Rationale for Immunomodulatory and Anti-inflammatory Effects of Ocimum sanctum: Radical Scavenging Potential and Effect on Nitric Oxide Production

Antioxidant activity of polar methanolic extract of aerial parts of Ocimum sanctum using multiple screens was studied. Various concentrations of the standardized extract were examined for the free radical scavenging activity, superoxide radical scavenging potential and effect on nitric oxide production in RAW 264.7 mouse monocytes cell line. The results indicated that the O. sanctum extract has strong antioxidant activity. In the assay for free radical scavenging activity measured in terms of scavenging of DPPH (1,1-diphenyl-2-picryl hydrazyl) radical, the extract exhibited the IC50 of 94.51 ± 6.47 μg/ml. NBT reduction assay was used to measure the superoxide reducing capacity of the extract and the extract inhibited the NBT reduction with IC50 of 71.17 ± 8.13 μg/ml. Effect on nitrite production was tested in Lipopolysaccharide (LPS) activated RAW 264.7 mouse monocytes cell line. In this assay, biphasic response was observed for the extract. At lower concentration it stimulated nitrite production while at higher doses the nitrite production was suppressed (EC50 4.89 ± 0.47 μg/ml and IC50 66.67 ± 0.91 μg/ml). These results suggest that the antioxidant activity of O. sanctum may be partly responsible for its reported immunomodulatory and anti-inflammatory effects. INTRODUCTION The role of free radical reactions in the disease pathology is well established suggesting that these reactions are necessary for normal metabolism and are detrimental to health as well (Harman, 1981). The diseases caused by these free radical reactions are atherosclerosis, ischaemic heart disease, ageing, inflammation, diabetes, immunosuppression, neurodegenerative diseases and some other diseased conditions (Harman, 1988; Maxwell, 1995). Ocimum sanctum, commonly used as Holy basil or sacred basil, is well known in India. Of all the Ocimum species, O. sanctum is most widely studied for its pharmacological properties. Traditionally, it has been used as diaphoretic, expectorant and hepatoprotective. It has been studied for its immunomodulatory (Godhwani et al., 1988), anti-hyperglycemic (Vats et al., 2002), antioxidant (Devi, 2001), antiinflammatory (Godhwani et al., 1987; Kelm et al., 2000), anti-stress and radioprotective effects (Gupta and Vishwanathan, 1955). Its anti-ulcer (Singh and Mujumdar, 1999) and anthelmintic (Asha et al., 2001) activities have also been reported. However, all these activities are carried out in vivo and the mechanism of action is still elusive. Since free radical involvement in various disorders is suggested, this paper is an attempt to give basis for various activities of O. sanctum. In the present paper, we studied standardized polar extract of O. sanctum for free radical scavenging activity, superoxide radical scavenging potential and effect on nitric oxide (NO) production. Proc. WOCMAP III. Vol. 4: Targeted Screening of MAPs, Economics & Law Eds. C. Franz, Á. Máthé, L.E. Craker and Z.E. Gardner Acta Hort. 678, ISHS 2005 160 MATERIALS AND METHODS Plant Material For the purpose of present study, plant material was collected during the month of June 2001 from NIPER medicinal garden and surroundings. The Taxonomist, NIPER medicinal plants garden confirmed the identity. Specimen sample is deposited in Natural Products Department Herbarium. Chemicals 1,1-diphenyl-2-picryl hydazyl radical, dextran, zymosan and nitroblue tetrazolium (NBT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA); RPMI-1640 media, glutamine, gentamicin and fetal bovine serum (FBS) were purchased from Himedia Ltd. (Mumbai, India). Escherichia coli 026:B6 lipopolysaccharide was purchased from Difco (Detroit, MI, USA). Free Radical Scavenging Activity Free radical scavenging capacity of the plant materials was determined using DPPH. A methanolic DPPH solution (0.15%) was mixed with different concentrations of extracts and fractions and after 10 min. the absorbance was read at 515 nm using spectrophotometer (Perkin Elmer Lambda 20 UV visible spectrophotometer). Inhibition curve was plotted and IC50 value was obtained (Viturro et al., 1999). NBT Reduction Assay The assay was carried out by the modified procedure described earlier (Richardson et al., 1998). Fresh heparinised blood from healthy volunteers was used for the isolation of polymorphonuclear cells (PMNs). By dextran sedimentation method (6% dextran) PMNs were isolated and suspended in RPMI-1640 medium supplemented with 10% heat inactivated FBS. From this suspension, 1 x 10 cells were taken and incubated with different concentrations of the extract for 5 min. To this, NBT (0.5 mg/ml; 200 μl), opsonised zymosan (4 mg/ml; 100 μl) were added and incubated at 37°C for 30 min. After incubation, the reaction was quenched by 1 N HCl. The formazan formed can be extracted with organic solvent and the absorbance is read at 520 nm. Control was run simultaneously using medium instead of extract. Ascorbic acid was used as the standard. The percentage inhibition of NBT reduction was calculated by the formula % inhibition of NBT reduction = (Control sample) x 100