Traditionally in Bangladesh,
Diclofenac sodium, morphine sulphate, naloxone, carrageenan, histamine phosphate, indomethacin, gallic acid, quercetin, pentobarbitone, and Folin-Ciocalteu phenol reagent were collected from Sigma Chemical Co. (St. Louis, MO, USA). Ethanol and formalin were purchased from E. Merck, Germany. Analytical grade Tween 80, sodium carbonate, aluminum chloride, and potassium acetate were bought from E. Merck, India, Ltd.
For laboratory animal studies, Swiss albino mice (20–25 g) and Wister rats (175–202 g) of both sexes were used. These animals were collected from the animal research branch of International Center for Diarrheal Disease Research, Bangladesh (ICDDR, B). They were kept under standard laboratory environments maintained at 25 ± 2°C and under 12/12 h light/dark cycle and fed with standard diet and water ad libitum during adaptation period. The animals were put away from any food/water overnight before the experiments. During the experimental period, these animals were treated according to the “Ethical Principles and Guidelines for Scientific Experiments on Animals (1995)” formulated by the Swiss Academy of Medical Sciences and the Swiss Academy of Sciences. The experimental procedure was approved by the Bangladesh Council of Scientific and Industrial Research (BCSIR) ethics committee (BCSIR/IAEC/08/11-12).
Phytochemical screening of EFHB was carried out by using standard quantitative procedures to investigate the presence of alkaloid, steroid, reducing sugar, saponin, tannin, and flavonoid [
Total phenolic content of EFHB was determined by modified Folin-Ciocalteu method [
Total flavonoid content of EFHB was determined by AlCl3 colorimetric method [
Acute toxicity test of EFHB was investigated in order to determine the dose(s) to be used in different tests in the laboratory animals. Rats were kept in fasting condition for 16 h. 500, 1000, 2000, 3000, and 4000 mg/kg EFHB dose were orally administered to fasting rats. After that, they were allowed free access to food and water and all the rats were under careful observation over a period of 72 h and the number of deaths within this period was recorded [
In the acetic acid-induced writhing model in mice, different groups of animals (five mice/group, total twenty-five) were treated with 1% Tween 80 in normal saline (10 ml/kg, p.o.), EFHB (100, 200, and 400 mg/kg, p.o.), and diclofenac sodium (25 mg/kg, i.p.) 30 min before intraperitoneal injection of 0.7% acetic acid. After an interval of 5 min, writhing (constriction of the abdomen, turning of trunk, and extension of hind legs) was observed for 10 min [
In the formalin-induced paw licking test in mice, overnight fasted animals (five mice/group, total thirty) were treated with 1% Tween 80 in normal saline (10 ml/kg, p.o.), EFHB (100, 200, and 400 mg/kg, p.o.), morphine (5 mg/kg, s.c.), and diclofenac-Na (25 mg/kg, s.c.). Formalin (20
In this assay, mice were placed on a heated (50 ± 0.05°C) metal plate and the time elapsed until the appearance of reactions (lifting or licking of the paws) to the thermal stimulus was recorded as an index of nociception [
In this experiments, twenty-five mice were homogeneously divided into five different groups where mice tails (2 cm) pretreated with 1% Tween-80 in normal saline (10 ml/kg, p.o.), EFHB (100, 200, and 400 mg/kg, p.o.), and morphine (5 mg/kg, s.c.) were immersed in warm water (55 ± 1°C). The latency between tail submersion and deflection of the tail was recorded and the pretreatment latency was recorded at 30, 60, 90, and 120 min. This latency period was taken as the index of antinociception. Moreover, an opioid nonselective antagonist naloxone (2 mg/kg) was injected (i.p.) in 25 other mice (5 mice/group) in 15 min prior to the administration of either morphine (10 mg/kg, s.c.) or EFHB and observed as explained above [
Carrageenan-induced rat hind paw oedema was used as the animal model of acute inflammation. In this experiment, twenty-five rats divided into five groups (five rats/group) were treated with 1% Tween 80 in normal saline (10 ml/kg, p.o.); EFHB (100, 200, and 400 mg/kg, p.o.); and indomethacin (10 mg/kg, p.o.). Acute inflammation was induced in groups by subplantar injection of 0.1 ml of carrageenan (1% suspension in Tween 80) in the right paw of rats 1 hour after all of the oral administration. The paw oedema volume was measured with micrometer screw gauze at 1, 2, 3, 4, and 5 h after the administration of the tested materials [
In this experiment, after dividing twenty-five rats into five groups (five rats/group), they were treated with 1% Tween 80 in normal saline (10 ml/kg, p.o.), EFHB (100, 200, and 400 mg/kg, p.o.), and indomethacin (10 mg/kg, p.o.). Acute inflammation was induced by subplantar injection of 0.1 ml of histamine with 1% suspension in Tween-80 in the right hind paw of the rats 30 min after the oral administration of the tested materials. The paw volume was measured with micrometer screw gauze at 1, 2, 3, 4, and 5 h after the administration of the tested materials [
In pentobarbital induced hypnosis test, twenty-five mice divided into five groups (five rats/group) were treated with 1% Tween 80 in normal saline (10 ml/kg), EFHB (100, 200, and 400 mg/kg), and diazepam (1 mg/kg). Each mouse was placed in a quadrangular observation box (36 × 36 cm2). After 30 min, pentobarbitone (40 mg/kg) was given to mice to induce sleep. The total sleeping time was monitored for all experimental groups. Mice were observed for the duration of sleep (i.e., time difference between the loss and recovery of reflex) and the latent period (i.e., time difference between pentobarbitone administration and loss of reflex) [
Open field experimental method is routinely used to evaluate emotional and locomotors activities in rodents [
Hole cross experimental method was run in a cage (
All of the experimental results are reported as mean ± SEM. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Dunnett’s test [
At 100, 200, and 400 mg/kg EFHB showed 37.76, 50.35, and 62.24 percentage of writhing episode inhibition, respectively. Standard drug diclofenac sodium (25 mg/kg) showed 75.52%
EFHB demonstrated significant inhibition of licking responses in early (44.57%, 50.36%, and 59.29%) and late phase (51.64%, 60.17%, and 71.61%) at the doses of 100, 200, and 400 mg/kg, respectively. Standard morphine produced marked inhibition in both the early phase (70.76%) and late phase (99.13%) whereas diclofenac sodium produced inhibition of licking responses (96.32%) in the late phase only (Table
EFHB significantly increased the reaction time to the thermal stimulus in a dose dependent manner (3.91, 3.97, and 5.13 s after 120 min at 100, 200, and 400 mg/kg, resp.) which was comparable to the standard drug morphine (5.27 s after 120 min). Naloxone produced a substantial antagonistic effect on the antinociceptive activity of both EFHB and morphine (Table
EFHB substantially increased the latency period to hot-water induced thermal stimuli (3.71, 4.52, and 5.26 s after 120 min at 100, 200, and 400 mg/kg dose) whereas standard drug morphine at 5 mg/kg showed 3.74 s latency period after 120 min. Naloxone counteracts the antinociceptive activity of both EFHB and morphine (Table
EFHB showed a significant reduction in the carrageenan-induced paw volume after 5 h as compared with the control group. EFHB (200 and 400 mg/kg) inhibited the inflammation to the extent of 44.94% and 59.49% while the reference drug, indomethacin, reduced the inflammation by 67.72% (Table
At the higher doses (200 and 400 mg/kg), EFHB inhibited the inflammation to the extent of 46.43% and 60.12% which was statistically significant (
A substantial increase in duration of sleep was observed in the dosages of 200 and 400 mg/kg (53.70 ± 1.77 and 60.80 ± 2.01 min, resp., as compared to 73.50 ± 2.20 min in the control group at
EFHB showed a noticeable decrease in locomotion in the test animals with increasing time. At 240 min, the number of movements was gradually decreased up to 37.50
The EFHB displayed suppression of motor activity and exploratory behavior in the test animal. The locomotor activity lowering effect was evident from the 2nd observation period (30 min) and continued up to 7th observation period (240 min). At 240 min, number of holes crossed was gradually decreased up to 6.10
We did not observe any mortality up to the dose of 4 g/kg b.w. of EFHB (p.o.) in the test animals which ensured the safety of the experimental dose.
Acetic acid generally produces pain by increasing the level of PGE2 and PGF2
Formalin test was performed since the results of the writhing test alone could not ascertain the source of antinociception. It evaluates antinociceptive property in two distinct phases. In the early/first phase, neurogenic pain is induced by direct chemical stimulation of the sensory afferent fibers; particularly c-fibers. The involvement of substance P and bradykinin has also been reported. Pain is induced by prostaglandins, histamine, bradykinin, and serotonin in the late phase [
The hot plate and tail immersion tests are widely used for assessing central antinociceptive activity. They are distinguished by their tendency to respond to the pain stimuli, as the hot plate demonstrates supraspinal reflex mediated by
The inflammatory response induced by carrageenan is characterized by a biphasic response with marked oedema formation due to the rapid production of inflammatory mediators. These mediators are subsequently sustained by the release of prostaglandins and nitric oxide which is produced by cyclooxygenase (COX-2) and nitric oxide synthase (iNOS), respectively [
Total phenolic and flavonoids content of EFHB.
Treatment | Total phenolic content |
Total flavonoids content |
---|---|---|
EFHB | 258.37 ± 6.68 | 144.29 ± 7.89 |
Here, each value is presented as the mean ± SEM
Effects of EFHB on acetic acid-induced writhing test in mice.
Treatment | Dose (mg/kg, p.o.) | Number of writhes | Inhibition (%) |
---|---|---|---|
Vehicle | 10 (ml/kg) | 14.3 ± 0.79 | — |
Diclofenac sodium | 25 |
|
75.52 |
EFHB | 100 |
|
37.76 |
EFHB | 200 |
|
50.35 |
EFHB | 400 |
|
62.24 |
Here, each value is presented as the mean ± SEM
Effects of EFHB on formalin-induced paw licking test in mice.
Treatment | Dose (mg/kg) | Licking of the hind paw | |||
---|---|---|---|---|---|
Early phase (0–5 min) | % inhibition | Late phase (15–30 min) | % inhibition | ||
Vehicle | 10 (ml/kg) | 98.50 ± 4.27 | — | 103.20 ± 4.93 | — |
Morphine | 5 |
|
70.76 | 0.90 ± 0.11 | 99.89 |
Diclofenac sodium | 10 |
|
36.85 |
|
96.25 |
EFHB | 100 |
|
44.46 |
|
51.64 |
EFHB | 200 |
|
50.36 |
|
60.17 |
EFHB | 400 |
|
59.29 |
|
71.61 |
Here, each value is presented as the mean ± SEM
Effects of EFHB on hot plate test in mice.
Treatment | Dose (mg/kg) | Latency period (s) (% MPE) | |||||
---|---|---|---|---|---|---|---|
0 min | 30 min | 45 min | 60 min | 90 min | 120 min | ||
Vehicle | 10 (ml/kg) | 2.19 ± 0.19 | 2.40 ± 0.16 | 2.42 ± 0.23 | 2.47 ± 0.34 | 2.39 ± 0.38 | 2.69 ± 0.45 |
Morphine | 5 | 2.38 ± 0.21 | 10.63 ± 0.65 (41.15 |
10.31 ± 0.57 (39.31 |
10.98 ± 0.69 (42.71 |
9.92 ± 0.54 (34.86 |
5.27 ± 0.30 (11.08 |
EFHB | 100 | 2.24 ± 0.22 | 2.71 ± 0.19 (2.64 |
3.45 ± 0.53 (6.78 |
3.87 ± 0.75 (9.74 |
4.23 ± 0.49 (11.21 |
3.91 ± 0.56 (9.74 |
EFHB | 200 | 2.29 ± 0.23 | 2.93 ± 1.56 (3.83 |
3.71 ± 0.61 (8.23 |
4.51 ± 0.60 (12.73 |
5.31 ± 0.53 (17.24 |
3.97 ± 0.60 (9.35 |
EFHB | 400 | 2.37 ± 0.19 | 3.89 ± 0.43 (8.60 |
5.60 ± 0.59 (18.31 |
7.67 ± 0.58 (18.16 |
6.78 ± 0.54 (19.30 |
5.13 ± 0.35 (12.26 |
NLX | 2 | 2.65 ± 0.35 | 2.51 ± 0.51 | 2.65 ± 0.34 | 2.39 ± 0.26 | 2.58 ± 0.38 | 1.77 ± 0.28 |
NLX + morphine | 2 + 5 | 2.90 ± 0.31 | 2.59 ± 0.43 (−2.02 |
3.92 ± 0.27 (0.74 |
2.76 ± 0.27 (−0.72 |
2.31 ± 0.22 (−3.64 |
2.88 ± 0.26 (−0.63 |
NLX + EFHB | 2 + 100 | 2.97 ± 0.32 | 2.30 ± 0.27 (−3.42) | 2.21 ± 0.47 (−4.01 |
2.27 ± 0.64 (−1.35 |
2.65 ± 0.83 (−1.07 |
2.97 ± 0.31 (−0.04 |
NLX + EFHB | 2 + 200 | 2.35 ± 0.20 | 2.19 ± 0.14 (1.11 |
2.18 ± 0.56 (−0.33 |
2.59 ± 0.62 (1.21 |
2.66 ± 0.79 (−0.27 |
2.18 ± 0.26 (−0.99 |
NLX + EFHB | 2 + 400 | 2.71 ± 0.71 | 2.87 ± 0.30 (2.07) | 3.22 ± 0.44 (3.34 |
3.10 ± 0.58 (2.44 |
2.91 ± 0.48 (1.28 |
2.27 ± 0.18 (−3.49 |
Here, each value is presented as the mean ± SEM
Effects of EFHB on tail immersion plate test in mice.
Treatment | Dose (mg/kg) | Latency period (s) (% MPE) | |||||
---|---|---|---|---|---|---|---|
0 min | 30 min | 45 min | 60 min | 90 min | 120 min | ||
Vehicle | 10 (ml/kg) | 2.15 ± 0.29 | 2.19 ± 0.31 | 2.74 ± 0.29 | 2.25 ± 0.34 | 2.33 ± 0.31 | 2.44 ± 0.27 |
Morphine | 5 | 2.28 ± 0.15 | 10.03 ± 0.73 (41.15 |
12.51 ± 0.62 (42.71 |
11.82 ± 0.46 (42.71 |
8.25 ± 0.44 (34.86 |
3.74 ± 0.2 (11.08 |
EFHB | 100 | 2.90 ± 0.28 | 4.75 ± 0.48 (2.64 |
6.56 ± 0.31 (9.74 |
5.86 ± 0.43 (9.74 |
4.38 ± 0.35 (11.21 |
3.71 ± 0.48 (9.74 |
EFHB | 200 | 2.27 ± 0.21 | 6.87 ± 0.56 (3.83 |
8.73 ± 0.54 (12.73 |
6.94 ± 0.39 (12.73 |
5.05 ± 0.31 (17.24 |
4.52 ± 0.35 (9.35 |
EFHB | 400 | 2.70 ± 0.22 | 8.85 ± 0.41 (8.60 |
9.83 ± 0.43 (18.16 |
8.06 ± 0.30 (18.16 |
6.25 ± 0.29 (19.30 |
5.16 ± 0.42 (12.26 |
NLX | 2 | 2.91 ± 0.32 | 2.80 ± 0.21 | 2.76 ± 0.28 | 2.82 ± 0.21 | 2.98 ± 0.26 | 2.70 ± 0.23 |
NLX + morphine | 2 + 5 | 2.63 ± 0.24 | 3.95 ± 0.26 (−2.02 |
3.81 ± 0.21 (−0.72 |
3.95 ± 0.27 (−0.72 |
3.91 ± 0.22 (−3.64 |
3.62 ± 0.37 (−0.63 |
NLX + EFHB | 2 + 100 | 3.05 ± 0.28 | 3.46 ± 0.34 (−3.42) | 3.20 ± 0.35 (−1.35 |
3.50 ± 0.18 (−1.35 |
2.95 ± 0.28 (−1.07 |
2.69 ± 0.41 (−0.04 |
NLX + EFHB | 2 + 200 | 2.98 ± 0.24 | 3.65 ± 0.28 (1.11 |
3.71 ± 0.23 (1.21 |
3.75 ± 0.24 (1.21 |
3.24 ± 0.49 (−0.27 |
3.01 ± 0.38 (−0.99 |
NLX + EFHB | 2 + 400 | 2.81 ± 0.18 | 4.02 ± 0.25 (2.07) | 4.96 ± 0.39 (2.44 |
5.11 ± 0.36 (2.44 |
3.75 ± 0.37 (1.28 |
3.11 ± 0.27 (−3.49 |
Here, each value is presented as the mean ± SEM
Effects of EFHB on carrageenan-induced oedema paw volume in Wistar rats.
Treatment | Dose (mg/kg) | Right hind paw volume (% inhibition) | ||||
---|---|---|---|---|---|---|
1 h | 2 h | 3 h | 4 h | 5 h | ||
Vehicle | 10 (ml/kg) | 1.03 ± 0.11 | 1.27 ± 0.16 | 1.38 ± 0.16 | 1.44 ± 0.19 | 1.58 ± 0.12 |
Indomethacin | 10 | 0.48 ± 0.16 (53.22% |
0.54 ± 0.19 (57.80% |
0.54 ± 0.13 (60.76% |
0.56 ± 0.18 (61.33% |
0.51 ± 0.18 (67.72% |
EFHB | 100 | 0.82 ± 0.14 (19.69% |
0.94 ± 0.14 (26.30% |
0.98 ± 0.19 (29.07% |
0.97 ± 0.15 (32.64% |
1.02 ± 0.16 (35.44% |
EFHB | 200 | 0.76 ± 0.10 (25.54% |
0.78 ± 0.22 (38.90% |
0.84 ± 0.14 (39.24% |
0.85 ± 0.11 (40.97% |
0.87 ± 0.17 (44.94% |
EFHB | 400 | 0.55 ± 0.17 (46.60% |
0.62 ± 0.18 (51.18% |
0.66 ± 0.17 (52.17% |
0.65 ± 0.21 (54.86% |
0.64 ± 0.11 (59.49% |
Here, each value is presented as the mean ± SEM
Effect of EFHB on histamine-induced oedema paw volume in Wistar rats.
Treatment | Dose (mg/kg) | Right hind paw volume (% inhibition) | ||||
---|---|---|---|---|---|---|
1 h | 2 h | 3 h | 4 h | 5 h | ||
Vehicle | 10 (ml/kg) | 1.15 ± 0.29 | 1.35 ± 0.26 | 1.46 ± 0.21 | 1.57 ± 0.21 | 1.68 ± 0.24 |
Indomethacin | 10 | 0.58 ± 0.27 (49.39% |
0.61 ± 0.18 (54.37% |
0.62 ± 0.14 (57.42% |
0.56 ± 0.11 (64.33% |
0.51 ± 0.11 (69.64% |
EFHB | 100 | 0.82 ± 0.19 (28.10% |
1.02 ± 0.13 (24.65% |
0.98 ± 0.16 (32.97% |
1.03 ± 0.18 (34.39% |
1.07 ± 0.12 (36.31% |
EFHB | 200 | 0.80 ± 0.17 (29.84% |
0.82 ± 0.15 (39.56% |
0.88 ± 0.21 (39.84% |
0.91 ± 0.15 (42.03% |
0.90 ± 0.16 (46.43% |
EFHB | 400 | 0.60 ± 0.14 (47.99% |
0.68 ± 0.17 (49.63% |
0.69 ± 0.15 (52.74% |
0.68 ± 0.21 (56.68% |
0.67 ± 0.13 (60.12% |
Here, each value is presented as the mean ± SEM
Effect of EFHB on pentobarbital-induced hypnosis in mice.
Treatment | Dose (mg/kg) | Time of onset of sleep (min) | Total sleeping time (min) |
---|---|---|---|
Vehicle | 10 (ml/kg) | 16.50 ± 0.81 | 37.80 ± 1.13 |
Diazepam | 1 (i.p.) |
|
|
EFHB | 100 |
|
|
EFHB | 200 |
|
|
EFHB | 400 |
|
|
Here, each value is presented as the mean ± SEM
Effects of EFHB on open field test in mice.
Treatment | Dose (mg/kg) | Number of movements | ||||||
---|---|---|---|---|---|---|---|---|
0 min | 30 min | 60 min | 90 min | 120 min | 180 min | 240 min | ||
Vehicle | 10 (ml/kg) | 112.30 ± 2.60 | 109.80 ± 2.15 | 110.40 ± 1.37 | 105.50 ± 1.31 | 101.80 ± 1.55 | 104.40 ± 0.95 | 106.30 ± 1.28 |
Diazepam | 1 (i.p.) |
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EFHB | 100 |
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EFHB | 200 |
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EFHB | 400 |
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Here, each value is presented as the mean ± SEM
Effects of EFHB on hole cross test in mice.
Treatment | Dose (mg/kg) | Number of movements | ||||||
---|---|---|---|---|---|---|---|---|
0 min | 30 min | 60 min | 90 min | 120 min | 180 min | 240 min | ||
Vehicle | 10 (ml/kg) | 11.10 ± 0.51 | 10.30 ± 0.68 | 10.20 ± 0.77 | 9.70 ± 0.34 | 9.20 ± 0.36 | 8.70 ± 0.57 | 7.50 ± 0.61 |
Diazepam | 1 (i.p.) |
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EFHB | 100 |
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EFHB | 200 |
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EFHB | 400 |
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Here, each value is presented as the mean ± SEM
A CNS depressant works either by decreasing the onset or by increasing the duration of sleep or both. EFHB increased the pentobarbitone-induced sedative effect in a dose dependent manner. Barbiturates naturally work on the cerebral cortex to generate their action [
Present study showed potent antinociceptive, anti-inflammatory, and sedative activities of EFHB. All activities were dose dependent and statistically significant. Presence of
The authors are responsible for the writing and contents of the paper.
The authors declare no conflicts of interest.
The authors are thankful to BCSIR Laboratories, Bangladesh Council of Scientific and Industrial Research, Dr. Qudrat-E-Khuda Road, Dhaka 1205, Bangladesh, for allowing them to use their research facilities.