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PHCOG MAG.: Research Article
Effects of Millettia pachycarpa on the trace metals and
tegumental enzymes of Raillietina echinobothrida
Kholhring Lalchhandama 1*, Bishnupada Roy 2 and Biman Kumar Dutta3
1Department of Zoology, Pachhunga University College, Mizoram University, Aizawl 796 001, Mizoram, India.
2 Department of Zoology, North Eastern Hill University, Shillong 793 022, Meghalaya, India.
3 Department of Ecology and Environmental Science, Assam University, Silchar 788 011, Assam, India.
* Author for Correspondence: madama@bsnl.in
ABSTRACT
The root bark extract of Millettia pachycarpa Bentham is known to certain Mizo tribes of north-east India as a
curative to gastrointestinal infestations. An in vitro treatment of the poultry gastrointestinal cestode Raillietina
echinobothrida Megnin with the crude ethanolic extract of the plant part reportedly indicate remarkable
cestocidal effects on the survival and morphological structures of the worm. In an attempt to further explore the
anthelmintic activity of the plant and with a view to understand the primary mode of action, the parasites were
exposed to 20 mg mL-1 of the different extracts (viz ethanol, methanol and acetone) of M. pachycarpa root bark till
they reached paralytic state. Similar dose of albendazole was used as a reference standard drug. The levels of the
vital trace elements such as calcium, magnesium, potassium and sodium, and the activities of tegumental enzymes
such as acid phosphatase (AcPase) and alkaline phosphatase (AlkPase) of the worms were assessed. The plant
extracts clearly indicated significant reduction (P < 0.05) in the levels of the trace metals compared to control
worms. The enzymatic activities of AcPase and AlkPase were also significantly inhibited. Among the three extracts,
the ethanol extract was the most potent in causing these biochemical alterations, and the effects were
comparable to those of albendazole. The overall results show that the plant extracts exert anthelmintic activity by
acting trans-tegumentally, inhibiting the major enzymes to induce tegumental damages, and depleting the trace
metals to bring about flaccid paralysis and mortality of the cestodes.
KEY WORDS - Acid phosphatase (AcPase); alkaline phosphatase (AlkPase); anthelmintic; Millettia pachycarpa;
Raillietina echinobothrida; trace metals.
INTRODUCTION
Millettia pachycarpa Bentham (family Fabaceae) is a
leguminous perennial climbing tree endemic to south-
east Asia, where it is acclaimed with a wide range of
medicinal applications in various traditional practices.
The juicy extracts of the root bark and leaf are
commonly used in the treatment of infertility, and as a
blood tonic and anticancer agent (1). A large number
of bioactvie compounds have been identified from it,
of which isoflavones such as erysenegalensein E,
isoerysenegalensein E, 6,8-diprenylorobol, millewanins
G and H, furowanin A and B, and auriculasin were all
demonstrated to have antiestrogenic activity (2–4),
confirming the anticancer potentials of the plant.
Following the ethnomedicinal usage of the Mizo tribes
of north-east India, we have earlier shown that the
crude ethanolic extract of the root bark exhibited a
potent anthelmintic activity against mature Raillietina
echinobothrida Megnin, the intestinal cestode of
domestic fowl (5). The plant extract caused dose-
dependent paralysis and mortality, associated with
inexorable degenerative effects on the tegument all
over the body surface of the cestode. The present
study is an attempt to disclose the probable primary
route of action of the plant extracts by investigating
the biochemical changes in the helminth physiology
such as the activities of the vital tegumental enzymes
and trace metals. Trace metals have been posited to
play a significant role in the physiology, growth and
development, the sequestration of free radicals and in
the cellular antioxidant defense system, metabolism,
host-parasite interactions and immuno-tolerance of
helminth parasites (6).
Despite successful experimental validation of a large
number of medicinal plants traditionally used in
different parts of the world as anthelmintic agents
against a variety of helminth parasites, the global
crisis of helminthic infestation is far from being
ameliorated (7–9). This is primarily due to the fact that
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though medicinal plants exhibit anthelmintic
properties, their chemical nature, safety and, above
all, mode of action remain poorly understood.
Particularly when applied on large-scale clinical trials,
plant extracts can exert undesirably serious
consequences. Several lines of recent evidences have
posited that even some highly acclaimed medicinal
plants and their products are highly toxic to the host
and without any appreciable practicable value in
clinical and veterinary applications (10–14). Thus,
there still remains a veritable hindrance for medicinal
plants at large to find their way as subtle alternatives
to pharmaceutical drugs.
Moreover, like the commercial drugs themselves,
majority of anthelmintic plants are helminth specific,
showing activity against a particular species or group
of the parasites (15, 16). Thus the choice of available
medicinal plant is restricted with the type of
helminthic infection. Besides, plant extracts are
prepared using different solvents so that the form of
extraction can also reflect the anthelmintic efficacy
(17–19). Therefore, it is ever more crucial to
comprehend the precise mode of activity of the well-
established anthelmintic plants within the parasite
tissue, and which particular type of extract should be
sought after for each plant in order to discover the
active principle; thus the aim of the study is to assess
the changes, if any, on the levels of vital trace metals
and activity of the tegumental phosphatases of R.
echinobothrida upon treatment with the extracts of M.
pachycarpa root bark.
MATERIALS AND METHODS
Preparation of Plant Extract
Collection, authentication and preparation of the
crude extract of Millettia pachycarpa was reported
previously (5). The fresh root barks were peeled off,
thoroughly washed with deionized water, cut into
small pieces, macerated and dried in a hot air oven at
50ºC. The dried parts were pulverized to fine powder
and a pre-weighed amount was refluxed with ethanol
(100g/L) for 8 h at 60ºC, following the methods of
Tandon et al. (15) and Roy (20). Refluxing was
repeated thrice. The solution obtained was filtered
through Whatman filter paper (No. 1) and then
evaporated to complete dryness at 50ºC. The alcoholic
extract was obtained as a deep brown powder. The net
yield from such extraction was 7.1%.
A portion of the alcoholic extract was macerated with
methanol (100g/L) in a fractionating flask, with several
changes of the solvent and then vigorously mixed in a
rotary shaker for 24 hours. The resultant solution was
filtered and refluxed as before. After complete
evaporation of the solvent, solid precipitates were
obtained as the methanol extract. The total yield was
2.5%. Similarly, another portion of the alcoholic
extract was mixed with acetone to get the acetone
extract with a net yield of 1.3%. The different extracts
were refrigerated at 4ºC until further use. Similar
extractions were also attempted using ethyl acetate,
diethyl ether, chloroform and benzene, but the crude
extract was strictly insoluble in these organic solvents,
thus, no extracts were obtained.
1 hour before the actual experimental assay, 20 mg
mL-1 of the ethanol, methanol and acetone extracts
were separately prepared by dissolving them in 0.9%
neutral phosphate buffered saline (PBS, pH 7-7.3),
supplemented with 1% dimethylsulfoxide (DMSO). The
solutions were then maintained at 37 ± 1ºC in a glass-
chambered incubator.
Chemicals and Drugs
All the chemicals used were of standard analytical
grades, obtained either from Merck or S.D. Fine-
Chemicals Limited, India, except where otherwise
stated. Ethanol was supplied by Bengal Chemicals,
Kolkata, India, and the reference drug albendazole is a
product of GlaxoSmithKline Pharmaceutical Limited,
India.
In Vitro Treatments of Parasites
Native bred live local fowls (Gallus domesticus
Linnaeus) were purchased from the local abattoir in
Aizawl, Mizoram, India. They were sacrificed and on
immediate autopsy, live worms, R. echinobothrida
Megnin, were recovered from the intestines. Only the
live adult worms with more or less body length (7.4 ±
0.8 cm) were selected and collected in 0.9% PBS.
Batches of fresh worms were directly introduced to the
media containing 20 mg mL-1 of the ethanol, methanol
and acetone extracts of M. pachycarpa dissolved in PBS
with 1% DMSO. Similar treatment was performed for
albendazole at its commercial dosage (20 mg mL-1) as
a reference drug, and one group is maintained in a
medium containing only PBS with 1% DMSO as control
experiment. Each incubation medium consisted of 5
replicates.
Measurement of Trace Metals
Persistence on the motility of the worms were
observed, time taken for the onset of paralysis was
recorded, as previously described (15, 20). The onset
of paralysis was defined as complete loss of motor
activity even after physical stimulation of the worms in
culture. The parasites subjected to treatments were
collected the moment they indicated paralysis.
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Sentient worms in the control medium were directly
taken for comparison with the treated groups. They
were washed with double distilled water and quick-
dried in an incubator at 50°C. 2 g of the powdered dry
worms taken from each incubation medium was
digested in 10 ml of concentrated HNO3 overnight at
50°C. The fully digested solution was evaporated on a
hot plate at 70°C. 10 ml of deionized water was then
added and filtered through Whatman filter paper (110
mm Φ). The volume was finally made to 100 ml with
deionized water, which was used for quantitative
analysis of trace elements using an atomic absorption
spectrophotometer (model Chemito AAS-201, India) at
the absorbance wavelengths of 422.6nm for calcium,
285.2 nm for magnesium, 589.0 nm for sodium, and
766.5 nm for potassium.
Estimation of Tegumental Enzyme Activities
The acid phosphatase (AcPase; EC 3.1.3.2) and alkaline
phosphatase (AlkPase; EC 3.1.3.1) activities were
estimated using p-nitrophenol product from an enzyme
source following the method of Plummer (21) with
slight modifications in the concentration of the buffer
and substrate. For AcPase, a 10% (w/v) of the cestode
was homogenized in sodium acetate buffer using a
Potte r-Elvehjem homogenizer and centrifuged at
5,000 rpm at 4°C for 20 minutes in a cooling
centrif uge (REMI C4, India). The supernatant
obtained was used as the enzyme source for estimation
of AcPase activity. For AlkPase, a 10% (w/v) of the
cestode was homogenized in glycine buffer and
centrifuged at 5,000 rpm at 4°C for 20 minutes. The
supernatant obtained was used as the enzyme source
for estimation of AlkPase activity.
For both the enzymes, p-nitrophenyl phosphate was
used as the substrate. Incubation was carried out at 37
± 1°C and the reaction was stopped after 15 min
through addition of 0.02 N NaOH. The absorbances of
both the blank and incubated solutions were measured
at 405 nm in UV-VIS Spectrophotometer (Systronics
model 119, India). The enzyme activity was calculated
from a linear standard graph of p-nitrophenol. One unit
of AcPase or AlkPase activity was defined as that
amount which catalyzed the formation of 1 mM of p-
nitrophenol/h at 37°C.
For all the enzymatic assays, the total protein content
was estimated following the method of Lowry et al.
(22) using bovine serum albumin as the standard
protein and Folin-Ciocalteau reagent as the substrate.
Data Analysis
All data were presented as means plus or minus the
standard error (SEM) of the mean. Comparison of the
mean values between the treated and control groups
was made using unpaired Student’s t-test, and the
level of significant probability considered at P < 0.05.
RESULTS
The quantitative observations of vital trace metals in
R. echinobothrida, those in control experiment and
those treated with albendazole and the ethanol,
methanol and acetone extracts of M. pachycarpa are
shown in Table 1. The data clearly indicate that the
cestodes exposed to 20 mg mL-1 each of albendazole
and all the three extracts of M. pachycarpa resulted in
significant reduction in the concentration of vital trace
metals. The concentrations of calcium, magnesium,
sodium and potassium at the basal level of the control
worms maintained in 0.9% PBS with 1% DMSO were
296.2 ± 5.7, 953.0 ± 4.8, 435.7 ± 2.2 and 132.8 ± 1.1
µg/g dry tissue weight, respectively.
The most effective reduction was caused by
albendazole reducing the levels of the trace metals to
128.6 ± 4.7, 629.5 ± 1.2, 268.3 ± 2.8 and 87.0 ± 2.1
µg/g dry tissue weight, respectively. Of the three
extracts of the plant, ethanol appeared to exert the
most abrupt effect resulting in a decrease to 174.6 ±
3.7, 653.1 ± 3.6, 326.4 ± 2.9 and 92.3 ± 3.4 µg/g dry
tissue weight, respectively; and the least effect was
with the acetone extract. Therefore, albendazole and
all the three extracts of M. pachycarpa caused highly
significant reduction in the levels of vital trace metals
in R. echinobothrida.
The enzymatic activities of AcPase and AlkPase in R.
echinobothrida maintained as untreated control, and
those treated with albendazole and the three extracts
of M. pachycarpa are presented in Table 2. Cestodes in
control group indicated high activity of AcPase and
AlkPase, 10.3 ± 1.8/1.7 ± 0.2 and 37.6 ± 1.2/3.4 ± 0.4
total activity/specific activity, respectively. The result
also indicates that between the two enzymes, AlkPase
is the dominant tegumental enzyme in R.
echinobothrida.
The AcPase activity was decreased by 44.7% upon
treatment with 20 mg mL-1 of albendazole. The same
treatment also resulted in a 48.7% inhibition of AlkPase
activity. Among the three extracts of M. pachycarpa,
highest inhibition on the tegumental enzymes was
observed for the ethanol extract, which indicated
37.9% and 47.3% inhibition of AcPase and AlkPase
activities, respectively. Worms treated with the
methanol extract showed 35% and 43.9% reduction of
the enzymes, respectively. While the acetone extract
was noted to affect inhibition of AcPase by 31.1% and
AlkPase by 42.8%. Therefore, the three extracts of M.
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pachycarpa significantly inhibited the activities of the
tegumental enzymes in R. echinobothrida.
DISCUSSION
The presence of trace metals such as cadmium,
calcium, cobalt, copper, iron, lead, nickel,
magnesium, manganese, nickel, potassium, selenium
and zinc has been adequately reported in different
helminth parasites, ranging from trematodes (23, 24),
nematodes (24–26) to cestodes (26, 27), including R.
echinobothrida (28, 29). These trace elements were
documented to play a significant role in the
physiology, growth and development, the
sequest ration of free radicals and in the cellular
antioxidant defense system, metabolism and immuno-
tolerance of parasites (6). For instance, it has been
shown that glucose transport is coupled to sodium
cations in cestodes like H. diminuta (30). This
condition also reflects the manifold role of calcium
inside the cell, as its presence regulates the sodium
level, maintenance of inter-cellular ionic bridges,
neuro-motor functions and several other activities
within the cell (6).
Moreo ver, di fferent t race metals are attributed to
pla y critical roles in host-parasite interacti on s.
Deficiencies of iron, molybdenum, copper, and zinc in
host tissues have been associated with higher worm
burdens, as have excessive intakes of molybdenum,
iron, and copper (31). The possibility is emerging that
there may be an optimum trace element level in the
diet above which and below which the parasite is
advantaged. Moreover, there is some data to suggest
that specific trace elements may be directly toxic to
the parasite (32). Thus, it is understood that not only
is there competition for elements between the
helminths inside the gut but there is also competition
for these elements between the host and the parasites
(33).
From the present investigation, it can be recognized
that calcium and magnesium are present in high
proportion in R. echinobothrida, 296.2 and 953 µg/g
dry tissue weight, respectively, supporting the data of
Das et al. (27). Additionally, sodium and potassium
were also detected at the concentrations of 435.7 and
132.8 µg/g dry tissue weight, respectively. The
cestodes treated with albendazole as a reference drug,
and the ethanol, methanol and acetone extracts of M.
pachycarpa root bark evidently caused considerable
decrease in the physiological concentration of these
trace metals. Therefore, drastic decline in the levels
of trace metals probably lead to gradual loss of
physical and metabolic activities within the cells,
eventually resulting in paralysis, and death. The result
comprehensively conforms to that of Lalchhandama et
al. (28) on the effects of Acacia oxyphylla but with a
comparatively higher efficacy.
Anthelmintic drugs are known to enter target parasites
by either oral ingestion or by diffusion through the
external surface (34). The cuticle in nematodes or
tegument in cestodes and trematodes is metabolically
active, and morphologically specialized interface to
perform selective absorption of nutrients, secretion of
glycoproteins for immunoprotection, osmoregulation
and (insofar as it supports sense organs) sensory
perception (35, 36). Trans-cuticular or trans-
tegumental passive diffusion is, therefore, the
principal mechanism of anthelmintic entry into the
helminths (36). Consequently, it has been sufficiently
accounted that one of the hallmark effects of any
anthelmintic is destruction of the worm’s surface (37–
39).
Albendazole and other benzimidazoles are construed to
enter the cestode body by passive diffusion through
the tegument in which they bind selectively and with
high affinity to the microtubule proteins, tubulins,
causing disruption of the microtubule dynamic
equilibrium, and with that, cell lysis (35, 40). By
binding specifically to free β-tubulin, BZs inhibit the
polymerization of α- and β-tubulin molecules and the
microtubule-dependent uptake of glucose, ensuing
starvation the worms are paralyzed, killed and
expelled (41).
The occurrence of vital enzymes, viz acid
pho sphatase (Ac Pase) and alkaline phosp hatase
(AlkP ase) has been resolutely de monstrat ed in a
number of hel minth parasites, both
histo chemically and biochemically (42–45),
including R. echinoboth rida (46). These enzymes
have been unequivocally revealed to be int im at ely
ass ociated with the tegument and subtegument al
regions of cestode s and trematodes, as well as the
cut icle of nematode s ( 47–49). The present study
also revealed a comparatively higher degree of activity
of AlkPase over AcPase in control R. echinobothrida. It
has been positively demonstrated that AcPase and
AlkPase are the two vital enzymes of the tegument and
subtegumental regions in cestodes, with AlkPase as the
dominant enzyme (4 2, 44, 46). Previous investigations
had demonstrated that the enzymes are highly
abundant in those parasite compartments crucially
involved in interacting with the host (50). Due to its
abundance at the host-parasite interface and its high
activity, it is conceivable that they represent
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Table 1. Effects of albendazole and extracts of M. pachycarpa root bark on the levels of trace elements in R. echinobothrida.
Incubation medium
Concentration (µg/g dry tissue weight) of
Calcium Magnesium Sodium Potassium
Control (PBS+DMSO) 296.2 ± 5.7a 953.0 ± 4.8a 435.7 ± 2.2 a 132.8 ± 1.1 a
Albendazole (20 mg mL-1) 128.6 ±4.7a 629.5 ± 1.2a 268.3 ± 2.8a 87.0 ± 2.1a
M. pachycarpa
extract
(20 mg mL-1)
Ethanol 174.6 ± 3.7a 653.1 ± 3.6a 326.4 ± 2.9a 92.3 ± 3.4a
Methanol 211.3 ± 3.6a 682.3 ± 2.5a 315.6 ± 2.1a 103.6 ± 5.7a
Acetone 246.2 ± 2.1a 675.7 ± 5.2a 349.2 ± 1.2a 115.0 ± 3.8a
Values are expressed as mean ± SD (n = 5). a P value significant at < 0.05 in treated group compared to control group.
Table 2. Effects of albendazole and extracts of M. pachycarpa root bark on the activity of the tegumental enzymes of R.
echinobothrida.
Incubation medium
Enzyme activity (total
1
/specific
activity2) Percentage (%) decrease of
AcPase AlkPase AcPase AlkPase
Control (PBS+DMSO)
10.3
±
1.8/
1.7 ± 0.2
37.6
±
1.2/
3.4 ± 0.4
Albendazole (20 mg mL-1)
5.7
±
1.1/
1.3 ± 0.4a
19.3
±
0.8/
1.4 ± 0.5a 44.7 48.7
M. pachycarpa
extract
(20 mg mL-1)
Ethanol
6.4
±
0.7/
0.9 ± 0.2a
19.8
±
1.9/
1.8 ± 0.5a 37.9 47.3
Methanol
6.7
±
0.8/
1.0 ± 0.6a
21.1
±
0.6/
1.7 ± 0.5a 35.0 43.9
Acetone
7.1
±
0.4/
1.2 ±0.3a
21.5
±
0.4/
1.7 ± 0.4a 31.1 42.8
Values are expressed as mean ± SD (n = 5). 1 Total enzyme activity is defined as the amount of enzyme that consumes 1.0 µm
substrate/g wet wt tissue/h. 2 Specific activity expressed as unit/mg protein/h. a P value significant at < 0.05 in treated group
compared to control group.
molecules of considerable importance for parasitic
helminths, as it may be involved in the acquisition of
nutrients (43) as well as in the modulation of
phosphorylation-dependent events at the host-parasite
boundary: for instance, those interactions initiated by
host-effector cells (50).
Extract s from certain med ic inal plants, including
But ea mon os perma, Emb elia rib es, and Roltle si a
tinctoria reportedly influenced drastic decrease
in the activities of both AcPase and AlkPase in the
trematode, Paramph istomum cervi (51). The root
tuber peel extract and geniste in from Flemingia
vestita similarly caused significant reduction of
the enzymes in R. echinob ot hrida (46), and in the
fluke, Fasciolopsis bu ski ( 52), co mp arable to
those of the stand ard pharmaceuticals, prizquan-
-tel and oxyclozan ide, r espectively.
Pha rmaceutical drugs such as albendazole,
flube ndazole, isatin, hexacho lo rophene,
lev amisole, luxabendazole, mebendazole,
praziqu antel and thiab endazole reported ly induce
detectable alterations in the activities of the
tegumental enzymes in different helmint hs ( 45,
53– 58). In the present study, R. echinobothrida
exposed to albendazole and the ethanol,
methanol and acet one extracts of M. pachycarpa
were found to be significantly inh ib ited in their
AcP ase and AlkPase activities. Similar inhibitions
were observed for the cestocidal effects of A.
oxyph ylla (28). Comparable results were reported
in a human tapeworm Echinoc occus multi locularis
met acestode in which acute inhibition of AlkPase
act iv ity by 23% following tre atment wit h isatin
was observed (5 4) . Th e ce stocidal e ffects of
alb endazole on E. multilocularis, characterized by
progressive degeneration and destruction of the
tegumental tissue wa s directly associated with
AlkPase activity, indicating that the phosphatase
act iv ity is an ideal parame ter for performing first-
rou nd i n vit ro tests o n the efficacy of a large
number of antiparasitic compounds ( 45). Similar
res ult was also obtained for the bot h the AlkPase
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and AcPase activity in H. d imunita, where a
defin it e correlation between pho sphatase activity
and glucose uptake was observed (59). Thus, the
obs erve d red uction in the two tegumental
pho sphatases also might be associated with
destruction of the tegumental surface (5) and
pos sibly, inhibition or reduced uptake of glucose
of R. echinob ot hr ida leading to gradual loss of
mot or activity due to deprivation of energy
source, cul minating in to paral ys is, and
ult imately, death.
CONCLUSION
The present investigation revealed that the
anthelmintic activity of albendazole and M.
pachycarpa root bark on R. echinobothrida involved
alterations in the enzymatic activities of AlkPase and
AcPase, and levels of trace metals such as calcium,
magnesium, potassium and sodium. The study
substantially demonstrated that the extracts of M.
pachycarpa root bark caused significant inhibition of
the tegumental enzymes and reduction of vital trace
metals; and the efficacy is in the order
albendazole>ethanol extract>methanol
extract>acetone extract. This is further suggestive that
in order to pinpoint the specific principal ingredient of
the plant as an anthelmintic, the ethanol extract
would be a good choice to start with. However, the
complete cellular and molecular events underpinning
the anthelmintic activity of the plant is yet
incompletely defined from this study and need further
investigation.
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Phcog Mag. Vol 4, Issue 16, Oct-Dec, 2008
Submitted on : 24
th
January, 2008
Revised on : : 24th February, 2008
Accepted on: : 2nd March, 2008
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