Ism-1
Pale yellow amorphous powder, IR (KBr, Cm
−1), 3391, 3263, 1659, 1519. UV λ
MeOH max nm: 342 and 347;
1H-NMR (500 MHz, CD
3OD): δ 7.59 (d, 1H,
J
6′,5′ = 8.0 Hz, H-6′), 7.10 (br s, 1H, H-2′), 7.09 (d, 1H,
J
5′,6′ = 8.0 Hz, H-5′), 6.63 (s, 1H, H-2), 6.52 (s, 1H, H-8), 3.92 (s, 3H, H-4′/OCH
3), 3.90 (s, 3H, H-3′/OCH
3), 3.86 (s, 3H, H-6/OCH
3), δ EI-MS:
m/z 344 (M
+), 329, 326, 301 [
32].
Ism-2
Light yellow amorphous powder, 1H-NMR (500 MHz, acetone-
d
6) δ 12.98 (s, 1H, H-5/OH), 7.96 (d, 2H,
J
2′,3′/6′,5′ = 8.0 Hz, H-2/H-6), 7.03 (d, 2H,
J
3′,2′/5′,6′ = 8.0 Hz, H-3/H-5), 6.68 (s, 1H, H-3), 6.66 (d, 1H,
J
8,6 = 2.0 Hz, H-8), 6.32 (d, 1H,
J
6,8 = 2.0 Hz, H-6), 3.92 (s, 3H, H-4′/OCH
3). EI MS
m/z (%) 284 (M
+), 255 [
33].
Ism-3
Slight yellow powder, IR (KBr, Cm
−1), 3500, 1662, 1614, 1512. UV (AlCl
3 + MeOH): λmax: 484 (2.86), 272 (2.79) and 1H-NMR (500 MHz, MeOD) δ 6.18 (1H,
d, J = 2.0 Hz, H-6), 6.39 (1H,
d, J = 2.0 Hz, H-8), 6.88 (1H,
d, J = 8.3 Hz, H-5′), 7.62 (1H,
dd,
J = 8.3; 2.1 Hz, H-6′), 7.74 (1H,
d,
J = 2.1 Hz, H-2′, ESI-MS:
m/z 300.9 [M-H]-, 602.6 [2 M-H] [
34].
Ism-4
White, waxy powder, IR (KBr, Cm
−1), 3373.6, 2940.7, 2867.9, 1641.6, 1457.3, 1381.6, 1038.7, 881.6. UV λ CHCl
3 max nm: 220. 1H-NMR (500 MHz, CDCl3) δ (ppm) = 0.74 (1H,
s, H-18), 0.87 (1H,
d,
J = 6.9 Hz, H-27), 0.88 (1H,
d,
J = 6.9 Hz, H-26), 0.89 (1H,
t,
J = 7.4 Hz, H-29), 0.93 (1H,
d,
J = 6.5 Hz, H-21), 0.97 (1H,
m, H-24), 0.98 (1H,
m, H-9), 1.04 (1H,
m, H-14), 1.06 (1H,
s, H-19), 1.07 (1H,
m, H-22b), 1.11 (1H,
tm,
J = 11.2 Hz, H-15b), 1.13 (1H,
m, H-1b), 1.16 (1H,
t,
J = 10.0 Hz, H-17), 1.21 (1H,
m, H-23), 1.21 (1H,
m, H-12b), 1.30 (1H,
m, H-16b), 1.31 (1H,
m, H-28), 1.36 (1H,
m, H-22a), 1.40 (1H,
m, H-20), 1.50 (1H,
qd,
J = 10.8; 4.6 Hz, H-11b), 1.50 (1H,
m, H-7), 1.55 (1H,
m, H-11a), 1.56 (1H,
m, H-2b), 1.63 (1H,
m, H-15a), 1.71 (1H,
m, H-25), 1.88 (1H,
m, H-2a), 1.89 (1H,
m, H-16a), 1.90 (1H,
m, H-1a), 2.03 (1H,
td,
J = 12.1; 2.4 Hz, H-8), 2.06 (1H,
dt,
J = 12.8; 3.6 Hz, H-12a), 2.30 (1H,
td,
J = 11.0; 2.0 Hz, H-4b), 2.34 (1H,
ddd,
J = 13.0; 5.0; 2.0 Hz, H-4a), 3.58 (1H,
tt,
J = 11.3; 5.3 Hz, H- 3), 5.40 (1H,
dd,
J = 5.2; 2.3 Hz, H-6); ESI-MS:
m/z:437 [M + Na] + [
35].
The crude methanolic extract and sub-fractions in test doses of 500, 1000, 1500 and 2000 mg/kg body weight showed no adverse effects on the behavioral responses in the tested mice following 14 days observation. No mortality or weight change observed. Therefore, a highest dose of 300 mg/kg given to mice in this study was considered to be safe.
In performing the acetic acid induced writhing test for the determination of antinociceptive effect, the samples showed a significant analgesic effect. The crude methanolic extract, chloroform and ethyl acetate fraction showed a dose dependent response and significantly inhibited the acetic acid induced writhings with a maximum value of 62.64 % (
P < 0.01,
n = 6), 71.88 % (
P < 0.001,
n = 6) and 66.93 % (
P < 0.001,
n = 6), respectively as shown in Table
1. Whereas, the residual aqueous fraction showed no significant activity (data not shown). All the results were compared against standard (diclofenac sodium, 50 mg/kg) with 84.96 % response.
Table 1
Acetic acid induced analgesic activity of test samples of Artemisia macrocephala
Control (2 % Tween 80) | 67.59 ± 1.02 | --- |
Crd 150 mg 300 mg | 31.63±1.25** | 53.20 |
25.25±1.05** | 62.64 |
Chf 100 mg 200 mg | 27.25±1.20** | 59.68 |
19.00 ± 1.35*** | 71.88 |
EtOA 100 mg 200 mg | 28.45±1.15** | 57.90 |
22.35±1.20** | 66.93 |
Diclofenac sodium (50 mg) | 10.16±0.70*** | 84.96 |
After the administration of the crude extract and its sub-fractions to the animals treated with formalin, it showed a dose dependent response and significantly inhibited both the phases with 37.93 % (**
P < 0.01, n = 6), 51.34 % (***
P < 0.001, n = 6) and 45.33 (**
P < 0.01, n = 6) in the first phase and 56.87 % (***
P < 0.001, n = 6), 73.54 % (***
P < 0.001, n = 6) and 66.63 % (**
P < 0.01, n = 6) of crude methanolic extract (300 mg/kg), chloroform and ethyl acetate fraction (200 mg/kg) respectively, for the second phase. While the aqueous fraction showed no any prominent response (data not shown).
Animals treated with morphine (5.0 mg) significantly inhibited both the phases with 86.86 % (
***
P < 0.001,
n = 6) and 96.11 % (
***
P < 0.001,
n = 6) for first phase and second phase respectively as shown in Table
2. A reversal of inhibitory potential was observed in animals pre-treated with naloxone.
Table 2
To show effects of Artemisia macrocephala on formalin-induced paw-licking response
Control (2 % Tween 80) | 50.03 ± 1.63 | 72.00 ± 1.30 | ---- | |
Crd | 150 mg | 34.22±1.12** | 37.25±1.619** | 31.60 | 48.26 |
| 300 mg | 31.05±1.125** | 31.05±1.668*** | 37.93 | 56.87 |
Chf | 100 mg | 28.25±1.65** | 30.50±1.425** | 43.53 | 57.63 |
| 200 mg | 24.34±1.411*** | 19.05±1.039*** | 51.34 | 73.54 |
EtOA | 100 mg | 28.96±1.55** | 30.95±1.441*** | 42.11 | 57.01 |
| 200 mg | 27.35 ±1.05** | 24.02 ±0.95** | 45.33 | 66.63 |
Indomethacin (10 mg) | 39.83±1.55** | 18.66±1.542*** | 20.38 | 74.08 |
Morphine (5 mg) | | 6.41±1.165*** | 2.83±1.260*** | 86.86 | 96.11 |
N + Crd | 150 mg | 47.15±1.75 | 63.60±1.542 | 5.75 | 11.66 |
| 300 mg | 47.70±1.50 | 64.50±1.428 | 4.65 | 10.41 |
N + Chf | 100 mg | 47.80±1.25 | 65.35±1.30 | 4.45 | 9.30 |
| 200 mg | 48.05±1.33 | 66.65±1.45 | 3.95 | 7.43 |
N + EtOA | 100 mg | 47.75±1.23 | 64.85±1.40 | 4.55 | 9.93 |
| 200 mg | 47.95±1.25 | 65.95±1.416 | 4.15 | 8.40 |
N+ Indomethacin (10 mg) | 44.00±1.84** | 25.00 ±1.539*** | 12.05 | 65.27 |
N + Morphine (5 mg) | 47.66±1.52 | 71.83±1.142 | 2.39 | 1.37 |
The results show that naloxone caused a prominent reversal effect of the analgesic potential of morphine in both the phases. Indomethacin (10 mg/kg) mildly inhibited the first phase with 20.38 % (***
P < 0.001, n = 6) and significantly inhibited the second phase with 74.08 % (***
P < 0.001, n = 6).
The samples (crude methanolic extract 300 mg/kg, chloroform 200 mg/kg and ethyl acetate fraction 200 mg/kg) when tested for its effectiveness in tail flick model, significantly increased the latency time to 66.54 % (
**
P < 0.01,
n = 6), 82.94 % (
**
P < 0.01,
n = 6) and 70.53 % (
***
P < 0.001,
n = 6) respectively at 60 min at which morphine (opioid analgesic, centrally acting), showed 85.07 % (
***
P < 0.001,
n = 6) activity resembling more to that of the chloroform fraction. Naloxone treated animals significantly reduced the analgesic potentials of morphine and the test samples (Table
3).
Table 3
To show analgesic activity of Artemisia macrocephala (tail flick method) and standard
Control (2 % Tween 80) | 0.79±0.035 | 0.89±0.025 | 0.97±0.027 | 0.94±0.036 | 0.87±0.027 | 0.93±0.045 |
Crd 150 mg 300 mg | 0.97±0.130 (18.55 %) | 1.24±0.124* (28.22 %) | 1.69±0.139 (42.60 %) | 2.10±0.122** (55.23 %) | 2.34±0.134** (62.82 %) | 2.05±0.124** (54.63 %) |
1.09±0.130* (27.52 %) | 1.40±0.121* (36.42 %) | 1.92±0.129** (49.47 %) | 2.77±0.130** (66.06 %) | 3.18±0.165*** (72.64 %) | 3.03±0.140*** (69.30 %) |
Chf 100 mg 200 mg | 1.10±0.995* (28.18 %) | 1.35±0.399* (34.07 %) | 1.98±0.236** (51.01 %) | 3.37±0.220** (72.10 %) | 3.75±0.213*** (76.80 %) | 3.31±0.237*** (71.90 %) |
1.16±0.029* (31.89 %) | 1.41±0.021* (36.87 %) | 2.10±0.042** (53.80 %) | 5.29±0.060** (82.23 %) | 4.33±0.041*** (79.90 %) | 4.47±0.057*** (79.19 %) |
EtOA 100 mg 200 mg | 1.04±0.214* (24.03 %) | 1.29±0.241* (31.00 %) | 1.87±0.361** (48.12 %) | 3.10±0.421** (69.67 %) | 3.38±0.302** (74.26 %) | 2.76±0.280*** (66.30 %) |
1.10±0.201* (28.18 %) | 1.33±0.202* (33.08 %) | 1.91±0.350** (49.21 %) | 3.19±0.435** (70.53 %) | 3.30±0.313*** (73.63 %) | 2.79±0.291*** (66.66 %) |
Standard (Morphine 5 mg) | 1.52 ±0.024 (48.02 %) | 2.03 ±0.066 (56.15 %) | 3.89±0.038 (75.06 %) | 6.30±0.054 (85.07 %) | 4.40±0.050 (80.22 %) | 4.30±0.074 (78.37 %) |
N +Crd 150 mg 300 mg | 0.79±0 .1 0 8 | 0 .95±0 .044 | 0 . 99±0.045 | 0.99±0.064 | 0.95±0.026 | 0.81±0.042 |
0.84±0.046 | 0.98±0.031 | 1.14±0.047 | 0.99±0.035 | 0.96±0.051 | 0.92±0.034 |
N +Chf 100 mg 200 mg | 0.93±0.048 | 0.92±0.036 | 1.10±0.031 | 1.10±0.047 | 0.90±0.038 | 0.93±0.061 |
0.87±0.037 | 0.91±0.065 | 1.50±0.049 | 0.99±0.038 | 0.89±0.029 | 0.95±0.044 |
N +EtOA 100 mg 200 mg | 0 . 89±0.03 3 | 0 .93±0 .0 5 1 | 1.20±0.051 | 1.11±0.034 | 0.91±0.061 | 0.90±0.056 |
0.85±0.037 | 0.90±0.065 | 0.99±0.049 | 1.12±0.038 | 0.91±0.029 | 0.99±0.044 |
Morphine (5 mg) + Naloxone (2 mg) | 0.77±0.022 | 0.87±0.036 | 0.94±0.040 | 0.91±0.034 | 0.91±0.030 | 0.91±0.042 |