Plant material
The bark, stem and leaves of L. sarmentosa and A. rotundifolia roxb were collected from the Sonadia deep of Cox’s Bazar. The plant material was originally identified and authenticated by Bangladesh National Herbarium, Mirpur, Dhaka. The voucher specimen numbers were DACB: 38312 and DACB: 38310 for L. sarmentosa and A. rotundifolia roxb respectively.
Preparation of extracts
The bark, stem and leaves of L. sarmentosa and A. rotundifolia roxb were washed thrice with speedy running tape water and twice with distilled water to remove the adhered salts, soils and other associated animals, parasites and then dried in shade at temperature between 21-30°C for 15 days. After the process of drying, these were grounded in blender into fine powders. Then 450 gm of each fine powdered crude plant was subjected for cold extraction process by macerating with 2100 ml of 98% methanol at room temperature for a week. At the end of 7 days, each macerate was filtrate with whatman No 1 filter paper and evaporated using a rotatory evaporator (Bűchi 011, USA) so as to get semi-solid extract. Residue left at the bottom of the beaker is the crude methanol extract of L. sarmentosa and A. rotundifolia roxb. The filtrate finally obtained was air dried and kept in refrigerator maintained at 4°C for further test.
Experimental animals
In the experiment, Swiss Albino Mice (25-30 g) and Wister Albino Rats (180-210 g) of either sex were used. They were collected from Jahangir Nagar University of Dhaka, Bangladesh. Animals of both category were kept in cages of polypropylene and feed on water ad libitum and standard laboratory pellet diet. Animals were uncovered to alternate cycle of 12 h light and dark while temperature of 25 ± 2°C and relative humidity of 55 ± 10% were also maintained. Animals were permitted for 7 days to adapt to the laboratory conditions before the start of experiment. This present project work was cleared by the Institutional Animal Ethical Committee [IAEC].
Acute toxicity studies
According to the procedures set by organization for economic co-operation and development guidelines (OECD), acute toxicity studies were accomplished for the plant extracts of AELS and AEAR [64]. Swiss Albino Mice (25-30 g) and Wister Albino Rats (180-210 g) of same age and either sex were taken into account to evaluate the acute toxicity level of proposed plant extract. For the experiment they were fasted whole night and only water ad libitum was allowed. Thus, different groups having six mice in each were orally administered with different prepared aqueous extracts of 100, 200, 400, 800 and 1000 mg/kg p.o respectively. After dosing, the rate of mortality and common behavior of these groups were witnessed continuously for initial 4 h, and intermittently for 6 h and then again at 24 h and 48 h respectively. In these experiment, doses of 1000 mg/kg show significant reduction in survival of mice (LD50- around 1000 mg/kg/b.wt). Hence, 100/200/400 mg/kg/b.wt of AELS and AEAR was considered for the present studies. Common behaviors of these animals such as convulsion, pilo erection, aggressiveness, loss of lighting reflex, analgesia, grooming, skin color, sedation, hypnosis, diarrhea and respiratory rate were also observed after dose administration [65].
Chemicals and drugs
Carragennan (Sigma Aldrich Inc., St Louis, MO, USA), Brewer’s Yeast (Arkopharma, Carros, France), Acetic acid (Merck Germany), Methanol (Merck Germany), Standard Diclofenac sodium, Indomethacin, aspirin, pentazocine and acetyl salicylic acid (Beximco Pharmaceuticals Ltd., Bangladesh). Chemicals used for present work were of analytical grade and arranged in the form of suspensions using a few drops of suspending agent diluted with distilled water.
Methods for the evaluation of anti-pyretic effect
Brewer’s yeast induced pyrexia method
This model was run to study the anti-pyretic activity, slightly modifying the method described by Adams et al.[66]. Total 8 groups of six animals each are taken into individual cages. To bring fever, 20% aqueous suspension of Brewer’s yeast (10 ml/kg) were injected into all group of animal’s dorsum region. About 18 h after the injection of pyretic agent, the rectal temperature was measured individually by a digital thermometer (SK-1250 MC, Sato keiryoki Mfg). Only those animals that shows an increase in temperature of at least 0.6°C are certified for the experiment. At 18 hour of brewer’s yeast administration, the following doses of sample was directed.
Group I: Treated as control, received vehicle 10 ml/kg normal saline p.o.
Group II: Received standard drug acetyl salicylic acid (250 mg/kg p.o.) dissolved in distilled water
Group III: Received AELS 100 mg/kg p.o. suspended in 2% w/v gum acacia solution.
Group IV: Received AELS 200 mg/kg p.o. suspended in 2% w/v gum acacia solution.
Group V: Received AELS 400 mg/kg p.o. suspended in 2% w/v gum acacia solution
Group VI: Received AEAR 100 mg/kg p.o. suspended in 2% w/v gum acacia solution.
Group VII: Received AEAR 200 mg/kg p.o. suspended in 2% w/v gum acacia solution.
Group VIII: Received AEAR 400 mg/kg p.o. suspended in 2% w/v gum acacia solution.
After sample and drug administration, rectal temperature of each rate was recorded by a digital thermometer at 0, 1, 2 and 3 hr. Thus average rectal temperature of each group (n = 6) was calculated and compared with standard drug acetyl salicylic acid.
Methods for the evaluation of analgesic effect
Tail immersion method
In the present study, analgesia was evaluated following Luiz et al. method [67]. Total 8 groups of six animals each are taken into individual cages and the animals were allowed to adopt in the cage environment for half an hour before the start of experiment. All animals were held in position with the tail extending out in a suitable restrainer. Freshly filled hot water maintained at 55.0 ± 1.0°C were used to immerse the lower 5 cm portion of the tail and the time to withdrawal of the tail was noted as the reaction time or tail flick latency. Any animals fail to withdraw its tail from hot water within 10 sec is rejected from the experiment.
Group I: Control group animals received 10 ml/kg of 0.5% sodium lauryl sulphate (SLS).
Group II: animals received Standard drug Pentazocine at dose of 30 mg/kg i.p.
Group III: Received AELS 100 mg/kg in 0.5% SLS p.o.
Group IV: Received AELS 200 mg/kg in 0.5% SLS p.o.
Group V: Received AELS 400 mg/kg in 0.5% SLS p.o.
Group VI: Received AEAR 100 mg/kg in 0.5% SLS p.o.
Group VII: Received AEAR 200 mg/kg in 0.5% SLS p.o.
Group VIII: Received AEAR 400 mg/kg in 0.5% SLS p.o.
After administration of above scheduled dose, the reaction time was counted at 0, 15, 30 and 60 min respectively. The mean reaction time for each group was recorded and compared with the value of standard [68].
Acetic acid induced writhing method
Peripheral analgesic activity is widely assessed by using acetic acid induced writhing technique in which acetic acid solution was intraperitoneally injected and the number of stretching’s and writhings was counted as previously reported by Koster et al. and Hendershot & Forsaith et al.[69, 70]. Total 8 groups of six animals each are employed into individual cages.
Group I: Control group animals received 10 ml/kg of 2% tween.
Group II: Standard group animals received Aspirin at dose of 50 mg/kg i.p.
Group III: Received AELS 100 mg/kg in 2% tween p.o.
Group IV: Received AELS 200 mg/kg in 2% tween p.o.
Group V: Received AELS 400 mg/kg in 2% tween p.o.
Group VI: Received AEAR 100 mg/kg in 2% tween p.o.
Group VII: Received AEAR 200 mg/kg in 2% tween p.o.
Group VIII: Received AEAR 400 mg/kg in 2% tween p.o.
All the scheduled test doses were administered orally 1 hr prior to injection of acetic acid intraperitonealy (0.1 ml of 0.6% v/v). After the five minutes of acetic acid injection, the number of writhing movements was recorded for individual animals for a period of 15 min which was considered as withering of the abdominal muscles of animal accompanied by stretching with a jerk at the hind limb. The counted number of writhing in each treated animal group was then compared to that of a control group.
Methods for the evaluation of anti-inflammatory effect
Carrageenan induced hind paw edema model
Anti-inflammatory activity was evaluated in accordance of slightly revising the method described by Winter et al.[55]. Total 8 groups of six animals each are placed into individual cages.
Group I: Control group animals received 10 ml/kg of 1% propylene glycol.
Group II: Animals received standard Indomethacin at dose of 8 mg/kg i.p.
Group III: Received AELS 100 mg/kg in 1% propylene glycol p.o.
Group IV: Received AELS 200 mg/kg in 1% propylene glycol p.o.
Group V: Received AELS 400 mg/kg in 1% propylene glycol p.o.
Group VI: Received AEAR 100 mg/kg in 1% propylene glycol p.o.
Group VII: Received AEAR 200 mg/kg in 1% propylene glycol p.o.
Group VIII: Received AEAR 400 mg/kg in 1% propylene glycol p.o.
After 1 h of above drug treatment, 2% w/v Carrageenan solution (0.05 ml) were injected subcutaneously into sub plantar tissue of right hind paw of each animal and the contra lateral hind paws of same animal were also injected with 0.1 ml of saline as control. The increased paw volume was then plethysmographically measured at 0, 1, 2 and 3 h after injection of odematogenic agent. Actual edema volume was calculated from the difference between initial and subsequent reading and the percent inhibition activity was calculated by using the formula-
Cotton-pellet granuloma model
Cotton pellet induced granuloma model in rats are used to produce sub-acute inflammation [71, 72]. Total 8 groups of six animals each are placed into individual cages. Sterilized dental cotton rolls (Johnson and Johnson, New Brunswick, NJ, USA) were cut into 5-mm pieces and two sterilized cotton pellets weighing 10 ± 1 mg were subcutaneously implanted along the flanks of axillae and groins bilaterally under ether anesthesia.
Group I: Control group animals received 10 ml/kg of 1% propylene glycol.
Group II: Animals received Standard Diclofenac sodium at dose of 40 mg/kg p.o.
Group III: Received AELS 100 mg/kg in 1% propylene glycol p.o.
Group IV: Received AELS 200 mg/kg in 1% propylene glycol p.o.
Group V: Received AELS 400 mg/kg in 1% propylene glycol p.o.
Group VI: Received AEAR 100 mg/kg in 1% propylene glycol p.o.
Group VII: Received AEAR 200 mg/kg in 1% propylene glycol p.o.
Group VIII: Received AEAR 400 mg/kg in 1% propylene glycol p.o.
Following drugs were administered orally for 6 consecutive days. On the 7th day, the animals were sacrificed by cervical dislocation and the collected granulomas were dried in an oven for 24 hr at 60°C, then weighed and compared with control.
Statistical analysis
All of values were expressed as mean ± SEM. Each results were analyzed for statistical significance using one-way ANOVA followed by Dunnet’s‘t’ test with *P < 0.05, **P < 0.01 and ***P < 0.001were considered as significant value.