healthmedicinet

Plant material collection

Aerial parts (leaves and stems) of Polygonum hydropiper L were collected from the Santosh, Tangail district of Bangladesh in 2015. Botanical
identification was carried out at the National Herbarium, Mirpur, Bangladesh where
an accession No. 40213 has been deposited.

Preparation of the plant extract

Aerial parts were cut, air-dried and powdered in a grinding machine and stored in
an airtight container until further analyzed. Powdered dried leaves (100 g) of the
plant were extracted with ethanol (1.0 L) in flat bottom glass container, through
occasional shaking and stirring for 7 days. For the stem we also used the same amount
(100 g dissolved into 1.0 L ethanol). The whole mixture of both stem and leaf were
then filtered separately by Whatman No 1 filter paper and the filtrates were dried
at 40 °C in vacuum using a rotatory evaporator 21] to afford a blackish mass. Finally we got 4.80 g leaf and 2.27 g stem extract. All
above extraction procedures are repeated thrice and finally selected the extract randomly
for the experimental purposes. The crude extracts were then kept at 4 °C in sterile
universal bottles.

Chemicals and drugs

Glibenclamide, aspirin, and glucose were obtained from Square Pharmaceuticals Ltd.,
Bangladesh. Vincristine sulphate was collected from Beacon Pharmaceuticals Limited
Bangladesh. All other chemicals were of analytical grade.

Model animals

Swiss albino mice (male), which weighed between 28–32 g were used in the present study.
The animals were obtained from International Centre for Diarrhoeal Disease Research,
Bangladesh (ICDDR, B). The animals were acclimatized for three days prior to actual
experiments. The study was conducted following approval by the Institutional Animal
Ethical Committee of Mawlana Bhashani Science and Technology University, Tangail,
Bangladesh.

Brine shrimp cytotoxicity assay

Brine shrimp (Artemia salina) cytotoxicity assay was carried out to check the cytotoxic activity of the plant
extract. The assay was done according to Meyer’s process with some modification 22]. Simply, Brine shrimp nauplii were obtained by hatching brine shrimp eggs (O.S.I.
Brine Shrimp Egg Company Utah, U.S.A.) in artificial sea water (3.8 % sodium chloride
solution) for 48 h.

After hatching, active nauplii were collected and 10 nauplii were drawn through a
dropper and placed in each well of microtitre plate containing 200 ?l of seawater.
Then 100 ?l of plant extract (leaf) solution (extract dissolved in 20 % ethanol) was
added to make final concentration of plant extract as 10 ?g/ml, 15 ?g/ml, 20 ?g/ml
25 ?g/ml, 30 ?g/ml, 35 ?g/ml, 40 ?g/ml and 45 ?g/ml. Vincristine sulphate (Beacon
Pharmaceuticals Limited, Bangladesh) was used as positive and 20 % ethanol was used
as negative control respectively. After 24 h incubation dead and live nauplii were
counted under microscope. Each experiment was performed in three replicas. The percentage
of mortality was then determined. Lethal Concentration 50 (LC
₅₀
) value was obtained from the best-fit line by plotting concentration versus percentage
of mortality.

Oral glucose tolerance tests for evaluation of antihyperglycemic activity

Oral glucose tolerance tests were carried out as per the procedure previously described
by Joy and Kuttan (1999) 23] with minor modifications. Briefly, fasted mice were grouped into seven groups of
five mice each. The various groups received different treatments such as group 1 received
vehicle (1 % Tween 80 in water, 10 ml/kg body weight) and served as control, group
2 received standard drug (glibenclamide, 10 mg/kg body weight) and groups 3–6 received
extract (EEPHL) at doses of 50, 100, 200 and 400 mg per kg body weight. For the stem
extract (EEPHS) we only used 400 mg per kg body weight and that was treated for group
seven. All substances were orally administered. Following a period of two hours, all
mice were orally administered 2 g glucose/kg of body weight. Blood samples were collected
two hours after the glucose administration through puncturing heart.

Blood glucose levels were measured by standard glucose oxidase method 24]. The percent lowering of blood glucose levels were calculated according to the formula
described below.

Where We and Wc represents the blood glucose concentration in glibenclamide, EEPHL or EEPHS administered
mice (Groups 2–7), and control mice (Group 1), respectively.

Antinociceptive activity evaluation through abdominal writhing test

Antinociceptive activity of EEPHL was examined as previously described 25]. Briefly, mice were divided into seven groups of five mice each. Group 1 served as
control and was administered vehicle only. Groups 2 and 3 were orally administered
the standard non-narcotic analgesic drug aspirin at doses of 200 and 400 mg per kg
body weight, respectively. Groups 4–7 were administered EEPHL at doses of 50, 100,
200 and 400 mg per kg body weight, respectively. Following a period of 60 minutes
after oral administration of standard drug or EEPHL, all mice were intraperitoneally
injected with 1 % acetic acid at a dose of 10 ml per kg body weight. A period of 5 min
was given to each animal to ensure onset of chemically induced irritation of acetic
acid, following the period, the number of abdominal writhings was counted for 10 min.
The percent inhibitions of abdominal writhings were calculated according to the formula
given below.

Where We and Wc represents the number of writhings in aspirin or EEPHL administered mice (Groups
2–7), and control mice (Group 1), respectively.

Statistical analysis

Experimental values are expressed as mean?±?SEM. Independent Sample t-test was carried
out for statistical comparison. Statistical significance was considered to be indicated
by a p value??0.05 in all cases.