INTRODUCTION:

Herbal medicines derived from plant extracts are being increasingly utilized to treat a variety of clinical diseases, though relatively little knowledge about their mode of action is available. There is a growing interest in pharmacological evaluation of various plants used in Indian traditional system of medicine. The study of plant species with analgesic effects is still a fruitful research in search of new painkillers. Abutilon indicum Linn. (Sweet) belonging to the family Malvaceae is an erect pubescent sub shrub found throughout hotter regions of India and as a weed in the sub Himalayan tract and other hills. The dried whole plant is used as a febrifuge, anthelminthic, diuretic and anti-inflammatory (in uterine and urinary discharges, piles etc) in traditional medicines¹. The cooked leaves were eaten for bleeding piles. The plant contains alkaloids, flavanoids, sterols, mucilage, tannins, asparagines, gallic acid and sesquiterpenes. Gallic acid is attributed to possess analgesic activity¹. The alcoholic and aqueous extract of the plant possess hypoglycemic effect²; hepatoprotective effects³ and was studied for uterotropic and uterine peroxidase activities in ovariectomized rats⁴. The presence of the gallic acid in the whole plant including the leaf and the lacuna of analgesic activity unreported have attracted us to evaluate the antinociceptive potential, and hence the present study was carried out.

MATERIALS AND METHODS:

Plant material and Extraction:

The leaves of the plant Abutilon indicum were collected during the month of July from rural areas of Chennai, Tamil Nadu, India.

The identity of the leaf specimen was confirmed by Dr. P. Jayaraman, Botanist, Plant Anatomy Research Centre (Chennai, Tamil Nadu, India). A voucher specimen was deposited in the departmental herbarium of C. L. Baid Metha College of Pharmacy. The coarsely powdered, shade-dried leaves of A. indicum (150 g) were extracted with ethanol for 72 h in a Soxhlet extractor. The dark-green extract obtained was cooled, filtered, and the solvent recovered by distillation in vacuo. The drug residue stored in a desiccator was used for subsequent experiments. The w / w yield in terms of dry material was 19. 4%. The dried extract was suspended in 0.5 % carboxy methylcellulose for animal use.

Animals:

Healthy Swiss albino mice (20-25 g) of either sex were used for the study. The animals were housed in polypropylene cages, maintained under standard conditions (12-h light: 12-h dark cycle; 25 ± 3°C; 50-60% humidity). They were fed with standard pelleted diet (Hindustan Lever Pvt Ltd, Mumbai, India) and water ad libitum. The study was approved by the Institutional Animal Ethical Committee of CLBMCP, Chennai, India. (Ref No: IAEC / CLBMCP / 12 / 15)

Acute toxicity study:

Wistar albino mice of either sex weighing 25-30 g (3 animals/dose) selected by random sampling technique was performed as per OECD-423 guidelines (acute class method)⁵. The animals were fasted overnight, provided only water, after which extract was administered to the respective groups orally at the dose level of 5 mg / kg body weight by gastric intubation. If mortality was observed in 2 or 3 animals, then the dose administered was assigned as a toxic dose. If mortality was observed in one animal, then the same dose was repeated again to confirm the toxic dose. If mortality was not observed, the procedure was repeated for further higher doses such as 50, 300 and 2000 mg / kg body weight. The animals were observed for toxic symptoms such as behavioral changes, locomotion, convulsions and mortality for 72 h.

Table 1.Effect of Ethanol extract of A. indicum on acetic acid induced writhing in mice

Drug Treatment No. of writhes Inhibition (%)

Control (1%CMC) 23 ± 1.01 -----

Indomethacin (10 2.67 ± 0.91^*** 88.39

mg / kg)

EAI (100 mg /kg) 9.44 ± 1.34^** 58.89

EAI (300 mg /kg) 7.33 ± 1.88^** 68.81

Data represent mean ± S.E.M, n = 6, ^**p<0.01, ^***p<0.001. One-Way ANOVA followed by Dunnet‘t’ test.

Evaluation of Analgesic activity:

The following experimental protocol was used to study the analgesic activity. The animals were divided into four groups comprising of six animals each for both the studies.

Group I: Control (0.5 % Carboxy methyl cellulose, CMC)

Group II: Reference standard (Indomethacin 10 mg/kg i.p. and Morphine 7.5 mg/kg i.p.)

Group III: EAI (100 mg/kg p.o.)

Group IV: EAI (300 mg/kg p.o.)

Writhing Test:

Acetic acid (0.6 % v/v) was administered intraperitonially to all the groups at the dose of 1 ml/kg body weight 60 min after the administration of test compounds. Abdominal constriction and full extension of hind limbs, called a writhe was produced. Analgesia was recorded by counting the number of writhing after the injection of acetic acid for a period of 10 min. Significant reduction in the number of writhes by drug treatments as compared to vehicle control animals was considered as a positive analgesic response and the percentage inhibition of writhing was calculated⁶

Hot-plate Test:

In hot-plate test, mice were preselected on the hot plate at 55±0.5°C. Animals were then treated with standard (morphine, 7.5 mg / kg) and test compounds (EAI, 200 and 400 mg / kg). The reaction time for each mouse was determined on the hot plate at 60 min after treatment. To avoid possible injury, a cut-off period of 15 sec was followed while measuring the reaction time, the time taken in sec for fore paw licking or jumping was taken as reaction time⁷.

Tail Immersion test:

Basal reaction time of mice towards the heat source was recorded by immersing the lower 5 cm portion if the tail into a beaker of water maintained at 55±0.5°C. The time in sec for tail withdrawal from the water was taken as the reaction time, with a cut-off time of immersion set at 10 sec. The reaction time was measured 1 h after administration of EAI, 200 and 400 mg / kg and Morphine (7.5 mg /kg)⁸

Table 2. Effect of Ethanol extract of A. indicum on Hot plate test in mice

Drug treatment Reaction time (in sec) Inhibition (%)

Control (1%CMC) 2.83 ± 0.21 ----

Morphine (7.5 10.26 ± 1.15^*** 262.54

mg / kg)

EAI (100 mg /kg) 5.21 ± 0.17^** 83.03

EAI (300 mg /kg) 6.26 ± 0.42^** 121.01

Data represent mean ± S.E.M, n = 6, ^**p<0.01, ^***p<0.001. One-Way ANOVA followed by Dunnet‘t’ test.

Formalin test:

Mice were treated with the extracts at, 100 and 300 mg / kg i.p. with 20μl of formalin (in 0.9 % saline, subplantar) and the duration of paw licking or biting responses of the injected paw was taken as an indicator of pain response. Responses were measured for 5 min after formalin injection (first phase) and then after 15-30 min (second phase). Standard drug (Morphine, 7.5 mg /kg) and the extracts were administered 60 min and 30 min, respectively before formalin injection⁹.

Statistical analysis:

One-way analysis of variance (ANOVA) followed by Dunnet’s t-test was applied for determining the statistical significance of difference between experimental groups. Values were considered significant when p < 0.05.

RESULTS:

Acute toxicity study:

The extract did not produce any toxic symptoms of mortality up to the dose level of 2000 mg/kg body weight in mice, and hence the drugs were considered safe for further pharmacological screening. According to the OECD-423 guidelines for acute oral toxicity, the LD₅₀ dose of 2000 mg/kg and above is categorized as unclassified.

Antinociceptive activity:

EAI produced significant antinociception (p<0.01) which was evidenced from the reduction in the writhing movements evoked by the injection of acetic acid (0.6 %) at the tested doses (Table 1). The reaction time was significantly increased (p<0.01) by the administration of the extract at dose of 200 and 400 mg / kg in hot plate and hot water tail immersion tests (Table 2 and 3). There was a dose dependent inhibition of both phases of the formalin-induced pain responses in mice with a more potent effect on the

Table 3. Effect of Ethanol extract of A. indicum on Tail immersion test

Drug treatment Reaction time (in sec) Inhibition (%)

Control (1%CMC) 2.6 ± 0.54 ---

Morphine (7.5 6.0 ± 0.70^*** 130.76

mg / kg)

EAI (100 mg /kg) 3.4 ± 0.54^* 30.76

EAI (300 mg /kg) 4.8 ± 0.83^** 84.61

Data represent mean ± S.E.M, n = 6, ^*p<0.05, ^**p<0.01, ^***p<0.001. One-Way ANOVA followed by Dunnet‘t’ test.

second than the first phase. The effect was more prominent at a dose of 400 mg / kg (p<0.01) (Table 4).

DISCUSSION:

The potential analgesic effect of EAI was estimated using both chemical and thermal methods in mice. The analgesic test used was chosen in order to test different nociceptive stimuli, namely cutaneous thermic (hot plate, tail immersion), chemical visceral (writhing) stimuli and chronic (formalin-induced). Acetic acid-induced writhing test was used for detecting both central and peripheral analgesia, whereas hot plate and tail immersion tests are most sensitive towards centrally acting analgesics.

In acetic acid-induced abdominal writhing, a visceral pain model, the release of arachidonic acid via cyclooxygenase and prostaglandin plays a role in the nociceptive mechanism¹⁰. Administration of EAI produces a significant reduction in the number of abdominal constrictions and stretching of hind limbs induced by the injection of acetic acid in a dose-dependent manner. EAI 200 and 400 mg/kg exhibited a writhing inhibition of 58.39% and 68.81% respectively, while the standard drug had 88.39% inhibition.

The hot plate method and tail immersion methods were found to be suitable for evaluation of centrally acting analgesic¹¹. The centrally acting analgesic exerts their action through µ receptors indicating narcotic involvement¹². These analgesics generally increase the pain threshold of the animals towards thermal nociception. EAI significantly increased the reaction time of the animals towards the thermal source in a dose-dependent manner. In hot plate method, EAI 200 and 400 mg/kg showed a pain inhibition of 83.03% and 121.01% respectively, standard drug morphine producing 262.54 % inhibition. In tail immersion method, the drug exhibited greater activity at 60 min post drug administration, in which EAI 200 and 400 mg/kg exhibited an inhibition of 30.76 % and 84.61 % as against 130.76 % inhibition produced by morphine 7.5 mg / kg. In thermal methods, EAI exhibited greater percentage of

inhibition and comparable with the standard drug. This clearly indicates that the analgesic activity of EAI may be mediated by central mechanism.

In formalin test, the response to formalin is biphasic with an early and a late phase involving different mechanisms of nociception¹³. There was a significant, dose dependent inhibition of both phases of the formalin-induced pain response in mice, with a more potent effect on the second than the first phase. The formalin pain test is a very useful method for evaluating the mechanism of pain and analgesia. Drugs which, act mainly centrally, such as narcotic analgesics, inhibits both phases of pain in this model, while peripherally acting drugs, such as indomethacin or acetyl salicylic acid only inhibit the late phase¹⁴.The early phase (immediately after injection) seems to be caused by C-fibre activation due to the peripheral stimulus. The late phase (starting approximately 20 min after formalin injection) appears to depend on the combination of an inflammatory reaction, activation of N-Methyl D-Alanine (NMDA) and non-NMDA receptors and the Nitric Oxide (NO) cascade in the peripheral tissue ¹⁵ and functional changes in the dorsal horn of the spinal cord¹⁶. Both these functional changes appear to be related to excitatory amino acid (EAA) release in the spinal cord and activation of NMDA receptor subtypes. Thus, formalin test has been used to evaluate the antinociceptive effects of competitive and non-competitive NMDA receptor antagonist¹⁷.

From the results, it could be concluded that the extract exhibits analgesic activity by both central and peripheral mechanisms. Even if further studies are needed this seems to provide a rationale for the use of this plant in pain and inflammatory disorders.

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Received on 24.03.2009 Modified on 22.04.2009

Research J. Pharm. and Tech.2 (3): July-Sept. 2009,;Page 544-547