Anti-oxidant bioassay-directed extraction of the fresh leaves and stems of Ocimum sanctum and purifi- cation of the extract yielded the following compounds; ...
Phytomed icine, Vol. 7(1), pp. 7-13
Phytomedicine
© Urban & Fischer Verlag 2000 http ://www.urbanfischer.de/journalslphytomed
Antioxidant and cyclooxygenase inhibitory phenolic compounds from Ocimum sanctum Linn. M. A. Kelml , M. G. Nair!, G. M. Strasburg", and D. L. DeWitt 3 IBioactive Na tural Prod ucts Laboratory, Depart ment of Horticultur e and Na tional Food Safety and Toxicology Cent er 2Food Science and Human N utri tion 3Depart ment of Biochemistry, Michigan State University, Michigan, USA
Summary Anti-oxidant bioassay-directed extraction of the fresh leaves and stems of Ocimum sanctum and purification of the extract yielded the following compounds; cirsilineol [1], cirsimaritin [2], isothymusin [3], isothymonin [4], apigenin [5], rosmarinic acid [6], and appreciable quantities of eugenol. The structures of compounds 1-6 were establi shed using spectroscopic methods. Compounds 1 and 5 were isolated previously from O. sanctum whereas comp ounds 2 and 3 are here identified for the first time from O. sanctum. Eugenol, a major component of the volatile oil, and compounds 1,3,4, and 6 demonstrated good antioxidant activity at 10-JIM concentrations. Anti-inflammatory activity or cyclooxygenase inhibitory activity of these compounds were observed . Eugenol demonstrated 97 % cycloox ygenase-l inhibitory activity when assayed at lOOO-JIM concentrations. Compounds 1,2, and 4-6 displayed 37, 50, 37, 65 , and 58 % cyclooxygenase-l inhibitory activity, respectively, when assayed at lOOO-JIM concentrations. Eugenol and compounds 1, 2, 5, and 6 demonstrated cyclooxygenase-2 inhibitory activity at slightly higher levels when assayed at lOOO-pM concentrations. The activities of compounds 1-6 were comparable to ibuprofen, naproxen, and aspirin at 10-, 10-, and lOOO-pM concentrations, respectively. These results support traditional uses of O. sanctum and identify the compounds respon sible. Key words: Ocimum sanctum, antioxidant, antiinflammatory, cyclooxygen ase enzymes.
Introduction Flavonoids represent one of the most ubiqu itou s classes of polyph cnolic secondary compounds found in higher plants. Many common fruit s, vegetables, herbs, and plant products such as wine, juices, and dried fruits are rich sources of flavonoids. More importantl y, many of these compounds have demon str ated rather potent antioxidant activity by block ing and/or scavenging free radical s (Saija et aI., 1995 ). The formation of oxygen free radicals is a normal phenom enon carr ied out in aerobic cells. The interaction of these free radicals with lipids produ ces hydroperoxides and peroxides, which in turn may act adversely with biological systems, resulting in cancer. By way of the free-radical scavenging mechanism, flavonoids effectively negate the deleteri -
sition metal ions such as Fe2 +, which can act as free rad ical initiators. Altern atively, flavon oids can prevent the form ation of free radi cals by chelation or complexation with the transition metal-free rad ical initiator. Concerning structure-activity relationships (SAR) of flavan oids, hydro xyl gro ups at the C, and C 7 positions and the C2-C3 double bond were shown to be necessary for high inhibitor y activity against xanthine oxidase (Cos et aI., 1998). Xanthine oxidase forms superox ide radicals and hydro gen pero xide and is involved in the oxidation of hypoxanthine to xant hine to uric acid. It was also found that a hydr oxyl group at C3' and at C3 were essential for high superox ide scavenging activity (Cos et aI., 1998). In addition, substitution pat-
o u s effects of h ydropero xides and peroxides . Free r adi-
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cal formation can also result fro m the presence of tran-
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Time (min) Fig. 1. Antioxidant activities of O. sanctum compounds and some commercial antioxidants at lO-pM concentration. The rate of peroxidation was monitored by a decrease in fluorescence intensity as a function of time. Relative intensity represents the fluorescence intensity at a given time divided by the initial intensity at the start of the assay. Values represent the means of duplicate measurements.
activity by preventing lipid peroxidation (Arora et al., 1998; Arora et al., 1997). However, it must be emphasized that the current study only discusses, and is in no way complete regarding the SAR of the isolated flavonoids. The formation of prostaglandins from arachidonic acid by prostaglandin synthase is a well-studied process. The formation and subsequent effect of prostaglandins can result in the stimulation of inflammation and associated pain (Stryer, 1988). Prostaglandin synthase has both cyclooxygenase and hydroperoxidase components; however, it is well understood that two distinct isoforms of cyclooxygenase (COX) exist, namely, COX-1 and COX-2, which are both involved in the conversion of arachidonic acid to prostaglandins (Lipsky et al., 1998). COX-1 constitutively expresses in nearly all cells and provides prostaglandins which protect the GI tract from ulceration, inhibit platelet oxygenation, and maintain bloodflow in compromised kidneys. COX-2 is normally absent from cells, but expression is induced in association with inflammation. Both COXc 1 and COX-2 are inhibited by traditional nonsteroidal anti-inflammatory drugs (NSAIDS) such as aspirin, ibuprofen, naproxen, sulindac, diclofenac, etc. Many of these drugs are taken to relieve inflammation pain such as musculoskeletal pain, including arthritis and tendonitis, as well as other general aches and pains. Since traditional NSAIDS are not specific in the inhibition of both COX forms, the inhibition of COX1 seems to be associated with gastrointestinal damage, renal dysfunction, and platelet abnormalities (Simon et al., 1998). Therefore, recent research has been directed toward the discovery of NSAIDS with specific inhibition towards the enzyme COX-2.
As stated earlier, flavonoids are widespread among plants of the human diet, and many of these have low toxicity in mammals. Their action as antiinflammatory agents and their low toxicity make them prime candidates to plant drug research. The antiinflammatory activity of flavonoids is believed to be controlled, at least in part, by the addition of cyclo-oxygenase (Kim et al., 1998). Flavonoids such as 3-hydroxyflavone, galangin, quercetin, and kaempferol demonstrated good cyclooxygenase inhibitory activity in assays utilizing rat mixed peritoneal leukocytes (Hoult et al., 1994). Other researchers have reported similar findings (Kim et al., 1998; Todera et al., 1994; Middleton et al., 1992). In a study examining at SAR of flavonoids, it was determined that flavonoids lacking 3', 4'-dihydroxy substitution, as well as fewer overall hydroxy groups, had greater cyclooxygenase inhibitory activity (Moroney et al., 1988). More specifically, it was demonstrated that B-ring hydroxyl substitution decreased a flavonoid's ability to inhibit cyclooxygenase. Baumann et al. (1979) reported that ortho dihydroxy-substituted phenols can act as initiators of cyclooxgenase by acting as cofactors in prostaglandin generation, specifically in cell-free assays. Many of the previous studies on Ocimum sanctum Linn. (Lamiaceae) have focused largely on the biological activity of crude extracts. A triterpene, ursolic acid, isolated from O. sanctum has been shown to be effective in protecting against lipid peroxidation (Balenehru and Nagarajan, 1991). We have reported the mosquitocidal compounds eugenol and (E)-6-hydroxy-4,6-dimethyl-3-heptene-2-one from O. sanctum (Markham, 1982). In the present study, we report for the first time the extraction, purification, and structure identification of antioxidant and antiinflammatory compounds from the fresh leaves and stems of O. sanctum. Also, the importance of the A-ring substitution relative to antioxidantlfree radical scavenging activity will be considered.
Materials and Methods General Experimental Procedures
lH-nmr spectra were recorded at 300 and 500 MHz. 13C-nmr and DEPT spectra were recorded at 126 MHz. Chemical shifts were recorded in DMSO-d6 , CD 30D, and CDCl 3 and the values are reported in 8 (ppm) based on residual of DMSO 2.29; DMSO-d6 39.7 and CDCl 3 7.24; CDCl 3 77.0. Coupling constants,], are in Hz. ElMS were recorded at 70 eY. UV experiments were carried out on a Shimadzu UV-260 spectrophotometer. Shift reagents were prepared and used according to Markham. UV samples were prepared at 12.5-50 ppm concentrations. The particle size of silica
Antioxidant and cyclooxygenase inhib itory pheno lic compounds from Ocimum sanctum Linn.
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Fig. 2. h z vitro COX-1 inhibitory activity of eugenol, compounds 1-6, aspmn, ibuprofen naproxen. and comEugenol, pounds 1-6, and aspi rin were tested at 1000-pM and ibuprofen and naproxen at 10pM concentrations.
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