Veterinary Parasitology 171 (2010) 305–313

Contents lists available at ScienceDirect

Veterinary Parasitology
journal homepage: www.elsevier.com/locate/vetpar

In vivo anthelmintic activity of five alkaloids from Macleaya microcarpa

(Maxim) Fedde against Dactylogyrus intermedius in Carassius auratus
Gao-Xue Wang ∗ , Zhuang Zhou, Dong-Xin Jiang, Jing Han, Jian-Fu Wang,
Liang-Wei Zhao, Jun Li
Northwest A&F University, Xinong Road 22nd, Yangling, Shaanxi 712100, China

a r t i c l e i n f o a b s t r a c t

Article history: The present study aims to evaluate the anthelmintic properties of aerial part of Macleaya
Received 16 December 2008 microcarpa (Maxim) Fedde. Bioassay-guided fractionation and isolation of the compounds
Received in revised form 1 March 2010
with anthelmintic activity were performed on the ethanolic extract of M. microcarpa yield-
Accepted 26 March 2010
ing five bioactive alkaloids namely: sanguinarine, cryptopine, ␤-allocryptopine, protopine
and 6-methoxyl-dihydro-chelerythrine by comparing spectral data (UV, NMR, and EI-MS)
Keywords:
with literature values. According to in vivo anthelmintic assays, they were found to be
Dactylogyrus intermedius
100% effective at the concentrations of 0.7, 8.0, 8.0, 16.0 and 7.0 mg l−1 , and the median
Anthelmintic
Macleaya microcarpa (Maxim) Fedde effective concentration (EC50 ) values for the five compounds were 0.37, 3.31, 4.64, 8.13
Alkaloids and 3.63 mg l−1 , respectively. Additionally, the acute toxicity on goldfish for the five active
compounds was also investigated with median lethal concentrations (LC50 ) values of 1.13,
16.12, 15.88, 21.69 and 10.91 mg l−1 , respectively. The resulting therapeutic indices for san-
guinarine, cryptopine, ␤-allocryptopine, protopine and 6-methoxyl-dihydro-chelerythrine
were 3.03, 4.82, 3.40, 2.66 and 2.99 correspondingly. Correlations analysis between the
log P and EC50 , LC50 of the five alkaloids revealed that the activity of the five alkaloids was
well correlated with their hydrophobicity and r2 = 0.45 is for anthelmintic activity while
r2 = 0.47 is for acute toxicity for goldfish, respectively. These results provided evidence that
the studied plant extract, as well as the isolated compounds, especially sanguinarine, might
be potential plant-based medicines for the treatment of D. intermedius infection.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction infecting the gill of freshwater fish of Cyprinidae, which

Fish culture in China has been growing rapidly in the
past two decades and the aquatic products have ranked
top in the world in recent years. Since intensive aquacul-
ture was initiated over the past decades, captive species
frequently develop heavy infestations of gill monogenean
parasites, resulting in significant economic loss. Dactylo-
gyrus, a main genus of monogenea, are common parasites
∗ Corresponding author at: Northwest A&F University, Xinong Road
22nd, Yangling, Shaanxi 712100, China. Tel.: +86 29 87092102;
fax: +86 29 8709 2102.
E-mail addresses: wanggaoxue@yahoo.com.cn,
wanggaoxue@126.com (G.-X. Wang).
are egg layers with two to four eyespots, one pair of large
anchor hooks. Their eggs hatch into free-swimming larvae
and are carried to a new host by water currents and their
own ciliated movement (Chinabut and Lim, 1991, 1993,
1994; Kaewviyudth and Chinabut, 1999; Řehulková and
Gelnar, 2006). Heavily infected fish of Cyprinidae in captiv-
ity show the swollen and pale gills, increasing respiration
rate, and less tolerance in low oxygen environment, which
can lead to high mortality. Moreover, secondary infection
with other pathogens including fungus and bacteria can
also occur.
Therefore, the control of this parasite has become an
urgent need in fish farming. Several chemicals such as
formalin (Marshall, 1999), trichlorfon (Goven and Amend,

0304-4017/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetpar.2010.03.032
306 G.-X. Wang et al. / Veterinary Parasitology 171 (2010) 305–313

1982), a triazine derivative (Schmahl, 1993), praziquan-

tel (Schmahl and Mehlhorn, 1985) and mebendazole
(Buchmann et al., 1993) have been used for the control
of Dactylogyrus, especially the latter two chemicals, which
were widely used in China. But these chemicals can have
undesirable effect on other organisms and the environ-
mental damage. Ultimately they become ineffective in the
control of parasites due to the quick development of resis-
tance to these chemicals. Such considerations have led to
the search for alternative anthelmintic agents.
Recently, there has been growing interest regard-
ing the utilization plant-based agents for the control of
parasites. Generally, the plant-based products are more
non-resistible for frequent use as compared with the chem-
ical drugs. Moreover, the novel products might be potential
sources of new antiparasitic drug (Osbourn and Lanzotti,
2009). Ekanem et al. (2004) found that the methanolic
extracts of the seeds of Piper guineense (Piperaceae) were
active against monogenean parasites and three active com-
pounds were identified. The Indian almond leaf extract was
also reported to exhibit a complete elimination of Dacty-
logyrus sp. (Chansue, 2007). Wang et al. (2008) reported
that osthol and isopimpinellin from the fruit of Fructus
cnidii also showed effectiveness against D. intermedius.
In our previous work, an active compound sanguinarine
from Macleaya cordata was found to be effective against
D. intermedius (Wang et al., 2007). Macleaya microcarpa
(Maxim) Fedde, as another species in the genus of Macleaya
in Papaveraceae, is widely distributed in northwest and
southwest of China, and has reported various bioactivi-
ties, such as antitumor (Qin et al., 2004), algaecidal (Jana
et al., 2007) and insecticidal activity (Feng et al., 2008).
However, to the best of our knowledge, there is no report
concerning about the use of M. microcarpa in the control of
fish parasites. In this study, bioassay-guided fractionation
monitored by the in vivo anthelmintic assay was employed
to investigate the anthelmintic activity of M. microcarpa
against D. intermedius. Additionally, acute toxicity for the
ethanolic extract and active compounds from this plant
were also evaluated.
2. Materials and methods
2.1. Preparation of infected fish
Healthy goldfish, Carassius auratus, weighing
2.9 ± 0.3 g, were transferred into the aquarium
(l × w × h = 120 cm × 50 cm × 50 cm) containing 180-l
aerated groundwater at 23 ± 1 ◦ C. Fish were acclimatized
under laboratory conditions for 7 days and mebendazole
was added at 1.5 mg l−1 (Wang et al., 2008) for 48 h to kill
the parasites and then florfenicol (Fukui et al., 1987) at
5.0 mg l−1 for 4 h to eliminate the bacteria to ensure fish
health. The healthy goldfish were co-habitated at a ratio
of 20% with the ones infected with D. intermedius which
were reared in our laboratory. The infected goldfish were
prepared according to our previous study (Wang et al.,
2008). After 7–10 days of co-habitation, 10 goldfish were
then randomly selected and killed by spinal severance
and the lamella branchialis of each fish was biopsied to
check the presence and intensity of parasites on the gills
Fig. 1. The aerial part of M. microcarpa.
of infected fish under a light microscope at 4 × 10 magni-
fication. Goldfish were chosen for the in vivo anthelmintic
assay when the infection rate was 100% and the mean
number of parasites on the gills was about 30 per fish.
2.2. Activity-guided isolation of active ingredients
2.2.1. Selection of extraction solvent
Five samples of air-dried, powdered aerial part of M.
microcarpa (Fig. 1) identified by Prof. X.L. He (North-
west A&F University, Shaanxi, China), each weighing 50.0 g
were respectively extracted with petroleum ether, ethyl
acetate, chloroform, acetone and ethanol in 75 ◦ C water
bath for 2 h, this process was performed in three repli-
cates. The resulting suspensions were filtered and then
concentrated to dryness under reduced pressure to get
the extracts. The dried extracts were dissolved at a con-
centration of 0.5 g ml−1 (sample/solvent, m/v) in dimethyl
sulfoxide (DMSO) (for petroleum ether extract) and ethanol
(for other four extracts) and used for anthelmintic efficacy
tests. The control solutions were made up of water and
DMSO or ethanol with no plant extract. Both of the two
solvents (ethanol and DMSO) were tested previously with
no anthelmintic efficacy at the highest concentration in the
treatments.
2.2.2. Isolation of active compounds
Air-dried aerial part of M. microcarpa (2.0 kg) was
crushed and reduced to fine powder (100 mesh), extracted
with ethanol (20.0 l × 3 times) in 75 ◦ C water bath for
2 h, then filtered. The resulting filtrate was evaporated
under reduced pressure to yield the dried extract (252.0 g,
yield 12.6%). The extract was then subjected to column
chromatography (120 cm × 10 cm) on silica gel (2000 g,
100–200 mesh) eluting with petroleum ether:ethyl
acetate:methanol (1:0:0, 100:1:0, 50:1:0, 25:1:0, 10:1:0,
5:1:0, 2:1:0, 1:1:0, 10:10:1, 4:4:1, 2:2:1, 1:1:1, 2:2:3, 1:1:2,
1:1:4, 1:1:8, 1:1:20, 0:0:1, v/v/v) gradients providing 639
fractions (500 ml each). TLC analysis was performed on
 G.-X. Wang et al. / Veterinary Parasitology 171 (2010) 305–313
silica gel using the same solvent system as the mobile
phase. Spots on thin layer chromatography (TLC) plates
were visualized under UV light (254 and 365 nm) and frac-
tions showing similar chromatograms were combined to
yield 6 main fractions, i.e., Fraction A: 1–110 (13.2 g); Fr
B: 111–173 (10.6 g); Fr C: 174–278 (22.9 g); Fr D: 279–344
(34.2 g); Fr E: 355–484 (95.0 g); and Fr F: 485–639 (29.7 g).
All these 6 fractions were concentrated to dryness, and part
of them was dissolved in ethanol to prepare stock solutions
(all the combined fractions and subfractions mentioned
below were all dissolved in ethanol) which were reserved
for the anthelmintic efficacy assay. Among these fractions,
Fr D and Fr E proved to be the most active fractions and
were used for further isolation and purification.
Fr D (31.0 g) was subjected to column chromatography
(80 cm × 6 cm) on silica gel (525 g, 100–200 mesh) using
a mixture of petroleum ether–ethyl acetate–methanol
(10:1:0, 5:1:0, 2:1:0, 1:1:0, 4:4:1, 2:2:1, 1:1:1, 2:2:3,
1:1:10, 0:0:1, v/v/v) gradient as eluent to yield 59 frac-
tions (300 ml each). The fractions showing similar TLC
chromatograms were combined into 5 fractions, i.e., Frac-
tion Da: 1–7 (3.5 g); Fr Db: 8–19 (4.7 g); Fr Dc: 20–27
(3.9 g); Fr Dd: 28–54 (8.8 g); Fr De: 54–59 (2.9 g). These
five fractions were submitted to anthelmintic efficacy
assay and Fr Dd (2.5 g) exhibited the highest activity
which was then subjected to column chromatography
(40 cm × 2 cm) with Sephadex-LH20 (20 g, 200–300 mesh)
using chloroform–methanol (1:0, 1:1, 0:1, v/v) as solvent
system affording Fr Dd1 (2.0 g) and Fr Dd2 (0.2 g). The high-
est active fraction, Fr Dd1 (1.5 g), was chromatographed on
a silica gel column (40 cm × 2 cm; silica gel: 30 g, 100–200
mesh) with a chloroform–methanol solvent system (1:1,
v/v) and a reddish brown-colored crystal, compound 1
(230.0 mg) was obtained by successive recrystallization
(chloroform–methanol).
Similar to Fr D, Fr E (91.2 g) was chromatographed
on a silica gel column (120 cm × 8 cm; silica gel: 1000 g,
100–200 mesh) and eluted with an solvent mixture of
petroleum ether–ethyl acetate–methanol (100:1:0, 5:1:0,
5:2:0, 1:1:0, 10:10:1, 4:4:1, 2:2:1, 1:1:1, 2:2:3, 2:2:5,
1:1:5, 1:1:10, 0:0:1, v/v/v) affording 526 fractions (400 ml
each). TLC analysis was performed on silica gel using
the same solvent system yielding 7 major fractions, i.e.,
Fraction Ea: 1–18 fractions (1.1 g); Fr Eb: 19–113 (3.4 g);
Fr Ec: 114–254 (10.3 g); Fr Ed: 255–354 (4.2 g); Fr Ee:
355–477 (26.5 g); Fr Ef: 478–508 (25.2 g); Fr Eg: 509–526
(1.2 g). Anthelmintic efficacy assay exhibited that Fr Eb,
Fr Ee and Fr Ef were the active fractions and used for
further isolation. A white-colored lamellar crystal, com-
pound 2 (850.0 mg), was obtained from Fr Eb (1.2 g)
by successive recrystallization with chloroform–methanol
system (1:1, v/v). Fr Ee (24.1 g) was subjected to a basic
aluminum oxide column chromatography (80 cm × 6 cm,
aluminum oxide: 800 g, 300–400 mesh) with a solvent
system of chloroform–methanol (1:1, v/v) affording 39
fractions (300 ml each). On the basis of TLC analysis,
these fractions were pooled in four fractions, i.e., Frac-
tion Ee1: 1–11 fractions (3.7 g); Fr Ee2: 12–23 (3.4 g); Fr
Ee3: 24–30 (2.2 g); Fr Ee4: 31–39 (2.8 g). Fr Ee4 (2.3 g)
was ascertained the active fraction by anthelmintic efficacy
assay and compound 3 (850.0 mg), a buff-colored gran-
307
ular crystal, was obtained by successive recrystallization
with chloroform–methanol system (1:2, v/v). Meanwhile,
Fr Ef (20.1 g) was chromatographed on a silica gel col-
umn (80 cm × 6 cm, silica gel: 362 g, 300–400 mesh) eluting
with chloroform–methanol (1:0, 5:1, 2:1, 1:1, 1:2, 1:5, 0:1,
v/v) affording 136 fractions (200 ml each). Four main frac-
tions, i.e., Fraction Ef1: 1–28 fractions (2.8 g); Fr Ef2: 29–37
(1.1 g); Fr Ef3: 38–95 (3.4 g); Fr Ef4: 96–136 (4.3 g) were
pooled based on TLC analysis. Among them, Fr Ef3 (1.5 g)
and Fr Ef4 (2.8 g) exhibited the highest effects, compound
4 (white-colored lamellar crystal, 1250.0 mg), and com-
pound 5 (lilac-colored granular crystal, 950.0 mg) were
obtained as the active compounds from Fr Ef3 and Fr Ef4,
respectively.
2.3. Anthelmintic efficacy assay
Experiments were conducted in 5 l plastic pot, each
containing 2.0 l of the test solution, and 10 infected fish.
The five crude extracts (0.5 g ml−1 ) were used for the
preparation of the desired concentrations. All the fractions
were dissolved in ethanol to prepare at a concentration
of 0.5 g ml−1 and the active compounds were prepared at
50 mg ml−1 . Initial tests were performed to determine the
concentration boundaries to avoid the mortality of gold-
fish occurred at high concentration. The five crude extracts
and fractions as well as the active compounds were investi-
gated at different concentrations based on the initial tests.
The control groups were set up under the same experimen-
tal conditions with 0.1% DMSO or ethanol (the maximum
amount used in the present study was 0.03%), and a blank
control was also included. The treatments for the ethano-
lic extract and active fractions as well as compounds were
conducted in triplicates while the inactive ones were per-
formed once. And all the controls were conducted with
three replicates. In all treatments and controls, the dis-
solved oxygen ranged between 5.6 and 7.0 mg l−1 (70–84%
saturation), pH between 7.3 and 7.6 and the temperature
was constant at 23 ◦ C.
The active fractions (Fr D and Fr E) from ethanolic extract
were assayed at 5.0, 10.0, 15.0 mg l−1 while the inactive
fractions (Fr A, Fr B, Fr C, and Fr F) were tested at higher
concentrations of 20.0, 40.0, 60.0 mg l−1 . Following the
bioassay guided fractionation, the active fraction, Fr Dd,
was assayed at 3.0, 6.0 and 9.0 mg l−1 . And the derived inac-
tive fractions (Fr Da, Fr Db, Fr Dc and Fr De) were tested at
higher concentrations (5.0, 10.0, 15.0 mg l−1 ). Two fractions
(Fr Dd1 and Fr Dd2) derived from Fr Dd were assayed at 0.5,
0.7, 0.9 mg l−1 and 3.0, 6.0, 9.0 mg l−1 , respectively.
Similarly, seven fractions (fraction Ea–Eg) from the
active fraction, Fr E, were assayed at concentrations of 5.0,
10.0, and 15.0 mg l−1 . And the derived active compounds
were tested at a series of concentrations: 0.3, 0.4, 0.5, 0.6
and 0.7 mg l−1 for compound 1; 3.0, 4.0, 5.0, 6.0, 7.0 and
8.0 mg l−1 for compound 2; 4.0, 5.0, 6.0, 7.0 and 8.0 mg l−1
for compound 3; 8.0, 10.0, 12.0, 14.0 and 16.0 mg l−1 for
compound 4; 3.0, 4.0, 5.0, 6.0, 7.0 mg l−1 for compound 5.
After 48 h treatment, the surviving goldfish in the treat-
ment and control groups were examined as described
above, and the surviving D. intermedius in the gills were
counted under a light microscope at 4 × 10 magnification
308 G.-X. Wang et al. / Veterinary Parasitology 171 (2010) 305–313
to determine the mean number of parasites per fish. Finally,
the anthelmintic efficacy of each treatment was calculated
according to the following equation (Wang et al., 2007).
B−T
AE (%) = × 100
B
Where AE is anthelmintic efficacy, B is the mean number
of surviving D. intermedius in the blank control and T in the
treatment.
2.4. Acute toxicity of ethanolic extract and active
compounds to C. auratus
The acute toxicity of ethanolic extract and the active
compounds from M. microcarpa were assayed using the
healthy goldfish (mean weight 2.7 ± 0.45 g). The tests were
conducted in plastic pot of 5.0 l capacity, each containing
2.0 l of test solution, and 10 healthy goldfish. The fish were
not fed during the experiment. Dilutions were prepared
from the stock solutions as the following concentrations:
50.0, 63.0, 79.0, 100.0, 126.0, 158.0 and 200.0 mg l−1 for
ethanolic extract; 0.9, 1.0, 1.1, 1.2, 1.3 mg l−1 for compound
1; 14.0, 15.0, 16.0, 17.0, 18.0 mg l−1 for compound 2; 14.0,
15.0, 16.0, 17.0, 18.0 mg l−1 for compound 3; 19.0, 20.0,
21.0, 22.0, 23.0 mg l−1 for compound 4; 9.0, 10.0, 11.0,
12.0, 13.0 mg l−1 for compound 5. The tests were conducted
in triplicate, and controls (under the same test condi-
tions with no chemicals). The fish mortality was recorded
when there was no opercula movement and the goldfish
Fig. 2. Anthelmintic efficacy of petroleum ether, ethyl acetate, chloroform and acetone extract against D. intermedius at 48 h.
no longer responded to mechanical stimulus (touch by
glass rod). Any observed dead fish was removed from the
medium in time to avoid deterioration of the water quality.
Fish mortalities in the treatment and control groups were
(1) recorded after 48 h.
2.5. Data analysis
The anthelmintic efficacy of the active fractions and
compounds was expressed as a 100% effective concen-
tration, which is the lowest concentration resulted in a
complete removal of parasites. Concentration–mortality
regressions of the ethanolic extract and active compounds
were estimated by probit analysis using probit proce-
dure and the 48 h median effective concentration (EC50 )
with 95% confidence interval, which is the concentration
that eliminated 50% of the parasites compared with the
control were calculated. The 48 h median lethal concen-
tration (LC50 ) value with its 95% confidence interval for the
ethanolic extract and each compound were calculated by
log concentration–probit equation using the probit anal-
ysis, and control mortality was corrected using Abbott’s
formula (Abbott, 1925).
p − C
P= (2)
1−C
Where p is the corrected mortality, p is the treated mor-
tality and C is the mortality of control.
 G.-X. Wang et al. / Veterinary Parasitology 171 (2010) 305–313 309

3.2. Anthelmintic efficacy of fractions

Followed by bioassay-guided fractionation, each frac-

Fig. 3. Anthelmintic efficacy of ethanolic extract against D. intermedius at
48 h.
The therapeutic index (TI) for each compound was cal-
culated by comparing LC50 versus the EC50 (Wikipedia,
2010, encyclopedia)
LC50
TI =
EC50
The hydrophobicity expressed as log P (logarithm of the
partition between octanol and water) was also attempted
to investigate its effect on EC50 and LC50 for all the com-
pounds.
2.6. Identification of active compounds
The structure of the active compounds was identified
by melting point measure and spectral methods, such
as nuclear magnetic resonance hydrogen spectrum (1 H
NMR) and nuclear magnetic resonance carbon spectrum
(13 C NMR) (Bruker Avance 400 spectrometer with SiMe4
as internal standard, USA), electron ionization mass spec-
trometry (EI-MS, 70 eV VG-ZAB-HS, VG Company, England).
3. Results
3.1. Selection of extraction solvent
Petroleum ether, ethyl acetate, chloroform, acetone and
ethanol were evaluated for their efficacy in extracting
the active compounds from M. microcarpa. The results
of the anthelmintic efficacy of the five extracts against
D. intermedius (Figs. 2 and 3) showed that ethanolic
extract exhibited the highest anthelmintic efficacy of
100% at the lowest concentration of 70.0 mg l−1 . And
the calculated EC50 of ethanolic extract was 31.21 mg l−1
(20.15–36.69 mg l−1 ) (Table 1). Therefore, ethanol was
selected for the extraction of the plant sample.
tion was assayed for the anthelmintic efficacy and the
active ones were selected for the further isolation and
purification. Of the six fractions collected from the ethano-
lic extract, Fr D and Fr E exhibited 100% efficacy at 15.0 and
10.0 mg l−1 , respectively. However, goldfish began to die
when the concentration of Fr D reached 15.0 mg l−1 . The
other four fractions (Fr A, Fr B, Fr C and Fr F) had no sig-
nificant efficacy at 60.0 mg l−1 with the efficacy of 11.5%,
23.0%, 9.1% and 13.2%, respectively (Fig. 4). So, the active
compounds were contained in the two active fractions, Fr
D and Fr E. Figs. 5 and 6 summarized the anthelmintic effi-
cacies of factions from Fr D. Fr Dd and Fr Dd1 were active
fractions and the 100% effective concentrations were 3.0
and 0.7 mg l−1 , respectively. However, the fish began to die
when the concentration reached 9.0 and 0.9 mg l−1 , respec-
tively. Meanwhile, seven fractions were obtained from Fr
E. Among them, Fr Eb, Fr Ee and Fr Ef were considered to be
(3) the active ones with 100% effective concentrations of 10.0,
10.0 and 15.0 mg l−1 , respectively. However, the fish mor-
tality occurred when the concentration of Fr Ee reached
15.0 mg l−1 (Fig. 7).
3.3. Identification of active compounds
3.3.1. Compound 1
Sanguinarine, C20 H14 NO4 (Perez Gutierrez et al.,
2002), was obtained as reddish brown crystal with m.p.
242–243 ◦ C.
EI-MS (70 eV) m/z: 334.3 (M+2), 333.3 (M+1), 332.3
(M+ ), 331.3 (M−1), 315.3 (M-CH3), 304.3, 274.3, 260.3.
1 H NMR (CD OD, TMS, 400 MHz) ı 7.56 (1H, s, H-1), 8.15
3
(1H, s, H-4), 9.94 (1H, s, H-6), 8.62 (1H, d, J = 9.2 Hz, H-9),
7.95 (1H, d, J = 9.2 Hz, H-10), 8.53 (1H, d, J = 9.2 Hz, H-11),
8.21 (1H, d, J = 9.2 Hz, H-12), 6.27 (2H, s, –OCH2 O-2,3), 6.58
(2H, s, –OCH2 O-7,8), 4.94(3H, s, N-Me).
13 C NMR (CD OD, TMS, 100 MHz) ı 104.3 (C-1), 148.2
3
(C-2), 149.5 (C-3), 105.0 (C-4), 121.2 (C-4a), 133.2 (C-4b),
150.8 (C-6), 111.3 (C-6a), 150.9 (C-7), 150.8 (C-8), 121.9
(C-9), 118.3 (C-10), 129.1 (C-10a), 127.6 (C-10b), 119.6 (C-
11), 132.9 (C-12), 134.2 (C-12a), 105.0 (–OCH2 O-2,3), 106.6
(–OCH2 O-7,8), 52.8 (N-Me).
3.3.2. Compound 2
Cryptopine, C21 H23 NO5 (Guineudeau and Shamma,
1982), was obtained as white lamellar crystal with m.p.
220–221 ◦ C.

Table 1
The anthelmintic efficacy and acute toxicity of ethanolic extract and five alkaloids.

Sample Anthelmintic efficacy Acute toxicity EI

−1 −1
Slope ± SE EC50 (mg l ) (95% CL) Slope ± SE LC50 (mg l ) (95% CL)

Ethanolic extract −3.99 ± 1.16 31.21 (20.15–36.69) 5.09 ± 1.52 121.70 (101.81–167.06) 3.90
Sanguinarine 7.78 ± 1.39 0.37 (0.33–0.40) 11.50 ± 3.62 1.13 (1.04–1.27) 3.03
Cryptopine 5.57 ± 1.08 3.31 (2.70–3.72) 21.78 ± 7.55 16.12 (15.38–17.75) 4.82
␤-Allocryptopine 7.56 ± 1.56 4.64 (4.11–5.02) 22.89 ± 7.50 15.88 (15.15–17.06) 3.40
Protopine 8.26 ± 1.83 8.13 (6.81–8.90) 29.56 ± 10.10 21.69 (20.84–22.81) 2.66
6-Methoxyl-dihydro-chelerythrine 6.07 ± 1.26 3.63 (3.12–4.01) 13.55 ± 3.75 10.91 (10.09–11.79) 2.99
310 G.-X. Wang et al. / Veterinary Parasitology 171 (2010) 305–313

Fig. 4. Anthelmintic efficacy of six fractions against D. intermedius at 48 h.

Fig. 5. Anthelmintic efficacy of five fractions from Fr D against D. intermedius at 48 h.

EI-MS (70 eV) m/z: 370.1 (M+1), 369.0 (M+ ), 353.9 (M- 13 C
NMR (CD3 OD, TMS, 75 MHz) ı 113.4 (C-1), 146.3
CH3), 333, 178.9, 147.9. (C-2), 149.3 (C-3), 112.2 (C-4), 134.6 (C-4a), 32.3(C-
1 H NMR (CD OD, TMS, 300 MHz) ı 7.00(1H, s, H-1), 6.67
3 5), 57.4 (C-6), 50.3 (C-8), 117.5 (C-8a), 146.0 (C-9),
(1H, s, H-4), 2.95 (2H, br, H-5), 2.61 (2H, br, H-6), 3.62 (2H, 147.1 (C-10), 106.8 (C-11), 124.9 (C-12), 129.4 (C-
br, H-8), 6.69 (1H, d, J = 7.5 Hz, H-11), 6.71 (1H, d, J = 7.5 Hz, 12a), 46.1 (C-13), 195.2 (C-14), 131.2 (C-14a), 55.9
H-12), 3.89 (6H, s, –OCH3 × 2), 5.93 (2H, s, –OCH2 O-9,10), (2-OMe), 57.6 (3-OMe), 100.8 (–OCH2 O-9,10), 41.4 (N-
1.89(3H, s, N-Me). Me).

Fig. 6. Anthelmintic efficacy of two fractions from Fr Dd against D. intermedius at 48 h.

 G.-X. Wang et al. / Veterinary Parasitology 171 (2010) 305–313
Fig. 7. Anthelmintic efficacy of seven fractions from Fr E against D. intermedius at 48 h.
3.3.3. Compound 3
␤-Allocryptopine, C21 H23 NO5 (Guineudeau and
Shamma, 1982), was obtained as buff granular crystal with
m.p. 169–171 ◦ C.
EI-MS (70 eV) m/z: 369.0 (M+ ), 354.0, 283.0, 206.0, 164.0.
1 H NMR (CD OD, TMS, 300 MHz) ı 6.95 (1H, s, H-1), 6.63
3
(1H, s, H-4), 2.90 (2H, br, H-5), 2.70 (2H, br, H-6), 3.40 (2H,
br, H-8), 6.79 (1H, d, J = 9.0 Hz, H-11), 6.91 (1H, d, J = 9.0 Hz,
H-12), 3.81 (6H, s, –OCH3 × 2), 5.94 (2H, s, –OCH2 O-2,3),
1.86 (3H, s, N-Me).
13 C NMR(CD OD, TMS, 75 MHz) ı 109.2(C-1), 146.0 (C-
3 3.4. Anthelmintic efficacy of active compounds
2), 148.0 (C-3), 110.6 (C-4), 140.0 (C-4a), 32.3 (C-5), 57.5
(C-6), 50.1 (C-8), 128.5 (C-8a), 151.6 (C-9), 147.3 (C-10),
110.4 (C-11), 127.7 (C-12), 129.6 (C-12a), 46.2 (C-13), 193.0
(C-14), 132.8 (C-14a), 55.6 (9-OMe), 60.7 (10-OMe), 101.1
(–OCH2 O-2,3), 41.2 (N-Me).
3.3.4. Compound 4
Protopine, C20 H19 O5 N (Guineudeau and Shamma, 1982;
Wynne et al., 2004), was obtained as white lamellar crystal
with m.p. 205–206 ◦ C.
EI-MS (70 eV) m/z: 353 (M+ ), 338, 309, 281, 267, 209,
190, 163, 148 (100%), 134, 91.
1 H NMR (CDCl , TMS, 400 MHz): ı 6.908 (1H, s, H-1),
3 compounds
6.648 (1H, s, H-4), 2.555 (4H, br, H-5, 6), 1.911 (3H, s, N-
CH3 ), 3.591 (2H, br.s, H-8), 6.671 (1H, s, H-11), 6.683 (1H,
s, H-12), 3.915 (2H, br. s, H-13), 5.954 (2H, s, H-15), 5.928
(2H, s, H-14).
13 C NMR (100 MHz, CDCl3): ı 108.175 (C-1), 146.307 (C-
2), 147.994 (C-3), 110.460 (C-4), 136.068 (C-4a), 31.768 (C-
5), 57.755 (C-6), 41.455 (N-CH3 ), 50.791 (C-8), 117.798 (C-
8a), 145.983 (C-9), 145.877 (C-10), 106.736 (C-11), 125.027
(C-12), 128.929 (C-12a), 46.3 (C-13), 194.96 (C-14), 132.734
(C-14a), 101.200 (C-15), 100.855 (C-15).
3.3.5. Compound 5
6-Methoxyl-dihydro-chelerythrine, C22 H21 NO5 (Barry
et al., 1984; Qin et al., 2004) was obtained as heliotrope
with m.p. 203–205 ◦ C.
EI-MS (70 eV) m/z: 379 (M+ ), 348, 333.
1 H NMR (CD OD, TMS, 300 MHz) ı 7.12 (1H, s, H-1), 7.70
3
(1H, s, H-4), 5.55 (1H, s, H-6), 7.04 (1H, d, J = 9.3 Hz, H-9),
7.62 (1H, d, J = 9.3 Hz, H-10), 7.77 (1H, d, J = 8.7 Hz, H-11),
311
7.47 (1H, d, J = 8.7 Hz, H-12), 6.05 (2H, s, –OCH2 O-2,3), 3.96
(3H, s, 7-OMe), 3.92 (3H, s, 8-OMe), 3.46 (3H, s, 6-OMe),
2.76 (3H, s, N-Me).
13 C NMR(CD OD, TMS, 75 MHz) ı 104.6 (C-1), 147.9 (C-
3
2), 147.3 (C-3), 100.6 (C-4), 126.7 (C-4a), 138.3 (C-4b), 86.1
(C-6), 125.7 (C-6a), 146.6 (C-7), 152.1 (C-8), 112.9 (C-9),
118.9 (C-10), 124.8 (C-10a), 122.5 (C-10b), 120.0 (C-11),
123.4 (C-12), 131.0 (C-12a), 55.9 (8-OMe), 61.6 (7-OMe),
53.8 (6-OMe), 101.0 (–OCH2 O-2,3), 40.6 (N-Me).
Through bioassay-guided fractionation, five pure com-
pounds (sanguinarine, cryptopine ␤-allocryptopine, pro-
topine, and 6-methoxyl-dihydro-chelerythrine) obtained
from M. microcarpa were proven to be active. The
anthelmintic efficacy assay revealed that the 100% effec-
tive concentrations for the five alkaloids were 0.7, 8.0,
8.0, 16.0 and 7.0 mg l−1 , and the calculated EC50 values for
the five active compounds were 0.37, 3.31, 4.64, 8.13 and
3.63 mg l−1 , respectively (Table 1).
3.5. Acute toxicity of ethanolic extract and active
The 48 h LC50 values of goldfish exposed to the ethano-
lic extract and five active alkaloids were presented in
Table 1. By the log concentration–probit equation using
the probit procedure, the LC50 values of the ethano-
lic extract, sanguinarine, cryptopine, ␤-allocryptopine,
protopine and 6-methoxyl-dihydro-chelerythrine were
121.70, 1.13, 16.12, 15.88, 21.69, and 10.91 mg l−1 , respec-
tively. And the resulting 48 h therapeutic indices were 3.90,
3.03, 4.82, 3.40, 2.66 and 2.99, respectively.
3.6. Analysis of log P
Correlations analysis was made between the
log P and EC50 , LC50 values for the five com-
pounds. And two reasonable equations were built
with linear equation of EC50 = 0.9643 × log P + 1.314
(r2 = 0.45) and LC50 = 2.7303 × log P + 5.4958 (r2 = 0.47)
(Figs. 8 and 9). The toxicity of these compounds was well
312 G.-X. Wang et al. / Veterinary Parasitology 171 (2010) 305–313
Fig. 8. Correlations analysis between the log P and EC50 values for the five
alkaloids.
Fig. 9. Correlations analysis between the log P and LC50 values for the five
alkaloids.
correlated with their hydrophobicity expressed as
log P.
4. Discussion
The chromatographic separation of an extract and the
isolation of intended compounds can be monitored by
detection systems which are based on the physical prop-
erties of the compounds. This physicochemical approach
results in defined compounds, however, it is not possi-
ble to predict, whether these compounds contribute to the
biological activity of the extract (Jarry et al., 2006). There-
fore, the isolation of the specified active ingredients may be
monitored by assays which address the bioactivity of the
extract. In this work, five active alkaloids were isolated by
chromatographic separation and used in combination with
in vivo anthelmintic assay.
In the present study, in order to make full evaluation
of the aerial parts of M. microcarpa, five different solvents
with an increasing polarity (i.e., petroleum ether, ethyl
acetate, chloroform, acetone and ethanol) were applied
for the extraction of this plant. All the extracts were
tested for anthelmintic activity against D. intermedius.
As the ethanolic extract exhibited the best anthelmintic
activity, it was purified using various chromatograph tech-
niques to yield five alkaloids: sanguinarine 1, cryptopine 2,
␤-allocryptopine 3, protopine 4, and 6-methoxyl-dihydro-
chelerythrine 5, implying that the active constituents are
polar in nature. The other four extracts were not subjected
for the further analysis because they showed less activity
compared with ethanolic extract. Additionally, ethanol was
easier to penetrate the cellular membrane to extract the
intracellular ingredients.
The anthelmintic activity of the five alkaloids obtained
in this work and ethanolic extract were assayed on D.
intermedius (Table 1). Of these, sanguinarine was the
most potent compound against D. intermedius with EC50
value of 0.37 mg l−1 . Cryptopine, ␤-allocryptopine, and 6-
methoxyl-dihydro-chelerythrine showed a similar activity
(3.31, 4.64, 3.63 mg l−1 , respectively). Protopine had the
lowest activity with EC50 value of 8.13 mg l−1 . All of the
five isolated compounds showed much better activity than
the ethanolic extract (EC50 = 31.21 mg l−1 ), indicating that
they may or partly responsible for the anthelmintic activ-
ity of the ethanolic extract of M. microcarpa. To the best
of our knowledge, this is the first report concerning the
anthelmintic activity of the five alkaloids 1–5. Also this is
the first bioassay-guided fractionation work to obtain the
anthelmintic compounds from the aerial part of M. micro-
carpa; the present study extended the general knowledge
about the anthelmintic activity of the alkaloids. Addition-
ally, cryptopine was first isolated form genus of Macleaya
(Rahman et al., 1995; Sousek et al., 1999).
Several studies on the bioactivity of the alkaloids from
the genus of Macleaya have been reported. Sanguinarine,
a novel compound, showed wide bioactivities including
antibacterial (Mitscher et al., 1978; Godowski, 1989), anti-
carcinogenic (Ahsan et al., 2007; Chang et al., 2007) and
molluscicidal property (Singh and Singh, 1999). In this
work, it had been found to exhibit a 100% efficacy at the
concentration of 0.7 mg l−1 which is in agreement with
our previous study (Wang et al., 2007) and more effec-
tive than mebendazole (1.5 mg l−1 ) (Wang et al., 2008),
which is widely used in practice. Allocryptopine had
been reported with antifungal (Morteza-Semnani et al.,
2003) and nematocidal activity (Satou et al., 2002a,b).
Protopine was identified as an effective nematocidal
and antifungal agent (Satou et al., 2002b; Morteza-
Semnani et al., 2003) and 6-methoxy-dihydro-chelerythine
exhibited a significant anticarcinogenic property (Yin et
al., 2005). The five obtained active alkaloids included
benzo[c]phenanthridine alkaloid (sanguinarine and 6-
methoxy-dihydrosanguinarine) and isoquinoline alkaloid
(cryptopine, allocryptopine, and protopine). As can be
seen from the anthelmintic activity of the five alkaloids,
benzo[c]phenanthridine alkaloid, sanguinarine showed
significant higher anthelmintic efficacy than the isoquino-
line alkaloids, which might be due to the differential
skeleton of alkaloid and the substituted groups in certain
position. The sanguinarine is amongst the most effective
component in this study and it was reported to have the
ability to cause DNA single and double strand breaks result-
ing in DNA damage (Matkar et al., 2008), which may be
responsible for the anthelmintic activity, but the detailed
mechanism of action need to be further elucidated.
As we know, the hydrophobicity of compounds plays a
significant role in numerous biological responses. log P can
well describe the bioavailability of a chemical to organism,
reflecting the ability of a compound to form non-covalent
 G.-X. Wang et al. / Veterinary Parasitology 171 (2010) 305–313
interactions with its environment, or to permeate the phase
interfaces. Generally, larger log P indicates a stronger abil-
ity of the chemical to permeate the cell membrane of an
organism and, therefore, much more easily interact with
its target towards the organism. The correlation analysis for
log P and the compound activities in this work also clearly
indicate that the anthelmintic and lethal activity of the five
alkaloids could be significantly affected by their hydropho-
bic properties.
In conclusion, the results of the present study provide
a significant basis for use of the ethanolic extract from
the aerial part of M. microcarpa for the treatment of D.
intermedius in goldfish. The extract as well as the isolated
compounds found to be active in this study could be useful
for the development of new anthelmintic agents, especially
sanguinarine. However, further anthelmintic efficacy has to
be evaluated under field conditions, and toxicity test should
be extended to other fish species. Further pharmacological
studies toward these isolated compounds are needed for
the development of these compounds as new antiparasitic
agents.
Acknowledgements
This research was supported by key project program of
Xi’an City (GG06114) and key project of Northwest A&F
University (07ZR006). We thank Prof. X. Tian in Lanzhou
University for the chemical structure identification and Ms
Elizabeth Mary Anderson an English teacher at our univer-
sity for reviewing the paper.
References
Abbott, W.S., 1925. A method of computing the effectiveness of an insec-
ticide. J. Econ. Entomol. 18, 265–267.
Ahsan, H., Reagan-Shaw, S., Breur, J., Ahmad, N., 2007. Sanguinarine
induces apoptosis of human pancreatic carcinoma AsPC-1 and BxPC-
3 cells via modulations in Bcl-2 family proteins. Cancer Lett. 249,
198–208.
Barry, D.K., Moses, O.F., Maurice, S., Belkis, G., 1984. The benzophenan-
thridine alkaloids. J. Nat. Prod. 47, 1–43.
Buchmann, K., Slotved, H.C., Dana, D., 1993. Epidemiology of gill para-
site infections in Cypirnus carpio in Indonesia and possible control
methods. Aquaculture 118, 9–21.
Chang, M.C., Chan, C.P., Wang, Y.J., Lee, P.H., Chen, L.I., Tsai, Y.L., Lin,
B.R., Wang, Y.L., Jeng, J.H., 2007. Induction of necrosis and apoptosis
to KB cancer cells by sanguinarine is associated with reactive oxy-
gen species production and mitochondrial membrane depolarization.
Toxicol. Appl. Pharmacol. 128, 143–151.
Chansue, N., 2007. Effects of dried Indian almond leaf (Terminalia catappa
L.) extract on monogenean parasites in goldfish (Carassius auratus).
Vet. Med. Austria/Wien. Tierärztl. Mschr. 94, 269–273 (in Thai with
English abstract).
Chinabut, S., Lim, L.H.S., 1991. Four new species of Dactylogyrids (Mono-
genea) from Cirrhinus jullieni Sauvage, 1878 (Cyprinidae) in Thailand.
Raffles Bull. Zool. 40, 75–79.
Chinabut, S., Lim, L.H.S., 1993. Seven new species of Dactylogyrus Diesing,
1850 (Monogenea) from Puntius Hamilton (Cyprinidae) of Thailand.
Raffles Bull. Zool. 41, 47–59.
Chinabut, S., Lim, L.H.S., 1994. Five new species of Dactylogyrus
Diesing, 1850 (Monogenea) from Puntioplites protozysron (Bleeker)
(Cyprinidae) of Thailand. Raffles Bull. Zool. 42, 885–892.
Ekanem, A.P., Wang, M., Simon, J.E., Obiekezie1, A.I., Morah, F., 2004. In vivo
and in vitro activities of the seed extract of Piper guineense schum. and
thonn against skin and gill monogenean parasites of goldfish (Caras-
sius auratus auratus). Phytother. Res. 18, 793–797.
Feng, G., Zhang, J., Li, X.W., Feng, J.T., Zhang, X., 2008. Insecticidal activity
of alkaloids from Macleaya microcarpa against several species of insect
pests. Journal of Zhejiang University (Agric. Life Sci.) 34, 187–192 (in
Chinese with English abstract).
313
Fukui, H., Fujihara, Y., Kano, T., 1987. In vitro and in vivo antibacterial activ-
ities of florfenicol, a new fluorinated analog of thiamphenicol, against
fish pathogens. Fish Pathol. 22, 201–207.
Godowski, K.C., 1989. Antimicrobial action of sanguinarine. J. Clin. Dent.
1, 96–101.
Goven, B.A., Amend, D.F., 1982. Mebendazole/trichlorfon combination: a
new anthelmintic for removing monogenetic trematodes from fish. J.
Fish Biol. 20, 373–378.
Guineudeau, H., Shamma, M., 1982. The protopine alkaloids. J. Nat. Prod.
45, 237–246.
Jana, S., Eva, T., Hana, B., Hana, P., Pavel, M., 2007. HPLC quantification of
seven quaternary benzo[c]phenanthridine alkaloids in six species of
the family Papaveraceae. J. Pharm. Biomed. 44, 283–287.
Jarry, H., Spengler, B., Wuttke, W., Christoffel, V., 2006. In vitro assays for
bioactivity-guided isolation of endocrine active compounds in Vitex
agnus-castus. Maturitas 55S, S26–S36.
Kaewviyudth, S., Chinabut, S., 1999. Five new species of Dactylogyrus
(Monogenea) from cyprinid fishes in Thailand. Asian Fish. Sci. 12,
391–399.
Marshall, C.J., 1999. Use of Supaverm® for the treatment of monogenean
infestation in koi carp (Cyprinus carpio). Fish Vet. J. 4, 33–37.
Matkar, S.S., Wrischnik, L.A., Hellmann-Blumberg, U., 2008. Sanguinarine
causes DNA damage and p53-independent cell death in human colon
cancer cell lines. Chem. Biol. Interact. 172, 63–71.
Mitscher, L.A., Park, Y.H., Clark, D., Clark, G.W., Hammesfahr, P.D., Wu,
W.N., Beal, J.L., 1978. Antimicrobial agents from higher plants. An
investigation of Hunnemannia fumariaefolia pseudoalcoholates of san-
guinarine and chelerythrine. Lloydia (J. Nat. Prod.) 41, 145–150.
Morteza-Semnani, K., Amin, G., Shidfar, M.R., Hadizadeh, H., Shafiee, A.,
2003. Antifungal activity of the methanolic extract and alkaloids of
Glaucium oxylobum. Fitoterapia 74, 493–496.
Osbourn, A.E., Lanzotti, V. (Eds.), 2009. Plant-derived Natural Products.
Synthesis, Function, and Application. Springer, Verlag, Preface.
Perez Gutierrez, R.M., Vargas Solis, R., Diaz Gutierrez, G., Martinez-
Martinez, F.J., 2002. Identification of benzophenanthridine alkaloids
from Bocconia arborea by gas chromatography–mass spectrometry.
Phytochem. Anal. 13, 177–180.
Qin, H.L., Wang, P., Li, Z.H., 2004. The establishment of the control sub-
stance and 1 H nuclear magnetic resonance fingerprint of Macleaya
microcarpa (Maxim.) Fedde Chinese. J. Anal. Chem. 32, 1165–1170.
Rahman, A.U., Ahmad, S., Bhatti, M.K., Choudhary, M.I., 1995. Alkaloidal
constituents of Fumaria indica. Phytochemistry 40, 593–596.
Řehulková, E., Gelnar, M., 2006. Three new species of Dactylogyrus Diesing,
1850 (Monogenea: Dactylogyridae) from the gills of the bala shark-
minnow Balantiocheilos melanopterus (Cyprinidae) from Thailand.
Syst. Parasitol. 64, 215–223.
Satou, T., Akao, N., Matsuhashi, R., Koike, K., Fujita, K., Nikaido, T., 2002a.
Inhibitory effect of isoquinoline alkaloids on movement of second-
stage larvae of Toxocara canis. Biol. Pharm. Bull. 25, 1651–1654.
Satou, T., Koga, M., Matsuhashi, R., Koike, K., Tada, I., Nikaido, T., 2002b.
Assay of nematocidal activity of isoquinoline alkaloids using third-
stage larvae of Strongyloides ratti and S. venezuelensis. Vet. Parasitol.
104, 131–138.
Schmahl, G., 1993. Treatment of fish parasites. 10. Effects of a new tri-
azine derivative HOE 092V, on Monogenea: a light and transmission
electron microscopy study. Parasitol. Res. 79, 559–566.
Schmahl, G., Mehlhorn, H., 1985. Treatment of fish parasites. 1. Praziquan-
tel effective against Monogenea (Dactylogyrus vastator, Dactylogyrus
extensus, Diplozoon paradoxum). Z. Parasitenkd. 71, 727–737.
Singh, S., Singh, D.K., 1999. Molluscicidal activity of Abrus Precatorius Linn.
and Argemone Mexicana Linn. Chemosphere 38, 3319–3328.
Sousek, J., Guedon, D., Adam, T., Bochorakova, H., Taborska, E., Valka, I.,
Simanek, V., 1999. Alkaloids and organic acids content of eight Fumaria
species. Phytochem. Anal. 10, 6–11.
Wang, G.X., Zhou, Z., Cheng, C., Yao, J.Y., Yang, Z.W., 2008. Osthol and
isopimpinellin from Fructus cnidii for the control of Dactylogyrus inter-
medius in Carassius auratus. Vet. Parasitol. 158, 144–151.
Wang, G.X., Wang, J.F., Yuan, J.L., Shen, Y.H., Zheng, W., Li, L., 2007. Activ-
ity of sanguinarine from Macleaya cordata to Dactylogyrus and six
pathogenic bacteria in aquaculture. Acta Bot. Boreal. Occident. Sin.
27, 1650–1655 (in Chinese with English abstract).
Wikipedia, 2010. The Free Encyclopedia. http://en.wikipedia.org/wiki/
Therapeutic index.
Wynne, P.M., Vine, J.H., Amiet, R.G., 2004. Protopine alkaloids in horse
urine. J. Chromatogr. B 811, 85–91.
Yin, H.Y., Kim, Y.H., Moon, C.K., Lee, B.H., 2005. Reactive oxygen
species-mediated induction of apoptosis by a plant alkaloid 6-
methoxydihydrosanguinarine in HepG2 cells. Biochem. Pharmacol.
70, 242–248.