Background
Methods
Plant collection
Extract preparation
Animals
Acute toxicity studies
Pharmacological activities
Analgesic studies
Acetic acid induced writhing test
Hot plate test
Anti-inflammatory activity
In vitro anti-inflammatory activities
Inhibition of TNF-α activated NFkB assay
Inhibition of LPS-induced nitric oxide synthesis (nitrite assay)
Cytotoxicity studies for dose response
Cytotoxicity studies for timed response
In vivo anti-inflammatory studies
Carrageenan induced paw edema
Subcutaneous air pouch
Cell count
Inflammatory mediators
Gas chromatography-mass spectrometry of FXM
Statistical analysis
Results
Peripheral analgesic effect
Acetic acid induced writhing test
Groups | Drug (dose), route | No. of writhing (Mean ± SD) | % Inhibition |
---|---|---|---|
Saline | 2 ml, i.p. | 71.00 ± 2.52a
| 0 |
FXM | 100 mg/kg, p.o. | 30.50 ± 1.87g
| 57.04 ± 3.63 |
FXM | 200 mg/kg, p.o. | 22.00 ± 3.41h
| 69.01 ± 4.98 |
FXH | 100 mg/kg, p.o. | 63.50 ± 1.87b
| 10.56 ± 2.63 |
FXH | 200 mg/kg, p.o. | 51.00 ± 2.19de
| 28.16 ± 3.08 |
FXC | 100 mg/kg, p.o. | 39.16 ± 3.48f
| 44.83 ± 4.91 |
FXC | 200 mg/kg, p.o. | 16.16 ± 3.62ij
| 77.23 ± 5.64 |
FXE | 100 mg/kg, p.o. | 59.00 ± 1.41bc
| 16.90 ± 2.02 |
FXE | 200 mg/kg, p.o. | 48.00 ± 2.44e
| 32.39 ± 3.14 |
FXB | 100 mg/kg, p.o. | 55.66 ± 1.63cd
| 21.59 ± 2.30 |
FXB | 200 mg/kg, p.o. | 52.16 ± 1.16de
| 26.52 ± 1.64 |
FXA | 100 mg/kg, p.o. | 62.00 ± 1.78b
| 12.67 ± 2.51 |
FXA | 200 mg/kg, p.o. | 54.33 ± 1.75cd
| 23.47 ± 2.46 |
Diclofenac sodium | 10 mg/kg, i.p. | 14.16 ± 2.60j
| 80.04 ± 5.25 |
Aspirin | 10 mg/kg, i.p. | 19.16 ± 2.46hi
| 73.00 ± 4.87 |
Central analgesic effect
Hot plate method (Thermal stimulation)
Treatment | Dose/route | Mean latency time in seconds | Increase in latency time (seconds) after various dosages (Mean ± SD)/Percent analgesia | ||
---|---|---|---|---|---|
0 min | 30 min | 60 min | 120 min | ||
Saline | 2 ml, i.p. | 8.83 ± 0.75 | 10.33 ± 0.51 | 9.83 ± 0.40 | 9.50 ± 0.44 |
7.03 ± 2.41e
| 4.65 ± 2.87h
| 3.03 ± 2.69i
| |||
FXM | 100 mg/kg, p.o. | 9.33 ± 0.51 | 13.00 ± 0.89 | 15.50 ± 0.54 | 17.00 ± 0.63 |
17.61 ± 6.27c
| 29.80 ± 3.19e
| 37.10 ± 2.44cd
| |||
FXM | 200 mg/kg, p.o. | 9.66 ± 1.03 | 16.50 ± 0.54 | 21.33 ± 1.03 | 23.66 ± 1.03 |
33.46 ± 4.41b
| 57.35 ± 4.72c
| 68.72 ± 5.71b
| |||
FXH | 100 mg/kg, p.o. | 9.50 ± 0.54 | 10.33 ± 0.51 | 12.00 ± 0.89 | 13.66 ± 0.51 |
4.00 ± 3.59de
| 12.10 ± 5.78fgh
| 20.31 ± 1.77fgh
| |||
FXH | 200 mg/kg, p.o. | 9.50 ± 0.54 | 10.66 ± 0.51 | 13.66 ± 0.51 | 15.66 ± 0.51 |
5.63 ± 3.56de
| 20.31 ± 1.77ef
| 30.00 ± 4.08def
| |||
FXC | 100 mg/kg, p.o. | 9.16 ± 0.75 | 12.33 ± 0.51 | 15.00 ± 0.89 | 18.00 ± 0.89 |
15.13 ± 3.23cd
| 27.99 ± 3.46e
| 42.33 ± 4.83c
| |||
FXC | 200 mg/kg, p.o. | 9.00 ± 0.63 | 15.66 ± 0.51 | 18.66 ± 0.51 | 25.00 ± 0.89 |
31.69 ± 3.14b
| 45.99 ± 3.89d
| 76.13 ± 4.49ab
| |||
FXE | 100 mg/kg, p.o. | 9.66 ± 0.51 | 12.00 ± 0.89 | 12.33 ± 0.51 | 13.33 ± 0.51 |
11.46 ± 4.01cde
| 13.05 ± 3.72fgh
| 18.01 ± 2.37fgh
| |||
FXE | 200 mg/kg, p.o. | 9.66 ± 0.81 | 12.33 ± 0.51 | 14.66 ± 0.51 | 16.66 ± 0.89 |
13.02 ± 3.81cd
| 24.54 ± 2.44e
| 34.39 ± 3.24cde
| |||
FXB | 100 mg/kg, p.o. | 9.33 ± 1.03 | 10.33 ± 0.72 | 12.33 ± 0.51 | 14.00 ± 0.89 |
4.60 ± 2.11de
| 14.42 ± 2.43fg
| 22.53 ± 3.36fgh
| |||
FXB | 200 mg/kg, p.o. | 9.83 ± 0.75 | 11.16 ± 0.89 | 12.83 ± 0.51 | 15.00 ± 0.89 |
5.63 ± 3.42de
| 10.59 ± 6.40gh
| 25.53 ± 5.14efg
| |||
FXA | 100 mg/kg, p.o. | 9.50 ± 0.54 | 9.66 ± 0.51 | 11.66 ± 0.51 | 12.33 ± 0.51 |
3.75 ± 0.65c
| 10.47 ± 4.55gh
| 13.77 ± 3.41h
| |||
FXA | 200 mg/kg, p.o. | 9.00 ± 0.89 | 10.33 ± 0.51 | 12.00 ± 0.89 | 13.66 ± 1.03 |
6.13 ± 4.21cde
| 14.30 ± 1.61fg
| 22.25 ± 2.68fgh
| |||
Diclofenac sodium | 10 mg/kg, i.p. | 9.83 ± 0.75 | 21.66 ± 0.51 | 26.66 ± 1.51 | 26.66 ± 1.62 |
58.69 ± 1.45a
| 83.51 ± 4.21a
| 83.39 ± 5.07a
| |||
Morphine | 10 mg/kg, i.p. | 9.33 ± 0.51 | 20.16 ± 0.75 | 23.66 ± 0.81 | 23.00 ± 0.89 |
52.34 ± 4.59a
| 69.36 ± 3.84b
| 66.03 ± 5.13b
|
In vitro anti-inflammatory activities
Inhibition of TNF-α activated nuclear factor kappa-B (NFkB assay)
Inhibition of LPS-induced NO production (nitrite assay)
Time dose response studies
Carrageenan induced paw edema
Treatment | Dose/route | Mean paw volume before carrageenan injection | Increase in paw volume (ml) after carrageenan injection (Mean ± SD)/Percent inhibition of edema | |||
---|---|---|---|---|---|---|
+1 h | +2 h | +3 h | +4 h | |||
Saline | 2 ml, i.p. | 1.12 ± 0.08 | 2.58 ± 0.09 | 2.67 ± 0.09 | 2.21 ± 0.09 | 1.81 ± 0.11 |
FXM | 100 mg/kg, p.o. | 1.11 ± 0.08 | 2.52 ± 0.06 | 2.30 ± 0.09 | 2.12 ± 0.20 | 1.48 ± 0.08 |
5.88 ± 1.99c
| 19.72 ± 2.39cde
| 35.84 ± 12.89cd
| 45.28 ± 4.02bc
| |||
FXM | 200 mg/kg, p.o. | 1.17 ± 0.09 | 2.44 ± 0.04 | 2.05 ± 0.06 | 1.92 ± 0.04 | 1.45 ± 0.06 |
15.34 ± 7.95b
| 41.07 ± 5.00b
| 52.51 ± 8.30b
| 58.52 ± 8.19b
| |||
FXH | 100 mg/kg, p.o. | 1.15 ± 0.08 | 2.61 ± 0.08 | 2.56 ± 0.09 | 2.51 ± 0.11 | 1.71 ± 0.08 |
2.84 ± 2.42c
| 4.99 ± 3.22g
| 13.44 ± 5.41e
| 18.81 ± 2.21e
| |||
FXH | 200 mg/kg, p.o. | 1.11 ± 0.05 | 2.55 ± 0.05 | 2.33 ± 0.06 | 2.32 ± 0.05 | 1.61 ± 0.04 |
3.96 ± 2.28c
| 17.89 ± 2.03de
| 23.29 ± 4.53de
| 27.88 ± 6.75de
| |||
FXC | 100 mg/kg, p.o. | 1.14 ± 0.06 | 2.58 ± 0.06 | 2.21 ± 0.06 | 2.06 ± 0.13 | 1.53 ± 0.08 |
3.85 ± 1.85c
| 28.28 ± 3.11c
| 42.12 ± 7.49bc
| 43.57 ± 5.21bc
| |||
FXC | 200 mg/kg, p.o. | 1.09 ± 0.04 | 2.17 ± 0.04 | 1.71 ± 0.03 | 1.53 ± 0.04 | 1.25 ± 0.04 |
29.19 ± 4.60a
| 59.67 ± 3.23a
| 73.29 ± 2.65a
| 77.64 ± 3.04a
| |||
FXE | 100 mg/kg, p.o. | 1.12 ± 0.07 | 2.59 ± 0.06 | 2.47 ± 0.04 | 2.34 ± 0.06 | 1.55 ± 0.08 |
1.71 ± 0.82c
| 8.76 ± 3.66fg
| 22.32 ± 6.02de
| 36.22 ± 8.23cd
| |||
FXE | 200 mg/kg, p.o. | 1.12 ± 0.06 | 2.56 ± 0.07 | 2.25 ± 0.05 | 2.18 ± 0.05 | 1.49 ± 0.05 |
4.08 ± 1.78c
| 23.60 ± 2.10cd
| 32.38 ± 4.66cd
| 45.77 ± 4.22bc
| |||
FXB | 100 mg/kg, p.o. | 1.11 ± 0.06 | 2.59 ± 0.04 | 2.50 ± 0.05 | 2.32 ± 0.07 | 1.62 ± 0.07 |
1.49 ± 1.71c
| 6.25 ± 1.15fg
| 23.07 ± 4.45de
| 25.68 ± 3.08de
| |||
FXB | 200 mg/kg, p.o. | 1.11 ± 0.06 | 2.55 ± 0.07 | 2.30 ± 0.06 | 2.19 ± 0.08 | 1.52 ± 0.06 |
3.63 ± 1.31c
| 19.84 ± 3.34cde
| 31.08 ± 5.33cd
| 39.89 ± 7.36cd
| |||
FXA | 100 mg/kg, p.o. | 1.10 ± 0.06 | 2.58 ± 0.06 | 2.48 ± 0.09 | 2.50 ± 0.08 | 1.70 ± 0.05 |
1.60 ± 1.16c
| 7.05 ± 2.71fg
| 11.17 ± 2.82e
| 13.18 ± 3.04e
| |||
FXA | 200 mg/kg, p.o. | 1.12 ± 0.06 | 2.59 ± 0.05 | 2.39 ± 0.04 | 2.36 ± 0.07 | 1.63 ± 0.01 |
1.82 ± 1.64c
| 14.47 ± 4.31ef
| 20.80 ± 2.55de
| 24.94 ± 7.76de
| |||
Diclofenac sodium | 10 mg/kg, i.p. | 1.13 ± 0.08 | 2.57 ± 0.05 | 1.63 ± 0.05 | 1.46 ± 0.08 | 1.24 ± 0.04 |
4.08 ± 2.47c
| 67.32 ± 4.25a
| 80.54 ± 2.61a
| 85.23 ± 7.71a
|
Subcutaneous air pouch assay
Effect of FXC on the hematology of air pouch exudate
Dose/route | Neutrophils | Monocytes | Lymphocytes | WBCs | |
---|---|---|---|---|---|
Saline | 2 ml i.p. | 0.150 ± 0.009d
| 0.079 ± 0.010d
| 0.472 ± 0.040d
| 0.945 ± 0.0982d
|
Carrageenan | 10 ml/kg p.o. | 6.027 ± 0.350a
| 1.972 ± 0.136a
| 12.665 ± 1.456a
| 23.422 ± 1.556a
|
Diclofenac sodium | 10 mg/kg p.o. | 4.494 ± 0.172c
| 1.445 ± 0.114bc
| 10.978 ± 1.231ab
| 17.521 ± 1.201b
|
FXC | 100 mg/kg p.o. | 5.082 ± 0.238b
| 1.665 ± 0.125b
| 9.240 ± 0.857b
| 16.932 ± 0.974b
|
FXC | 200 mg/kg p.o. | 4.285 ± 0.153c
| 1.353 ± 0.122c
| 5.698 ± 0.483c
| 12.199 ± 1.571c
|
Effect of FXC on inflammatory mediators of air pouch exudate
Estimated values/(Percent inhibition) | |||||
---|---|---|---|---|---|
Dose/route | TNF-α (pg/ml) | IL-6 (pg/ml) | NO (μM/ml) | PGE2 (pmole/ml) | |
Saline | 2 ml i.p. | 5.50 ± 1.04d
| 27.66 ± 5.00c
| 0.443 ± 0.058d
| 1301.8 ± 90.87b
|
Carrageenan | 10 ml/kg p.o. | 964.50 ± 95.525a
| 6291.3 ± 562.77a
| 0.784 ± 0.105a
| 1525.7 ± 75.76a
|
Diclofenac sodium | 10 mg/kg p.o. | 418.50 ± 57.83c
| 4544.8 ± 235.87b
| 0.362 ± 0.043c
| 1339.7 ± 25.17b
|
56.44 ± 6.01 | 34.03 ± 3.42 | 56.11 ± 4.99 | 7.69 ± 1.73 | ||
FXC | 100 mg/kg p.o. | 592.33 ± 45.31b
| 5146.2 ± 257.58b
| 0.605 ± 0.060b
| 1404.7 ± 21.37ab
|
38.35 ± 4.71 | 25.30 ± 3.73 | 26.71 ± 7.33 | 3.21 ± 1.47 | ||
FXC | 200 mg/kg p.o. | 505.67 ± 36.037bc
| 4398.2 ± 159.46b
| 0.408 ± 0.049bc
| 1309.8 ± 30.46b
|
47.37 ± 3.75 | 36.16 ± 2.31 | 50.52 ± 5.96 | 9.74 ± 2.09 |
Gas chromatography-mass spectrometry of FXM
Compound name | Area % | Class | |
---|---|---|---|
1. | 7-(diphenylmethylene) -6-phenylbicyclo [3.2.0] hept-2-en-endo-6-ol | 2.32 | Alcohol |
2. | 1-[1-(Bromomethyl) 2 (2,2,2-trichloroethyl) cyclopent-5-y-l]-(1H,3H)-5-methyl-pyrimidine-2,4-dione | 1.15 | δ Lactam |
3. | 2,2-Dimethyl-5-(2′,2′-dideuterio-1′-indanyldene)-1,3-dioxane-4,6-dion | 1.00 | Lactone |
4. | 3-Heptene, (E)- (CAS) | 1.31 | Alkene |
5. | (S)-(−)-2-(Methoxymethoxy) propan-1-al | 3.89 | Aldehyde |
6. | (S)-5-(1-Hydroxy-1-methylethyl)-2-pyrrolidinone | 4.13 | γ Lactam |
7. | 1-(3,6,6-Trimethyl-1,6,7,7a-tetrahydrocyclopenta [c] pyran-1-yl)ethanone | 2.10 | Ketone |
8. | Phenol, 4-nitroso- (CAS) | 1.78 | Phenol |
9. | 1-(0-Methoxyphenyl)-3-Buten-1-ol | 1.04 | Alcohol |
10. | 5-Isopropyl-4 (trifluoromethyl)-1H-pyrimidin-2-one | 7.28 | δ Lactam |
11. | 9-Hydroxy-1-methyl-1,2,3,4-tetrahydro-8 h-pyrido (1,2-A) pyrazin-8-one | 0.85 | Ketone |
12. | á-ionol | 1.66 | Alcohol |
13. | Methyl (E)-5-(2 Oxocyclopentyl)-2-pentenoate ethylene ketal | 2.61 | Ester |
14. | 4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol | 5.44 | Phenol |
15. | (E)-3-(3′-hydroxy-2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl propionic acid methyl ester | 11.98 | Ester |
16. | Isoquinolinium bis (ethoxycarbonyl) methylide | 1.22 | Ester |
17. | 1-Cyclododecanone, 2-ethylidene | 1.54 | Ketone |
18. | 3,5-Dimethoxy-p-coumaric alcohol | 1.15 | Phenol |
19. | 2-Hexadecen-1-ol,3,7,11,15-tetramethyl-,[R-[R*,R*-(E)]] (CAS) or (Phytol) | 4.92 | Diterpene |
20. | 2,4,6-Trimethoxyisophthalonitrile | 2.64 | Nitrile |
21. | 1-Phenyl-2-(3′,4′,5′ trimethoxyphenyl) ethyne | 0.89 | Alkyne |
22. | 2H-Pyran-2-one, tetrahydro-6-tridecyl- (CAS) | 1.31 | Lactone |
23. | Pyrimidine, 5-methyl-2,4-bis[(trimethylsilyl)oxy]- (CAS) | 2.25 | Silylether |
24. | 2-palmitoyl glycerol | 1.32 | Fatty acid glycerol |
25. | Gusation A | 3.91 | Lactam |
26. | 2-linoleoyl glycerol | 1.69 | Fatty acid glycerol |
27. | Cholest-5-ene-1,3,16-triol, (1à,3á,16á)- (CAS) | 1.08 | Steroid |
28. | Squalene | 18.95 | Triterpene |
29. | Stigmasta-5,22-dien-3-ol, (3á,22E)- (CAS) | 5.83 | Steroid |
30. | 6,10,14,18,22-Tetracosapentaen-2-ol 3-bromo 2,6,10,15,19,23-hexamethyl-, (all-E)- | 2.74 | Terpenoid |