BIOASSAY GUIDED ISOLATION AND CHARACTERIZATION OF SECONDARY METABOLITES FROM ISODON RUGOSUS WALL. EX BENTH. AND THEIR ANTI-NOCICEPTIVE ACTIVITIES

Abstract

Isodon rugosus Wall.ex Benth. is widely employed in folk medicine to cure various types of pain conditions like dental, gastric, abdominal, ear pain and rheumatism. In this thesis, we have explored the analgesic potential of Isodon rugosus, to isolate the active compounds responsible for analgesic activity and to figure out its possible mechanism of action. The bioassay guided isolation was followed to obtain analgesic compound from Isodon rugosus. Preliminary phytochemical evaluation was carried out for the presence of various secondary metabolites in Isodon rugosus. Initially, the analgesic activity was performed using methanolic extract and subsequent fractions. The chloroform fraction was sorted out as most active following various analgesic activities. The isolation of analgesic compounds was carried out from chloroform fraction by using gravity column chromatography, in which thin layer chromatography and various stains were employed for confirmation of single and pure spots of compounds. At the end, three (3) compounds (AZ-1, AZ-2 and AZ-3) were isolated from chloroform fraction of Isodon rugosus in pure form. The pure compounds were then subjected to various analytical techniques for identification purposes including 1HNMR, 13CNMR and MS. The compound AZ-1 and AZ-2 were in sufficient quantity and were subjected to evaluate their analgesic potential following various standard protocols. In acetic acid induced writhing test, the compound AZ-1 showed 61% inhibition whileAZ-2 showed 4% inhibition of pain. In formalin test, AZ-1at 50 mg/kg displayed 69 and 63% inhibitions at phase-I and phase-II respectively. Similarly, the compound AZ-2at a similar dose, i.e. 50 mg/kg displayed 8 and 13% inhibitions at early and late phase respectively. In hot plate test,AZ-1 and AZ-2 exhibited average reaction times of 8 min and 3 min respectively. The possible mechanism of action of AZ-1 was figured out using different receptors including opioid, dopaminergic and adrenergic receptors. Moreover, the AZ-1 2 was also assessed against enzymes lipoxygenase and cyclooxygenase, which have a prominent role in nociception. The mechanistic study revealed that AZ-1 was actively involved in the anti-nociception through opioid receptors. The possible mechanism of analgesic action was confirmed to be the central pathway using opioid receptors, because the mice showed activity as that of morphine after the administration of opioid antagonist naloxone. The involvement of opioids receptors is evident from both hot plate and formalin assays in which nalaxone was used to figure out the involvement of opioids receptors. The AZ-1 was also involved in anti-nociception through adrenergic receptor and showed the reaction time of 5.35± 0.66, 5.17± 0.53, 6.53± 0.68, 5.90± 0.58 and 4.63±0.56 at the interval of 15, 30, 45, 60 and 90 min respectively. The involvement of adrenergic receptor shown by the pre-treatment of adrenergic antagonist (yohimbine) which reversed the activity of AZ-1. Moreover,AZ-1 was also active against cyclo-oxygenase and lipoxygenase enzymes. The positive control used was Zileuton in lipoxygenase assay, in which the AZ-1 demonstrated 49.40± 0.65, 42.37± 0.72, 36.87± 1.04, 31.80± 0.55 and 26.97± 1.49% inhibitions at concentrations of 1000, 500, 250, 125 and 62.5 μg/ml respectively having IC50of 1024 μg/ml. The celecoxib was used as positive control in cyclo-oxygenase in which AZ-1 showed 42.77± 0.82, 38.87± 0.70, 32.73± 0.95, 28.60± 1.19 and 21.70± 1.57% inhibitions at concentration of 1000, 500, 250, 125 and 62.5 μg/ml respectively with IC50 1423μg/ml. The antioxidant properties of Isodon rugosus was also explored using various free radicals assays such as DPPH, H2O2 and ABTS. In DPPH, H2O2 and ABTS assays, AZ-1 revealed 56.33± 0.89, 49.33± 0.33 and 59.33± 0.33% inhibitions respectively at 1000 μg/ml concentration each. Additionally, the essential oil of Isodon rugosus (Ir.Eo) was also isolated and was subjected to GC coupled MS analysis. A total of 141 compounds found in the essential oil of Isodon rugosus. 3 The essential oil was also subjected to anti-nociceptive and antioxidant activity to find out it role in pain. The Ir.Eo showed a comparative response of the sample (57.14%) with the positive control (70.29%) at the dose of 100 mg/kg. The Ir.Eo in combination with naloxone reduced 09.01and 07.95% pain in phase I and II respectively. Mean reaction time for a sample of Ir.Eo and naloxone at a dose ratio of 50 + 1 mg/kg body weight was figured out as 05.28 ±0.13 min. Similarly, in DPPH and ABTS assay Ir.Eo revealed IC50 of <0.1 μg/ml. In brief, AZ-1 was revealed as an active anti-nociceptive compound of Isodon rugosus. This compound should further be subjected to various investigations which can help in drug discovery.