Bulbul et al.

, International Current Pharmaceutical Journal, February 2013, 2(3): 57-62 International Current
http://www.icpjonline.com/documents/Vol2Issue3/04.pdf
Pharmaceutical Journal

ORIGINAL RESEARCH ARTICLE OPEN ACCESS

Phytochemical and pharmacological evaluations of Polygonum

lapathifolium stem extract for anthelmintic and antiemetic activity
*Latifa Bulbul1, Somen Mojumder Sushanta1, Md. Jahir Uddin2, Shahnaj Tanni3
Department of Pharmacy, Noakhali Science and Technology University, Noakhali, Bangladesh

ABSTRACT
The present study is an attempt to preliminary phytochemical investigation, anthelmintic and anti-emetic activity
studies on methanolic extract of Polygonum lapathifolium stems. The phytochemical screening shows the presence of
phytosterols, diterpens, amino acid & protein, alkaloid and flavonoids, those are responsible for antiviral, antibac-
terial, antiallergic, antihypertensive, antiarrythmic, hepatoprotective, anti-inflammatory effects in mammals. In
Anthelmintic activity test (using Pheretima posthuma model) five concentrations (20, 40, 60, 80 and 100 mg/ml in
distilled water) of extracts were used which involved the determination of time of paralysis (vermifuge) and time of
death (vermicidal activity) of the worms, where the concentration of 60, 80 &100 mg/ml showed a significant
anthelmintic activity. Observations were comparable with the standard drug at concentration of 10mg/ml of piperi-
zine citrate. On the other hand, in anti-emetic test, emesis was induced by the oral administration of copper sulphate
50mg/kg body weight to four days age of young chicks using chick emesis model. The antiemetic activity was
determined by calculating the mean decrease in number of retching in contrast with those of control disorders. The
extracts (150 mg /kg orally) showed statistically significant antiemetic effect (90.45% Inhibition) compared with
reference drug metoclopramide (50mg/kg intraperitoneally) which showed 82.48% Inhibition. From these observa-
tions, it was concluded that stems extracts have potential anthelmintic and anti-emetic properties.
The plant may further be explored for its various pharmacological activities.

Key Words: Polygonum lapathifolium, Pheretima posthuma, piperizine citrate, chick, metoclopramide, copper sulphate.

INTRODUCTION
INTRODUCTION ics are typically used to treat motion sickness and
The present study concentrates on the evaluation of the side effects of opioid analgesics, general anaes-
phytochemical screening, anthelmintic and anti- thetics, and chemotherapy directed against cancer
emetic activity of Polygonum lapathifolium stems (Fake et al., 2004; Sontakke and Thawani, 2003).
extract. Helminthic infection is most prevalent
disease in developing countries especially in P. lapathifolium (s.l.), generally known as Knotweed
Bangladesh. Mismanagement of anthelmintics, or belongs to the family Polygonaceae, is 2'-5' tall
development of resistance to various anthelmintic annual herb (Choudhary et al., 2011; Freckmann and
compounds increase its toxicity problems (Akhtar et Bowers, 2012). The plant grows in swampy areas,
al., 2000). Moreover synthetic drugs used in helmin- roadsides, floodplains andwaste places (Anjen et al.,
thiasis treatment have some potential side effects. 2003). The round stems are hairless and somewhat
Anthelmintics from natural sources could play a key swollen near the base of the leaves. The peduncles
role in the treatment of these parasite infections. and pedicels often seem glandular. The alternate
Hence there is an increasing demand towards leaves are up to 2'-10' long and broader than those
natural anthelmintics (Kumar et al., 2010). Antiemet- of the preceding. They are lanceolate, glabrous or
slightly pubescent, and have smooth margins. At
*Corresponding Author: the base of each petiole of the leaves, there is a
Latifa Bulbul, Lecturer membraneous sheath (ocrea) that wraps around the
Department of Pharmacy
stem (Britton and Brown, 1993). The upper stems
Noakhali Science and Technology University
Sonapur-3814, Noakhali, Bangladesh terminate in spike-like racemes of flowers. The small
E-mail: [email protected] flowers are densely crowded together along the
Contact No.: +880 1741 676 362

© 2013 Bulbul et al.; licensee Saki Publishing Club. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by-nd/3.0/), which permits unrestricted use (including commercial use), distribution and
reproduction of the work in any medium, provided the original work is properly cited and remain unaltered.
length of the raceme. They are usually white or fresh condition. It was sun-dried and then dried in
greenish white and less often light pink. Each flower an oven at reduced temperature (not more than
is about 1/8" long, consisting of 5 sepals and no 50°C) to make it suitable for grinding. The coarse
petals. Because the flowers usually don't open fully, powder was then stored in air-tight container with
the inner sepals are often difficult to observe. Each marking for identification and kept in cool, dark
seed is dark brown or black, rather flat and oval in and dry place for future use.
shape, and up to 2 mm. across having a smooth
shiny surface. The root system is shallow and Extraction of the plant material
fibrous (Peng, 1996). The genus primarily grows in The dried and ground plant powder of P. lapathifo-
northern temperate regions including Bangladesh, lium stems (350 gm) were soaked in 1.3liter
India, Britain, S. Africa (Choudhary et al., 2011; methanol. Plant powders were kept in separate
Freckmann and Bowers, 2012). The whole plant has desiccators at room temperature with occasional
antiseptic and astringent property. An infusion of stirring and shaking for 15 days. The extract was
root has been used in the treatment of stomach then filtered through filter-cloth. The filtrate was
complaints and fevers. The plant is also applied kept to dry in fresh and clean air to afford a green-
externally on burns. Young leaves and seeds are ish mass of biological investigation.
used as raw or cooked form. The plant produces a
soft white mass which is used for bathing and Worm collection and authentication
washing clothes (Freckmann and Bowers, 2012). Earthworms, Pheretima posthuma (Annelida), were
collected from moist soil at Noakhali Science and
The family Polygonaceae consists of several impor- Technology University, Noakhali Dhaka and
tant medicinal plants with wide range of biological washed with normal saline to remove soil and fecal
activities and interesting phytochemical constitu- matter. Earthworms were identified by Fisheries
ents. Various plants of Polygonaceae are used in the and Marin Science Dept, Noakhali Science and
management of GI complication and helminthiasis Technology University. The earthworms of 4-6 cm
treatment in traditional medicine (Hameed et al., in length and 0.2-0.3 cm in width were used for the
2008; Hussain et al., 2010). The selection of plant P. experimental protocol.
lapathifolium was based on its availability, therapeu-
tic value and the degree of research work, which is Animals
not done mostly in earlier. Keeping in mind about Young male chicks, 2- 4 days of age, weighing from
the adverse effects of synthetic drugs available in 32-52 gm were obtained from a poultry local store.
the market, P. lapathifolium extracts were used for After 24 hrs fasting, the antiemetic activity was
the screening of different pharmacological activities evaluated. All chicks were kept under laboratory
and active constituents present in the extract. conditions at room temperature with 12h light and
dark cycles. All animal experiments were carried
out in accordance with the acts of the Animal
MATERIALSAND
MATERIALS ANDMETHODS
METHODS Ethical Committee of NSTU Research Cell, Noakhali
Collection and identification Science and Technology University.
Plant sample of P. lapathifolium were collected from
Noakhali Science and Technology University Chemicals
campus, Sonapur, Noakhali, in September 2012. The Piperazine Citrate was purchased from GlaxoS-
plant was identified by the expert of Bangladesh mithKline (BD) Limited. Copper sulfate was
National Herbarium, Mirpur, Dhaka, Bangladesh purchased from Scharlau Chem-ie S.A. Barcelona,
(Identification number-37924). Spain. Metoclopramide hydrochloride was pur-
chased from. Dimethyl sulfoxide (DMSO), Polyoxy-
Preparation of the plant materials ethylene sorbitan monooleate (Tween 80) and
The stems of the plant were collected and washed methanol were purchased from Merck, Darm-stadt,
thoroughly with water several times. During Germany. Acetic anhydride, Sulphuric acid, lead
collection any type of adulteration was strictly acetate, Nitric acid, Copper acetate were also
prohibited. The steams of the plant were collected in purchased from Merck, Darm-stadt, Germany. All

58
other reagents were procured from Sigma Chemi- physiological similarity with intestinal roundworm
cals limited. parasites of human beings and they are belonging to
same group of Annelida. The earthworms were
Phytochemical Screening divided into different groups with equal size and
Preliminary phytochemical study was screened for each group containing six worms. 60 ml formula-
presence of alkaloid, phenols, phytosterols, Sapo- tions containing five different concentrations of
nins, proteins and aminoacids, flavonoids and methanolic extract of P. lapathifolium (20, 40, 60, 80
diterpenes where phenols and saponins are absent and 100 mg/ml in distilled water) were prepared.
in extracts. These were identified by characteristic Piperazine citrate (10mg/ml) was used as reference
colour changes using standard procedures (Hosa- standard while saline water served as a control. All
hally et al., 2012; Nasrin, 2013; Trease and Evans, the test solution and standard solution were
1989). prepared freshly before starting experiments. The
time for paralysis was noted when no movement of
Detection of alkaloids any sort could be observed except when the worms
Hager’s Test: Extracts were dissolved individually were shaken vigorously. The times of death of the
in dilute Hydrochloric acid and the solutions were worms were recorded after ascertaining that worms
filtered. Filtrates were treated with Hager’s reagent neither moved when shaken vigorously or when
(saturated picric acid solution). Presence of alkaloids dipped in warm water (50°C).
was confirmed by the formation of yellow colored
precipitate. Antiemetic activity
The 4 days old chicks were divided into four groups
Detection of phytosterols of five chicks each and each chick was kept in a
Libermann Burchard’s test: Extracts were treated large beaker at 25°C for 10 minutes. The antiemetic
with chloroform and filtered. The filtrates were effect was determined by calculating the mean
treated with few drops of acetic anhydride, boiled decrease in number of retching following the
and cooled. Conc. Sulphuric acid was added. protocols of Akita et al. (1998). The extracts of P.
Formation of brown ring at the junction indicates lapathifolium stems were dissolved in 0.9% saline
the presence of phytosterols. containing 5% DMSO and 1% Tween 80 and
administered at a dose of 150mg/kg orally and
Detection of flavonoids volume of 10ml/kg to the test animal on the basis of
Lead acetate test: Extracts were treated with 4-5 their body weights. Control group received only
drops of lead acetate solution. Formation of yellow saline 0.9%. After 10 minutes copper sulphate was
color precipitate indicates the presence of flavonoids. administered orally at 50mg/kg, then the number of
retching was observed during next ten minutes.
Detection of proteins and aminoacids Metoclopramide was used as a standard drug (50
Xanthoproteic test: The extracts were treated with 4- mg per kg body weight intraperitoneally. The
5 drops of conc. Nitric acid. Formation of yellow antiemetic effect was assessed as the decrease in
color indicates the presence of proteins. number of retches in the treated group in contrast to
the control. The inhibition (%) was calculated as
Detection of diterpenes follows:
Copper acetate test: Extracts were dissolved in 𝐴−𝐵
Inhibition (%) = × 100
water and treated with 3-4 drops of copper acetate 𝐴
solution. Formation of emerald green color indicates where A is the control frequency of retching and B is
the presence of diterpenes. the frequency of retching of the treated group.

Anthelminic activity Statistical analysis

The anthelmintic assay was carried as per the In case of anthelmintic activity test, the experimental
method of Ajaiyeoba et al. (2001) with minor data were calculated as were calculated as Mean ±
modifications. In this experiment, Pheretima posthu- SEM, evaluated by unpaired one way ANOVA.
ma were used because of its anatomical and Test values of P < 0.01 were considered statistically
significant. All numerical data are expressed as the

59
Table 1: Anthelmintic activity of methanolic extracts of P. lapathifolium stems.

Concentration (mg/ml) Time taken for paralysis Time taken for Death
Groups
Piperazine citrate Methanolic extract in min. (Mean±S.E.M) in min. (Mean±S.E.M)
Control - - - -
Reference standard 10 - 9.33±0.33 36.00±1.15
Group 1 - 20 19.33±1.45 38.00±1.73
Group 2 - 40 17.33±2.18 29.66±0.33
Group 3 - 60 9.00 ±1.45 23.66±2.66
Group 4 - 80 5.66±1.15 20.33±0.66
Group 5 - 100 3.67±1.02 15.04±1.64

mean ± standard error of mean (SEM). In case of anti- characterization of the active components to establish
emetic test, statistical analysis was carried out using an effective drug resource scientifically.
student’s t-test and differences between means were
considered to be significant when p < 0.05. Antiemetic activity
Result of the antiemetic activity of methanolic
extracts of P. lapathifolium stem is given in table 2.
RESULTSAND
RESULTS ANDDISCUSSION
DISCUSSION After administration of a dose of 50mg/kg BW
Phytochemical screening Metoclopramide and the extracts of stems, the
Preliminary phytochemical screening of the extract numbers of retches were reduced. The group of
revealed the presence of flavanoid, protein & chicks treated with Metoclopramide was found to
aminoacids, alkaloid, diterpenes and phytosterols have 11 retches as compared to the 62 retches of
compounds. control group, thus Metoclopramide reduced the
retches by 82.48%. The chicks treated with steams
Anthelmintic activity extract was found to have 6 retches and inhibited
Crude methanolic extracts of stem of inhibited the retches up to 90.45%. Therefore, the extract of
earthworms in a dose-dependent manner. In case of Steams inhibited emesis to an extant greater than
stem extract, the paralysis time at different concentra- Metoclopramide at 50 mg/kg (Figure 2). On the basis
tions, including 20 mg/ml, 40 mg/ml, 60 mg/ml, 80 of these results it may be concluded that extracts of
mg/ml and was 19.33,17.33, 9.00, 5.66, 3.67 minutes stems have anti-emetic potential and are compara-
respectively, whereas death time was 36, 38, 29.66, ble with that of Metoclopramide (the reference
23.66, 20.33 and 15.04 minutes respectively. The drug). Although the results are significant but the
paralysis and dead time for standard piperazine mode of action is not known.
citrate at a concentration of 10 mg/ml were 9.33 and
36.15 minutes, respectively (Table 1 and Figure 1). However, as the oral copper sulphate induces emesis
by peripheral action (Hosseinzadeh et al., 2008), and
From the phytochemical detectection in the metha- the extracts were able to effectively prevent its effect,
nolic extract acting as a major role to possess it could be implied that these extracts have a peri-
anthelmintic properties. From Phytochemical
investigation, the presence of flavon content which
may be sole responsible for activity (Paria et al., 2012) Table 2: Antiemetic activities of methanolic extracts of
From the table 1, mainly methanolic stems extract P. lapathifolium Stems.
containing 60, 80 and 100 mg/ml showed a significant
No. of retches
anthelmintic activity comparable with the standard Drug / dose % Inhibition
(Mean±S.E.M)
drug at concentration of 10mg/ml of piperizine citrate Control (10ml/kg) 62.00±1.58 -
produces dose-dependent paralysis ranging from loss Metoclopramide
of motility to loss of response to external stimuli, (50 mg/kg) 11±1.89 82.48%
which gradually progressed to death. Further P. lapathifolium Stems
attention has to be carried out for isolation and (150mg/kg) 6±2.85 90.45%
*S.E.M=Standard Error of Mean

60
 40 38.0
36.0 100
ParalysisTime
29.7 Death Time 90
30 80
Time (minute)

23.7
70 62

No. of retches
19.3 20.3
20 17.3
15.4 60
11.7 50
9.3
10 5.7 40
3.7
30
0 20 11
10
6
0
Control Metoclopramide Polygonum
L.Stem
Group Group

Figure 1: Paralysis Time and Death Time of P. posthuma Figure 2: Number of retches for control, standard and
for standard and stems extract. methanolic extracts of P. lapathifolium Stems.

pheral anti-emetic action. This study also justifies the P. lapathifolium stems extract and thus provides
traditional use of P. lapathifolium in G.I.T complaints. scientific basis for its use in folk medicine for the
From chemical point of view, stem of P. lapathifolium management of GI complication. Further studies are
contain alkaloids and showed highest activity as required to determine the exact mode of action and
compared to standard. Therefore, it may be said that the active compounds responsible for this effect.
alkaloidal contents may play some role in anti-emetic
effect (Hasan et al., 2012).
CONCLUSION
CONCLUSION
Chemotherapeutic agents or their metabolites can The results of this study suggest that the methanolic
directly activate the medullary chemoreceptor extracts of stem of P. lapathifolium have shown active
trigger zone or vomiting center or act peripherally in vitro anthelmintic effect against adult stage of
by causing cell damage in the gastro-intestinal tract Pheretima posthuma. On the other hand, the stem
and releasing serotonin from enterochromaffin cells extracts have protective effects against copper
of the small intestinal mucosa. The released seroto- sulfate induced–retching in young chickens,
nin activates 5-HT receptors on vagal and possibly by peripheral and central mechanisms.
splanchnic afferent fibers, which then carry sensory Further studies are required to recognize active
signals to the medulla, leading to the emetic constituents and the molecular mechanism of
response (Hosseinzadeh et al., 2008; Sontakke and anthelmintic and antiemetic activities.
Thawani, 2003). Metoclopramide, which has already
been known to elicit antiemetic activity through
acceleration of gastrointestinal tract movement ACKNOWLEDGEMENT
ACKNOWLEDGMENTS
(Ahmed et al., 2011), was found to be less effective The authors are thankful to all faculty members and
than stem extracts. The ability of extracts to signifi- the technical and non technical staffs of the Depart-
cantly attenuate the effect of copper sulphate- ment of Pharmacy, Noakhali Science and
induced emesis in chicks may in part be due to its Technology University, Bangladesh for their kind
effect on the visceral afferent sympathetic fibre in co-operation.
the GIT and possibly through agonistic effect at the
inhibitory receptor sites such as dopamine and
GABA in central nervous system (Zia-Ul-Haq et al.,
2012). The observed antiemetic activity of P. lapathifo-
lium stems extract may be attributed to its alkaloid
and terpenes constituents. Until now, no other
research papers are found to the antiemetic activity of

61
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