ANTI-INFLAMMATORY AND ANALGESIC ACTIVITY OF METHANOLIC EXTRACT OF WHOLE PLANT OF SIDA ACUTA (LINN.F.ND)

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Assessment of Analgesic activity
Acetic acid induced writhing test

Writhing is defined as stretch, torsion to one side and retraction of the abdomen and opisthotonous so that the belly of the mouse touches to the floor. The test was conducted on five groups of five mice each. Abdominal writhing episodes were induced in mice by i.p. injection of acetic acid (10 ml/kg of 0.6 % acetic acid). Mice were pretreated with vehicle or MESA or indomethacin 1 h prior to acetic acid injection and thereafter the observations were made. The number of stretching occurred for 30 min. immediately after the acetic acid injection was recorded. The mean number of stretching per group were calculated and represented as % inhibition of stretching movement with control group as below.
% Analgesic activity = (n-n/n) ×100
Where,   n is the average no of stretching of control group and
n′ is the average no of stretching in test group [2]

Hot plate analgesia in mice
The test was conducted on five mice groups of five mice each. The mice were placed on hot plate maintained at 55±1°C and time in seconds for the paw licking or jumping was recorded as the reaction time. Mice which have shown basal reaction time period below 6 seconds were selected and used for the screening. Mice were fasted for 12 h before test to be started. Mice were pretreated with vehicle or MESA or MESR or pentazocin (10 mg/kg i.p.). The mouse activity on hot plate was closely observed and the reaction time (latency period) was recorded in seconds as the time taken for the animal to react to the thermal pain by licking its paw or attempting to jump out. [22]

Statistical analysis
The data were analyzed by one way ANOVA followed by Dunnett’s multiple comparison post hoc test using Graph Pad Prism 5, software (Graph Pad Software Inc, USA). The difference of p<0.05 was considered significant in all the cases.

Results
Acute oral toxicity study

Acute oral toxicity studies revealed that the both the extracts MESA and MESR were safe up to a dose level of 2000 mg/kg of body weight (limit test) and LD50 is more than 2000mg/kg. No lethality or any toxic reactions or moribund state were observed up to the end of the study period.

Assessment of Anti-inflammatory activity
One-way repeat measure ANOVA showed significant (p<0.0001) influence of MESA and MESR on carrageenan-induced inflammation. Dunnett’s test indicated MESA at 400 mg/kg caused significant (p<0.01) decrease in paw oedema compared to vehicle while lower dose 100 mg/kg did not show any effect. MESA 400 mg/kg produced a reduction in paw oedema (58.28 %) as shown in table 1.

Assessment of Analgesic activity
Effect of MESA on acetic acid induced writhing

One-way ANOVA showed significant (p<0.001) influence of MESA on acetic acid induced writhing compared to control. Post hoc Dunnett’s test showed that MESA at 400 mg/kg caused significant inhibition of writhing (p>0.05). The maximum inhibition was noted for MESA 400mg/kg (83.78%). The effects were comparable with that of reference standard, indomethacin that showed 78.97% inhibition of writhing (p<0.001) as shown in Table 1.

Effect of MESA on hot plate analgesia
One-way repeat measure ANOVA exhibited significant (P<0.001) influence of MESA on hot plate reaction time. Dunnett’s test showed that for MESA at 400 mg/kg showed significant (P<0.01-0.001) increase in reaction time but the dose of 100 mg/kg did not produce any effect compared to control (P>0.05). The effects very much comparable with pentazocin 10 mg/kg that showed increase (P<0.001) in reaction time from 3.51 to 7.88 sec. at the end of 150 min as shown in table 2.

Table 1.  Effect of MESA and MESR on carrageenan-induced rat paw edema

Ø Treat
ment (mg/kg) 

Ø Change in Paw oedema (mm)

Ø
Percent Inhibition         at 3rd hr

Ø
Percent Inhibition         at 5th hr

ØAcetic acid-induced writhing response in mice

Ø 1st hr

Ø  3rd hr

Ø  5th hr

No. of writhing episodes
/30 min

% inhibition of writhing

Control

0.68

±0.022

0.76

±0.031

0.80

±0.042

-----

-----

66.60 ± 6.84

-

MESA
100

0.57

±0.035

0.62

±0.021

0.64

±0.014

18.42

20.00

63.20 ± 5.44

5.10%

MESA
200

0.36 *

±0.010

0.42 *

±0.042

0.48 *

±0.015

44.73

40.00

43.60 ± 4.17*

34.98%

MESA
400

0.32 *

±0.011

0.35 *

±0.008

0.38 *

±0.020

53.94

52.5

10.80 ± 1.24**

83.78%

IND

0.22*

±0.020

0.28*

±0.020

0.32*

±0.038

63.15

60.00

14.0 ± 1.81**

78.97%

Values are means± S.E.M for five animals.
*P<0.001,**P<0.001, compared to vehicle control

Table 2. Effect of MESA on hot plate analgesia

Treatment mg/kg

Hot plate reaction time (sec.)

 

0 min

30 min

60 min

90 min

120 min

150 min

MESA 100

3.822 ± 0.511

3.820 ± 0.482

3.964 ± 0.698

4.400 ± 0.847

5.094 ± 0.716

4.938 ± 0.541

MESA 200

4.124* ± 0.775

4.904* ± 0.868

4.896* ± 0.533

6.258* ± 1.056

5.890 * ± 0.905

4.980* ± 0.789

MESA 400

3.960** ± 0.487

4.902** ± 0.545

5.438** ± 0.484

5.912** ± 0.426

6.328** ± 0.699

5.956** ± 0.397

Pentazocin 5

3.518** ± 0.155

4.330** ± 0.129

5.622** ± 0.063

6.120** ± 0.077

7.062** ± 0.236

7.884** ± 0.078

 Values are mean ± S.E.M of five mice.
*P<0.01, **P<0.001, compared to control group.

Discussion
Inflammation is a tissue-reaction to infection, irritation or foreign substance. It is a a part of the host defence mechanism but when it becomes great it is a hopeless condition. Carrageenan induced  rat paw edema is commonly used as an experimental animal model for evaluation of anti-inflammatory potential of natural products and is believed to be biphasic.[22] The present study revealed that revealed that methanolic extract of whole plant of Sida acuta significantly suppressed the carrageenan challenge due to the inhibition of the enzyme cyclo-oxygenase and subsequent inhibition of prostaglandin synthesis. Various chemical constituents of plant origin such as beta-sitosterol, ephedrine, heraclenin, scopoletin are reported to have anti-inflammatory activity. The constriction response of abdomen produced by acetic acid is a sensitive procedure for peripheral analgesic agents. This response is believed to be mediated by the prostaglandin pathways.[23] The methanolic extract of Sida acuta have analgesic effects and thus indicates the presence of analgesic components that might influence the prostaglandin pathways. In the radiant heat tail flick test, the extract prolonged the stress tolerance capacity of the mice, indicating the possible involvement of a higher center.[24]

In conclusion, this work has demonstrated that extract from the whole plant of Sida acuta exhibited anti-inflammatory and analgesic activities. But the exact mechanism by which S. acuta exerts its anti-inflammatory and analgesic activity is not determined yet and needs further investigation to elucidate the other active compounds and underlying mechanism(s).

References
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