Bioactivity-guided Separation of Antinociceptive and Antioxidant Subfractions from Alkaline Chloroform Fraction of Fenugreek Seeds (Trigonella foenum-graecum L.) in an Animal Model

Antinociceptive effect of fenugreek seeds (Trigonella foenum-graecum L.) has been reported in different animal models in response to various chemical or thermal stimuli. In a recent study, alkaline chloroform fraction (AKC) of this plant has exhibited the greatest analgesic effect. In the present study, to isolate the active component(s) from the plant, the subfractions resulting from AKC column chromatography were evaluated in an animal model for anti-nociception effect. From the 17 separated fractions, 5 major fractions (F4, F6, F14, F15 and F16) were used for the formalin test at three different doses (2.5, 5 10 mg/kg). Antioxidant activity of the most active subfractions was studied too. Subsections F16 and F14 (5, 10 mg/kg) showed the greatest analgesic effect and reduced, which was similar to morphine and even stronger than morphine in some doses. The greatest antioxidant activity was observed by F14 (radical inhibition percentage of 17.34± 0.14 in DPPH assay, reduction power percentage of 74.05±4.23 in RPA versus green tea (91.68± 3.04 and 97.59± 6.24 in DPPH assay and RPA test respectively). The absorbance of F14 was 0.25±0.11 in the FTC method in comparison to ascorbic acid 10 μg/ml and 100 μg/ml (0.72±0.33 and 0.05±0.41 respectively). Separated subfractions exhibited more antinociceptive effect than AKC fraction, so further separation can lead to the acquisition of antinociceptive compound (s), while AKC fraction was found to be more potent antioxidant than separated sub-fractions in all three experiments. So, most likely, the anti-nociception effect of subfractions might be achieved via other mechanisms than antioxidant activity. Based on phytochemical screening, AKC and all sub-fractions especially F14, F15 and F16 were positive for the presence of alkaloids and only F14 was positive for flavonoids.