr.C3:81–86,Sfr.C4:87–105,Sfr.C5:106–120, Sfr.C6:121–132).Subfraction eluted with ethyl acetate/ methanol(5:1,v/v;Sfr.C4)poss the highest anthelmintic efficacy and was potential for the further paration.
Sfr.A2(6.0g)was subjected to open column chroma-tography on a silica gel column(silica gel:300–40
0mesh) and eluted with a solvent mixture of chloroform/methanol (100:1–20:1,v/v),finally eluted with methanol affording five subfractions(Sfr.A2–1:1–2,Sfr.A2–2:3–9,Sfr.A2–3: 10–17,Sfr.A2–4:18–23,Sfr.A2–5:24–31).White powder (compound1)was obtained from Sfr.A2–2by successive recrystallization with chloroform:methanol.Rechromatog-raphy of Sfr.C4(12.3g)was performed on a silica gel column(silica gel:200–300mesh)and eluted with a solvent mixture of chloroform/methanol(50:1–4:1,v/v) and finally eluted with methanol affording two subfractions, Sfr.C4–1and Sfr.C4–2.Colorless crystal(compound2)was obtained from the active Sfr.C4–2following the same procedure as Sfr.A2–2.The other fractions or subfractions with lower anthelmintic activity were not further charac-terized,although they may contain compounds that have high activity,but prent in low concentration.
In vivo bioassays
The in vivo bioassays protocol included the evaluation of the crude extracts(including petroleum ether,chloroform, ethyl acetate,ethanol,and water extract)and fractions isolated from chloroform extract and the pure compounds. Tests were conducted in each glass tank of5–l capacity, filled with2l aerated groundwater each containing the test samples and five previously infected fish.The derived fractions and pure compounds were dissolved in1ml DMSO and made up to100ml with distill
ed water which were ud for the preparation of the different concentrations of the test solutions.Initial tests were conducted to get a moderate concentration range in order to avoid the mortality of fish at high concentrations.The five crude extracts and fractions isolated from chloroform extract were conducted at a different ries of concentrations bad on the initial tests, respectively.All the experiments were performed in dupli-cate,except for the pure compounds1and2,which were conducted in triplicate,following the same protocol for the Fructus Arctii fractions,using five concentrations,0.6,1.0, 1.5, 2.0,and 2.5mg L−1and 4.0, 5.0, 6.0,7.0,and 8.0mg L−1correspondingly.A negative control was included using distilled water containing the same amount of DMSO as the test sample.Mebendazole was ud as a positive control with a different ries of concentrations of0.6,1.0,1.5,2.0,and2.5mg L−1.The water temper-ature was constant at23±1°C,dissolved oxygen between 6.1and7.6mg L−1(70–84%saturation),and the pH ranged from7.0to7.5.Mortality of fish was recorded throughout the experiments,during which no food was offered to the fish.
The surviving fish in all the treatment and control groups were biopsied under a light microscope at4×10magnifi-cation.The effectiveness of each treatment was confirmed by the comparison of the number of parasites in each treatment group with tho in the control group after 48-h treatment.Fin
ally,anthelmintic efficacy of each treat-ment and the positive control group was calculated using the follow equation(Wang et al.2008):
AE¼BÀT PðÞ
½ Â100%=Bð1Þ
where AE is anthelmintic efficacy,B is the mean number of surviving D.intermedius in the negative control and P in the positive control,and T is the treatment.
日皮小说Acute toxicity tests
Acute toxicity tests(LC50)of the two compounds(1and 2)were undertaken for goldfish.It was conducted in duplicate using ten individuals in each plastic pot of5–l of capacity,containing2l of filtered water at23°C.Control groups were t under the same test conditions without chemicals.The experiments were performed using con-centrations of7.5,8.0,8.5,9.0,and9.5mg L−1for compound1as well as concentrations of12.0,13.0,14.0, 15.0,and16.0mg L−1for compound2,respectively.The death of fish were recorded when the opercula movement and tail beat stopped and the fish no longer responded to mechanical stimulus.The obrved dead fish was removed from the water in time to avoid the deterioration of the water quality.
Statistical analysis
The homogeneity of the replicates of the samples was checked by the Mann –Whitney U test.Probit analysis was ud for calculating the LC 50and median effective concen-tration (EC 50)at the 95%confidence interval with upper confidence limit and lower confidence limit (Finney 1971).
Results
Selection of extraction solvent
Five solvents,petroleum ether,chloroform,ethyl acetate,ethanol,and water,were ud for the extraction of plant samples.The results of anthelmintic efficacies for five extracts are depicted in Fig.1,which indicated that the chloroform extract revealed a 100%efficacy at the lowest concentration of 240.0mg L −1after 48h of exposure.Followed by the ethanol and ethyl acetate extracts and the maximum anthelmintic efficacies were 71.3%(700.0mg L −1)and 81.9%(600.0mg L −1),respectively.The petroleum ether and water extracts exhibited the least activity with the maximum anthelmintic efficacy of 38.4%(180mg L −1)and 31.7%(800mg L −1),respectively.And the solvent (DMSO)acted as a control showed no anthelmintic activity when treated at the highest concentration.Therefore,chloroform was the optimal solvent for the extraction of plant samples.In viv
o bioassays
In order to begin the process of identifying the active compounds within the chloroform extract,the extract was divided into ven fractions which were tested for anthel-mintic activity.Of the ven fractions,Fr.A (13.6g)and Fr.C (70.3g)were found to be more effective than other fractions,with a complete elimination of parasites at concentrations of 6.0and 15.0mg L −1,respectively (Fig.2).The two fractions were lected for further purification.
Fractionation of the active fraction,Fr.A,yielded six subfractions.The anthelmintic efficacy assay revealed that Sfr.A 2(6.5g)exhibited the optimal anthelmintic activity with 100%efficacy at the do of 6.0mg L −1and showed to be toxicity to fish at 10.0mg L −1(Fig.3).Followed by the Fr.A 3and Fr.A 4,with the maximum anthelmintic efficacies of 65.6%and 51.3%for 48h,respectively.The other three subfractions,Fr.A 1,Fr.A 5,and Fr.A 6,had no significant activity.
Six subfractions were collected from the purification of Fr. C.The results of the anthelmintic efficacy of the subfractions were shown in Fig.4.Of the six subfractions,Fr.C 4(44.3g)revealed a 100%efficacy at the concentra-tion of 10.0mg L −1.However,fish mortality occurred when the concentrations were up to 12.0mg L −1.Fr.C 3and Fr.C 5revealed a promising efficacy with 79.2%an
d 79.7%at 15.0and 20.0mg L −1which resulted in fish mortality,respectively.The other three subfractions (Fr.C 1,Fr.C 2,and Fr.C 6)showed no significant efficacy up to 20mg L −1.Rechromatography of the active Sfr.A 2yielding five subfractions,among them,Sfr.A 2–2(1.1g)showed the highest activity when tested 5.0mg L −1(Fig.5)and was lected for further purification.Rechromatography of Fr.C 4affording two major subfractions and Fr.C 4-2(3.8g)showed a higher activity (Fig.5),and was ud for further isolation.Finally,compounds 1(0.6g)and 2(2.4g)were obtained from the subfractions Sfr.A 2–2and Sfr.C 4–2by successive recrystallization,respectively.
Both compounds 1and 2showed remarkable anthelmin-tic efficacy against D.intermedius with EC 50values of 0.62and 3.55mg L −1,respectively.While the calculated EC 50of the positive control,mebendazole,was 1.25mg L −1(Table 1).Acute toxicity
The LC 50values of the two compounds (1and 2)were determined from linear y ¼m þbx ðÞplots of the probit curves which were prented in Table 1.The
probit
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A n t h e l m i n t i c e f f i c a c y (%)
Fig.1Anthelmintic efficacy of five extracts against
D.intermedius after 48h.PEE petroleum ether extract,CIE chloroform extract,EAE ethyl acetate extract,EtE ethanol extract,WaE water extract
mortality percentage of the goldfish was plotted against log concentrations.
For the compound 1,the linear equation y ¼À16:07þ17:32x which was derived from the regression analysis of probit mortality of goldfish in test solution bioassay.The calculated LC 50was 8.47mg L −1with the 95%confidence interval of 7.95–9.02mg L −1.For the compound 2,the linear equation y ¼À13:29þ11:56x which was derived from the regression analysis of probit mortality with a LC 50of 14.14mg L −1and 95%confidence interval of 12.87–15.25mg L −1.There was no fish mortality occurred in control groups during the experiments.Structural determination of active compounds
The chemical structure confirmation of the compounds from the Fructus Arctii was accomplished by comparing UV ,infrared (IR),mass spectrometry (MS),1H nuclear magnetic resonance (NMR),and 13C NMR data obtained to tho published.
Compound 1was obtained as white powder;UV (MeOH)ν/nm:211,237(2.39),281(1.82).IR (KBr)ν/cm −1:3422.9,1762.8,1515.7;FAB-MS m/z:372;ESI-TOF m/z:767.3([2M+Na]+),395.1([M+Na]+),390.2([M+NH4]+),373.2([M+H]+).1H-NMR(CDCl 3,400MHz)δ
ppm:6.46(1H,d,H-2′),6.64(1H,d,H-2′),6.76(1H,d,H-5),6.84(1H,d,
H-5′),6.56(1H,dd,H-6),6.60(1H,dd,H-6′),2.65(1H,dd,H-7),2.47(1H,dd,H-7′),2.91(1H,dd,H-7′α),2.94(1H,dd,H-7′β),2.54(1H,m,H-8),2.54(1H,m,H-8′),3.89(1H,dd,H-9),4.14(1H,dd,H-9′),5.59(OH),3.85,3.83,3.82(OCH 3×3).13C-NMR (CDCl 3,100MHz),DEPT δppm:130.4(C-1),129.5(C-1′),111.3(C-2),111.5(C-2′),149.0(C-3),147.8(C-3′),146.7(C-4),144.5(C-4′),111.8(C-5),114.1(C-5′),120.3(C-6),122.0(C-6′),38.1(C-7),34.5(C-7′),40.9(C-8),46.6(C-8′),71.2(C-9),178.7(C-9′),55.3,55.4,55.5(OCH 3×3)agree well with the data reported (Rahman et al.1990;Nishibe et al.1984).So,the compound was identified as arctigenin.
Compound 2was isolated as colorless crystals;UV (MeOH)ν/nm:212,235(2.23),279(1.32).IR (KBr),ν/cm-1:3421.8,1762.8,1607.7,1515.7,1458.0,1240.8,1066.5,819.FAB-MS m/z:533(M-H);ESI-TOF m/z:557.1([M+Na]+),552.1([M+NH 4]+),373.1.1H-NMR (CDCl 3,400MHz)δppm:6.74(1H,s,H-2),6.49(1H,d,H-2′),6.75(1H,d,H-5),6.90(1H,d,H-5′),6.60(1H,d,H-6),6.56(1H,dd,H-6′),2.65(1H,m,H-7),2.48(1H,m,H-7′),2.83(1H,m,H-7′α),2.83(1H,m,H-7′β),2.55(1H,m,H-8),2.55(1H,m,H-8′),4.13(1H,t,H-9),3.84(1H,m,H-9′),4.83(1H,d,H-Glc-1), 3.84, 3.81, 3.75(OCH 3×3).13C-NMR(CDCl 3,100MHz)δppm:130.4(C-1),133.1(C-1′),112.1(C-2),113.3(C-2′),149.3(C-3),149.1(C-3′),147.9(C-4),145.1(C-4′),111.6(C-5),113.3(C-5′),120.7(C-6),121.8(C-6′
),
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20.040.060.080.0100.0F r .A (4.0)F r .A (6.0)F r .A (8.0)
F r .B
(10.0)F r .B (
15.0)F r .B (20.0)
F r .C (10.0)F r .C (15.0)F r .C (20.0)
F r .D
(10.0)F r .D (
15.0)F r .D (20.0)
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F r .E (10.0)F r .E (15.0)F r .E (20.0)
F r .F (30.0)F r .F (40.0)F r .F (50.0)
F r .
G (30.0)F r .G (40.0)F r .G (50.0)
Fraction concentration (mg L -1)
A n t h e l m i n t i c e f f i c a c y (%)
Fig.2Anthelmintic efficacy of ven fractions from chloroform extract against D.intermedius after 48
h
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20.040.060.080.0100.0F r . A 1 (5.0)F r . A 1 (10.0)F r . A 1 (20.0)
F r .
A 2 (4.0)F r . A 2 (6.0)F r .A 2 (8.0)F r . A 2 (10.0)F r . A 3 (5.0)F r . A 3 (10.0)F r . A 3 (15.0)F r . A 4 (5.0) F r .A 4 (10.0) F r .A 4 (20.0)F r . A 5 (5.0) F r .A 5 (10.0)F r . A 5 (20.0)F r . A 6 (5.0)F r . A 6 (10.0)F r . A 6 (20.0)
Fraction concentration (mg L -1)
A n t h e l m i n t i c e f f i c a c y (%)
Fig.3Anthelmintic efficacy of six fractions from Fr.A against D.intermedius after 48h.Star indicates when fish mortality firstly occurred
37.9(C-7),34.4(C-7′),41.1(C-8),46.4(C-8′),71.2(C-9),178.6(C-9′),101.9(Glc-1),73.2(Glc-2),76.7(Glc-3),
69.4(Glc-4),76.9(Glc-5),61.5(Glc-6),55.3,55.3,55.5(OCH 3×3).The data were in agreement with the reported literature values.Thus,the structure of compound was deter-mined as arctiin (Takasaki et al.2000;Shoeb et al.2004).The structures of the compounds isolated from Fructus Arctii are shown in Fig.6.
Discussion
Monogenean parasites have been recognized as a rious pathogen in the aquaculture of many marine and freshwater fish (Ogawa 2002),and their control can be difficult as exemplified by the extent and range of published data on potential treatments.Nonspecific treatments such as formalin have been reported as uful treatments to monogenean when administrated as a prolonged bath at low do or short-term bath at higher do (Thoney and Hargis 1991).However,the efficacy of formalin against D.intermedius has not been determined.Meanwhile,the side effects of formalin on the host were also reported in some species.For example,Diggles et al.(1993)reported higher do rate of formalin were toxic to dulfish in small-scale trials,with the majority of fish dying following a 250-ppm formalin bath of 1-h duration.The antiprotozoal chemical,toltrazuril,has
recently been shown to be effective against monogenean,Dactylogyrus ,when treated using a water
bath with 10mg L −1for 4h at 20°C (Schmahl et al.1988).A similar efficacy of praziquantel against Dactylogyrus when applied by bath treatment at 10mg L −1was also reported.Moreover,the effects of toltrazuril at a cellular level have also been investigated on monogenean,Dactylogyrus (Schmahl and Mehlhorn 1985).The toltrazuril affects the permeability of the cell membrane of the parasites,resulting in incread contraction of parasites,and the drug further caus vacuoli-zation and disinteration of monogenea teguments (Schmahl et al.1988),which were similar to that previously shown for praziquantel treatment (Schmahl and Taraschewski 1987).However,the repeat u of the chemical-bad substances for the control of monogenean parasites can be problematic,leading to the potential deleterious effects on both the environment and the human consumers of their products due to public health implications of residues (Committee on Mutagenicity of Chemicals in Food,Consumer Products and the Environment 1999).
There is growing interest in using plant extracts for the treatment of human and animal dia,but little informa-tion is available on the u of the plant-derived products in the treatment of aquaculture dias (Ekanem et al.2004).In the prent study,we made a screen test using the in vivo anthelmintic efficacy assay,resulting in the lection of the Fructus Arctii from many medical
plants.
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F r . C 6 (10.0)F r . C 6 (15.0)F r . C 6 (20.0)
Fraction concentration (mg L -1)
A n t h e l m i n t i c e f f i c a c y (%)
Fig.4Anthelmintic efficacy of six fractions from Fr.C against D.intermedius after 48h.Star indicates when fish mortality firstly occurred
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A
2-2 (5.0)F r
.A 2-2 (10.0)F r .A 2-3 (5.0)F r .A 2-3 (10.0)F F r .A 2-4 (5.0)r .A 2-4 (10.0)F r .A 2-5 (5.0)F r .A 2-5 (10.0)F r .C 4-1 (5.0)F r .C 4-1 (10.0)F r .C 4-2 (5.0)F r .C 4-2(10.0)Fraction concentration (mg L -1)
A n t h e l m i n t i c e f f i c a c y (%)
Fig.5Anthelmintic efficacy of five subfractions from Fr.A2and two subfractions from Fr.C 4against D.intermedius after 48h
