Plant material and extraction
Celosia cristata were collected from the Gowsul Azam Nursery, Kamalapur, Dhaka, Bangladesh on November 27, 2014. The collected plants were identified by Bushra Khan, Principal Scientific Officer, Bangladesh National Herbarium, Mirpur, Dhaka, Bangladesh and a voucher specimen (DACB: 41890) has been deposited for further reference. The plant material was shade dried and grounded. Then 205 g of powdered material were macerated by 1000 mL of methanol. The solution was occasionally stirred at 25 ± 2 °C for 7 days and then filtered using sterilized cotton and Buchner funnel. The filtrate was concentrated to evaporate solvent using rotary evaporator at 40 °C and 50 r.p.m. Finally, 14.26 g (yield 6.96 %) of dried extract was obtained and this crude extract was used for phytochemical screening, toxicity, and antinociceptive activity studies.
Chemicals and drugs
The chemicals and drugs used in the present study are methanol, toluene, ethyl acetate, dichloromethane, vanillin, sulphuric acid, Folin–Ciocalteu’s reagent, aluminum chloride, Na-K tartrate, formalin, acetic acid, L-glutamic acid, (Merck Co., Darmstadt, Germany), DPPH (2,2-Diphenyl-1-picrylhydrazyl), ascorbic acid, quercetin, stigmasterol, methylene blue, pentobarbital sodium (Sigma Co., St. Louis, MO, USA), digoxin (Aristopharma Ltd., Shampur, Dhaka, Bangladesh), atropine, morphine sulfate (Gonoshasthaya Pharmaceuticals Ltd., Savar, Dhaka, Bangladesh), diclofenac sodium (Novartis Bangladesh Ltd., Gazipur, Dhaka, Bangladesh), glibenclamide (Square Pharmaceuticals Ltd., Gazipur, Dhaka, Bangladesh), naloxone hydrochloride (Samarth Life Sciences Pvt. Ltd., Nalagarh, Himachal Pradesh, India).
Animals
Swiss albino male mice with 20–25 g body weight (b.w.) were collected from Animal Resources Branch of the International Center for Diarrhoeal Disease Research, Bangladesh (icddr,b) The animals were acclimatized for 14 days in laboratory condition before experiments. The animals were housed in 120 × 30 × 30 cm cages at standard laboratory environment (room temperature 25 ± 2 °C; relative humidity 55–60 %; 12 h light-dark cycle) and were provided with standard diet (icddr,b formulated) and tap water ad libitum. Flake wood shavings were used for bedding. Health status of animals was monitored every day. The animals were randomly selected and divided into control, positive control and experimental group (n = 5) for each experiment. The animals were abstained from food overnight only before experiments. All the experiments were carried out in accordance with The Swiss Academy of Medical Sciences and the Swiss Academy of Sciences formulated Ethical Principles and Guidelines for Scientific Experiments on Animals (1995) and performed under the approval of Ethics Committee of Stamford University Bangladesh (SUB/IAEC/14.07). The acute oral toxicity test was carried out following the guideline (420 – fixed dose procedure) of Organization for Economic Cooperation and Development (OECD). The animals were euthanized using pentobarbital in accordance with AVMA guidelines for the Euthanasia of Animals: 2013 Edition and made all efforts to alleviate animal suffering.
Drugs and treatments
The control group orally received vehicle (0.9 % sodium chloride) at the dose of 10 mL/kg (b.w.) 30 min before the experiments. The positive control group intraperitoneally (i.p.) received standard drug morphine in hot plate, tail immersion and formalin-induced licking test at the dose of 5 mg/kg and diclofenac sodium in acetic acid-induced writhing and glutamate-induced paw licking test at the dose 10 mg/kg 15 min before the experiments. MECC was administered orally at the doses of 50, 100, 200, 400 mg/kg (b.w.) 30 min before the experiments. The doses of MECC were selected from trial and previously reported effective doses of C. cristata [29]. To evaluate the involvement of opioid-mediated antinociceptive activity, naloxone was administered (i.p.) at the dose of 2 mg/kg 15 min before morphine sulfate or MECC administration in the hot plate and tail immersion test. Methylene blue (20 mg/kg) and glibenclamide (10 mg/kg) were intraperitoneally employed 15 min before vehicle or MECC (50, 100, 200 and 400 mg/kg) administration to evaluate the involvement of cyclic guanosine monophosphate (cGMP) and ATP-sensitive K+ channel pathway respectively. All the doses of drugs and MECC were prepared using the vehicle.
Phytochemical analysis
Phytochemical screening
MECC was qualitatively tested for the detection of proteins, carbohydrates, steroids, saponins, alkaloids, flavonoids, tannins, glycosides and resins following standard procedures [30]. The phytoconstituents of MECC was further authenticated by thin layer chromatography (TLC). Aliquots of (10 % w/v dissolved in methanol) MECC extract was spotted on pre-coated silica gel 60 F254 plates (Merck Co., Darmstadt, Germany). The phytochemical profile was obtained using different solvent systems as flavonoids, phenolics and steroids (toluene: ethyl acetate: acetic acid (9:1:1); glycosides (ethyl acetate: methanol (10:1); alkaloid (dichloromethane: methanol (8:1). A collection of standard phytochemicals such as quercetin, gallic acid, stigmasterol, digoxin, and atropine were co-chromatographed for flavonoids, phenolics, steroids, glycosides, alkaloids respectively. The developed chromatographed plates were visualized under visible (254 nm), UV light (356 nm) or using suitable spraying reagents (such as steroid by 1 % w/v vanillin-sulphuric acid solution, glycosides by sulphuric acid, alkaloid by Dragendorff’s reagent) [31]. Then retention factor (Rf) for each spot was determined as Rf = distance travelled by the solute/distance travelled by the solvent). Each experiment was carried out twice and the mean Rf values were compared with the standard markers.
Determination of phenolic content
The total phenolic content (TPC) was determined using Folin–Ciocalteu’s reagent [32]. 1 mL of MECC (200 μg/mL) was mixed with 0.5 mL of Folin–Ciocalteu’s reagent. 5 min later 4 mL of sodium carbonate (7.5 % w/v in distilled water) solution was added to the mixture. The solution was incubated at 20 °C for 60 min. The absorbance of the solution was read at 765 nm using spectrophotometer (Specord 250, Analytik Jena, Germany). A calibration curve (y = 0.0057x + 0.0146, R2 = 0.9985) was constructed by preparing gallic acid solutions (25 – 400 mg/L). The experiment was carried out in triplicate and the mean value of absorbance was calculated. Then TPC was determined in gallic acid equivalents (GAE) from the following formula: A = (C × V)/m, where, A denote total phenolic content of extract equivalent to gallic acid, C is the concentration of the gallic acid obtained from calibration curve (mg/mL), V denote volume of the extract (mL) and m is the plant extract weight (g).
Determination of flavonoid content
Total flavonoid content was determined using aluminum chloride (AlCl3) [33]. 1 ml of MECC (100 μg/mL) was taken and mixed with 2 mL of methanol. Then 0.1 mL of 10 % AlCl3 (w/v in distilled water), Na-K tartrate (1 M) solution and 2.8 mL distilled water was sequentially added to the extract solution. Then the mixture was vigorously shaken by vortex mixture (Clifton CM-1, Camlab, UK) and incubated at room temperature for 30 min. The absorbance of the solution was taken using spectrophotometer at 415 nm. The experiment was carried out in triplicate and the mean absorbance value was noted. The total flavonoid content was measured from the calibration curve of quercetin (y = 0.0165x + 0.1353, R2 = 0.9933) and expressed as mg of quercetin equivalent/g of plant extract.
DPPH free radical scavenging assay
To determine the scavenging effect of MECC on DPPH radical, a stock solution of 1.6 mg extract in 0.4 mL methanol was prepared. The test solution was prepared at the concentration of 1.5625, 3.125, 6.25, 12.5, 25, 50, 100, 200 and 400 μg/mL using methanol. A volume of 2 mL test solution was added to 2 mL of a methanol solution of DPPH (0.1 mM). The mixture was properly mixed and kept in a dark place at room temperature for 30 min. The absorbance of the solutions was read at 517 nm against blank. The experiment was carried out triplicate for each concentration. The percentage of inhibition of DPPH free radicals was calculated from following equation: % inhibition = [(absorbance of blank – absorbance of the sample)/absorbance of blank] × 100. The scavenging capacity of ascorbic acid in the same concentration was determined similarly as standard [34]. The concentration at which the 50 % DPPH free radicals were inhibited (IC50) was calculated using GraphPad prism, Version 6.05.
Acute toxicity test
The animals were divided into control and three experimental groups (n = 5). MECC was orally administered at the dose of 1000, 2000 and 4000 mg/kg to the experimental groups. The animals were kept in a distinct cage. After gavage, animals were provided free access to water ad libitum and food. Any allergic reactions (skin rashes, itching), discharges from eyes and mucous membrane, behavioral changes, food and water refusal, salivation, convulsion, tremors, diarrhea, and mortality of the animals were observed for 14 days. After the observation period, the body weight of surviving animals was recorded. Then, animals were sacrificed to examine any abnormalities and significant gross changes of the vital organs [35, 36].
Pharmacological tests
Hot plate test
Hot plate test was performed to determine the central analgesic activity of MECC. The test was carried out using Eddy’s hot plate (Kshitij Innovations, Haryana, India). Hot plate temperature was kept constant at 50 ± 0.5 °C. The mice were gently placed on the hot plate and jumping, withdrawal of paw(s), forepaw licking were considered as nociception. The mice were kept on the hot surface only for the 20 s (cut-off time) to avoid any thermal injury [37]. A latency was determined before treatment as a baseline for the study. After vehicle, standard drug morphine (i.p.) or MECC (p.o.) treatment the latency periods were measured at 30, 45, 60, 90 and 120 min. The maximal possible effect (MPE) in % of individual mice was measured by following formula: % MPE = [(post-treatment latency – pre-treatment latency)/(cut-off time – pre-treatment latency)] × 100.
Tail immersion test
The test was carried out based on the findings that morphine-like drugs extend the tail withdrawal time from hot water in mice [38]. Mice were gently restrained using ‘Chux’ and one to two cm of the tail was immersed into hot water set at 52 ± 1 °C. The latency between submersion and withdrawal of the tail was recorded and considered as the index of nociception. Mice that demonstrated withdrawal of tail between 1.5 and 3.5 s from the hot water were selected for the test. A 20s cut-off period was maintained to avoid tissue damage of tail of mice. The latency time was recorded before and after 30, 45, 60, 90 and 120 min of the vehicle, morphine (i.p.) or MECC (p.o.) treatment. The % MPE was calculated from pre and post treatment latency as described in hot plate test.
Acetic acid-induced writhing test
The acetic acid-induced writhing test was conducted to determine the central and peripheral antinociceptive effect of MECC against chemical induced nociception. Mice were treated (i.p.) with 0.6 % (v/v) acetic acid to induce writhing. Acetic acid was employed at the dose of 10 mL/kg (b.w.) 30 min after vehicle or MECC and 15 min of diclofenac sodium (i.p.) administration. Mice were then placed in separate box. After 5 min of acetic acid treatment, the number of writhing was recorded for 30 min [39]. The writhing was defined by contraction of the abdomen, arching of back, twisting of the trunk and/or pelvis ending with the elongation of limbs.
Formalin-induced licking test
A volume of 20 μl of formalin (2.5 % formalin (0.92 % formaldehyde) prepared in saline) was injected into the subplantar region of the right hind paw of mice to induce pain. Formalin was injected 15 min after morphine and 30 min after vehicle or MECC treatment. Responses like licking, biting, lifting, shaking of right hind paw were considered as nociception. The nociceptive responses were measured 0 to 5 min (early phase) and 15 to 30 min (late phase) following formalin treatment, representing to the neurogenic and inflammatory pain responses respectively [40].
Glutamate-induced paw licking and edema test
Mice were injected with 20 μl of 20 μ mol glutamate (prepared in saline, pH 7.4) in the sub-plantar area of the right hind paw. Mice received vehicle or MECC and diclofenac sodium 30 and 15 min before glutamate injection respectively. The right hind paw thickness of mice was measured before glutamate challenge using digital slide caliper (M: 091552; Shanghai Shenhan Measuring Tools Co., Ltd, Shanghai, China). The licking was measured for 15 min after glutamate challenge and accounted as indicative of nociception. Then paw thickness of right hind paw of every mouse was measured again and the degree of edema (Δ) in mm was calculated from the following formula: Δ = (paw thickness after treatment – paw thickness before treatment) [41].
Analysis of the possible mechanism of action of MECC
Involvement of the opioid system
In order to determine the participation of opioid system in the pain inhibition effect of MECC naloxone was intraperitoneally administered 15 min before MECC (50, 100, 200 and 400 mg/kg) or morphine sulfate administration in the hot plate and tail immersion test [42]. The latencies of the hot plate and tail immersion were sequentially recorded at pre-treatment, 30, 45, 60, 90, 120 min of after administration of MECC or morphine and assembled with the result of the hot plate and tail immersion test respectively. The same cut-off period of the 20 s was maintained for any thermal injury.
Involvement of the cyclic guanosine monophosphate (cGMP) pathway
Mice were pre-treated with methylene blue (MB), a non-specific guanylylcyclase/NO inhibitor to verify the possible involvement of cGMP in the pain inhibition action of MECC. MB (20 mg/kg) was administered (i.p.) 15 min before the employment of effective doses (100, 200, 400 mg/kg) of MECC. After 30 min of treatment, animals were intraperitoneally administered 0.6 % (v/v) acetic acid at the dose of 10 mL/kg to induce writhing. The nociceptive behavior was recorded for 30 min, starting after 5 min of acetic acid treatment. The abdominal writhing syndrome was counted as an indication of nociceptive behavior [43, 44].
Involvement of the ATP-sensitive K+ channel pathway
The participation of K+ channel in the antinociceptive action of the effective doses of MECC (100, 200 and 400 mg/kg was evaluated to the groups of mice, pre-treated with glibenclamide, an ATP-sensitive K+ channel inhibitor. Glibenclamide was intraperitoneally administered at the dose of 10 mg/kg 15 min before the treatment of MECC. After 30 min of above treatments, the mice of each group were challenged with 0.6 % acetic acid (i.p.) to induce writhing. After 5 min of an acetic acid challenge, the number of writhing was noted for 30 min as an indication of nociceptive response [44, 45].
Statistical analysis
Data are presented as mean ± standard error of mean (SEM). Statistical analysis of the results was performed using one-way analysis of variance (ANOVA). The significance of differences between groups was tested by Dunnett’s or Bonferroni’s test, as appropriate and p < 0.05 values were considered as significant. SPSS 22 software was used for performing the statistical analysis. IC50 values were calculated using GraphPad prism (version 6.01) software.