Plant material collection and identification
For the investigation, the leaves of L. glutinosa were collected by the authors from Potia, Chittagong, Bangladesh in July 2012. The plant was identified and authenticated by an expert botanist of Bangladesh National Herbarium (DACB), Mirpur, Dhaka (Accession No. 38277) and a voucher specimen was submitted at the herbarium for future reference.
Extract preparation
Weighed (630 g of the dried and powdered) sample was soaked in 2200 ml of 99% methanol in clean, sterilized, and flat-bottomed glass container. Afterwards, it was sealed and maintained for 15 days accompanying occasional stirring and agitation. The complete mixture was then subjected to coarse filtration on a piece of clean, white sterilized cotton material and Whatman® filter paper. The extract was obtained by evaporation using rotary evaporator (Bibby RE-200, Sterilin Ltd., UK) at 4 rpm and 65°C temperature. It rendered a gummy concentrate of greenish color. The gummy concentrate was designated as crude extract or methanolic extract. Then the crude methanolic extract was dried by freeze drier and preserved at +4°C (yield 0.79%).The concentrated methanolic extract was partitioned by modified Kupchan method [32]and the resultant aqueous soluble partitionates i.e., n-hexane (yield approx. 19.02%), ethyl acetate (yield approx. 26.54%), and chloroform (yield approx. 6.59%) soluble fractions were used for the experimental processes.
Chemicals
All the chemicals used in this study were of analytical grade, and purchased from Sigma Chemical Co. (St. Louis, MO, USA), and Merck (Darmstadt, Germany). To the commercially available lyophilized S-Kinase™ (Streptokinase) vial (Batch no: VEH 09, Popular Pharmaceutical Ltd., Bangladesh) of 1,500,000 I.U., 5 ml 0.9% sodium chloride (NaCl) was added and mixed properly. This solution was used as a stock from which 100 μl (30,000 I.U) was used for in vitro thrombolysis assay.
Test animals
For the screening of in vivo antipyretic, analgesic, and anti-inflammatory potential of hydroalcoholic extracts of L. glutinosa leaves, young Swiss-albino mice (aged 20–25 days) of either sex, average weight 20–25 g were used. They were collected from the Animal Resources Branch of ICDDR, B (International Centre for Diarrheal Disease and Research, Bangladesh). After collection, they were kept in favorable condition for one week for adaptation and fed rodent food and water ad libitum formulated by ICDDR,B. Throughout the experiments, all animals received human care according to the criteria outlined in the ‘Guide for the Care and Use of Laboratory Animals’, 8th edition, prepared by the National Academy of Sciences and published by the National Institute of Health (US).
Thrombolytic assay
In vitro clot lysis activity of the leaves was carried out according to the method of Prasad et al.[33] with minor modifications. With ethical considerations, and aseptic precaution, 7 ml of venous blood was drawn from healthy volunteers (n = 5) having no history of smoking, taking lipid lowering drugs, oral contraceptive or anticoagulant therapy and transferred to different pre weighed sterile micro-centrifuge tube (1 ml/tube). The micro-centrifuged tubes were subjected to incubation at 37°C for 45 min. After the formation of clot, serum was completely removed from the tubes (carried out without disturbing the clot formed) and each tube having clot was again weighed to determine the weight of the clot (clot weight = weight of clot containing tube – weight of tube alone).
To each micro-centrifuge tube containing pre-weighed clot, 100 μl solution of different extracts (n-hexane, ethyl acetate, chloroform and methanolic extract), concentration 1 mg/mL, were added accordingly. As a positive control, 100 μl of streptokinase and as a negative non thrombolytic control, 100 μl of sterilized distilled water were separately added to the control tubes numbered. Then all the tubes were incubated again at 37°C for 90 min and observed for clot lysis. After incubation, the obtained fluid was removed from the tubes and they were again weighed to observe the difference in weight after clot disruption. At last, difference obtained in weight was calculated and the result was expressed as percentage of clot lysis following the underneath equation.
Analgesic activity test
In the current investigation two different methods were employed for testing the possible peripheral and central analgesic effects of L. glutinosa leaves; namely acetic acid induced writhing test and hot plate test in mice respectively.
Hot plate test
The hot plate test was performed following the method of Asongalem et al.[34]. Pain reflex in response to the thermal stimulus was measured using a Le7406 hot plate (Panlab S2, Cornella, Barcelona, Spain). Before 30 min of the test, mice were placed in ten different groups comprising six (6) mice in each group and were intragastrically treated in the following manner: group I was treated with negative control (isotonic saline solution, 0.9%), group II with positive control (ketorolac) as reference drug (10 mg/kg body weight), and groups III–X with the plant extracts at dose of 250 and 500 mg/kg body weight. Mice were placed on a 55 ± 1°C hot plate in order to obtain their response to electrical heat-induced nociceptive pain stimulus judged by the presence of behaviors such as licking of the fore and hind paws or jumping. The pain response was measured at 30 min and at every hour thereafter for 4 h. The cut-off time used to prevent skin damage was 25 sec.
Acetic acid induced writhing test
The analgesic activity of the crude methanolic extract was studied using acetic acid induced writhing model in mice [10, 35]. The animals were divided into four groups including control (Group I), positive control (Group II) and two test groups (Group III-IV). The animals of group III and IV were administered test substance at the dose of 250 and 500 mg/kg body weight respectively. Positive control group received ketorolac (standard drug) at the dose of 10 mg/kg body weight and vehicle control group was treated with 1% Tween 80 in distilled water at the dose of 10 ml/kg body weight. Test samples, standard drugs and control vehicle were administered orally 30 min before intraperitoneal administration of 0.7% acetic acid. After 15 min of time interval, the writhing (constriction of abdomen, turning of trunk and extension of hind legs) was observed on mice for 5 min.
Anti-inflammatory activity test
Carrageenan induced mice hind paw edema was used as the animal model of acute inflammation according to the method of Lanhers et al. [36]. In this experiment, the mice were divided into four groups of five animals each. Group I (control) received 2% Tween 80 in normal saline (2 ml/kg). Group II (Positive control) received 10 mg/kg body wt. of ketorolac orally. Group III and IV received 250 and 500 mg/kg body wt. of the extract orally respectively. Acute inflammation was induced in all the four groups by sub plantar injection of 0.05 ml of its suspension of Carrageenan with 2% Tween 80 in normal saline in the right Paw of the mice 30 minutes after the oral administration of the tested materials. The paw volume was measured with a micrometer screw gauze at 1, 2, 3 and 4 h after the administration of the drug and the extract. The percentage inhibition of inflammatory effect of the extract was calculated using the following expression:
Where Vc is the average degree of inflammation by the control group and Vt is the average degree of inflammation by the test group.
Antipyretic activity test
The antipyretic activity was evaluated by Brewer’s yeast induced pyrexia in experimental animal [37]. Hyperpyrexia was induced by subcutaneous administration of 10 ml/kg body weight 20% aqueous suspension of brewer’s yeast. The selected animals were fasted overnight with water ad libitum before the experiments. Initial rectal temperature of animals was recorded using an Ellab thermometer (33.19 ± 0.40°C). After 18 h of subcutaneous administration, the animals that showed an increase of 0.3–0.5°C in rectal temperature were selected for the antipyretic activity. Crude methanolic extract of plant was given orally (500 mg/kg). Paracetamol (150 mg/kg orally) was used as reference drug, whereas, control group received distilled water (10 ml/kg) only. The rectal temperature was recorded at 1 h intervals for 3 h after treatment [38].
Statistical analysis
One way ANOVA with Dunnett’s post Hoc test for this experiment was carried out with SPSS 16.0 for Windows® software and the results obtained were compared with the control group. P values <0.001 were considered to be statistically significant.