Isolation of Two Flavonoids from Tanacetum microphyllum as PMA-Induced Ear Edema Inhibitors Jacqueline Martinez, Ana Maria Silva ´ n, Maria Jose ´ Abad,* Paulina Bermejo, and Angel Villar Department of Pharmacology, Faculty of Pharmacy, University Complutense, 28040 Madrid, Spain Monica So ¨llhuber Department of Organic Chemistry, Faculty of Pharmacy, University Complutense, 28040 Madrid, Spain Received December 13, 1995 X The CH 2 Cl 2 extract of Tanacetum microphyllum exhibited antiinflammatory activity on PMA- mouse ear model. Two antiinflammatory flavonoids, 5,7-dihydroxy-3,6,4′-trimethoxyflavone (santin) (1) and 5,7-dihydroxy-3,4′-dimethoxyflavone (ermanin) (2), were isolated. In view of the well-known side effects of classical cyclooxygenase inhibitors and glucocorticoids, antiphlo- gistic agents having other mechanisms of action are being intensively investigated. One of the possible new ways of antiinflammatory action is the inhibition of protein kinase C (PKC). Activation of PKC leads to a number of intracellular signal transduction pathways implicated in the pathogenesis of inflammation, includ- ing phospholipase A 2 -dependent arachidonic acid re- lease, eicosanoid production, and reactive oxygen me- tabolite formation. 1 Recent studies demonstrate that the topical admin- istration of phorbol myristate acetate (PMA) to mouse ears induces an inflammatory response resulting from PKC activation. 2 The effects of topical administration of selected antiinflammatory drugs and a variety of structurally related PKC inhibitors, have been docu- mented using this model. 3-6 Despite the fact that most research in developing antiinflammatory agents has been directed along these lines, recent trends include the search for compounds in sources that have, for various reasons, been explored considerably less, includ- ing higher plants. 7,8 Ethnomedicine provides a source of information about these plants, which is of great value in identifying possible pharmacologically active substances. In searching for natural products as potential anti- inflammatory agents, investigations have been con- ducted on Tanacetum microphyllum DC. (Compositae), an endemic species of the Iberian Peninsula, widely used in traditional medicine. Previously, pharmacologi- cal activity had been reported for extracts of this plant, 9 and the identification of three compounds with antiin- flammatory activity in vivo and in vitro: hydroxyachil- lin, a sesquiterpene lactone of the guaianolide type, 10 and two flavonoids, centaureidin and 5,3′-dihydroxy-4′- methoxy-7-carbomethoxyflavonol. 11,12 As part of a mechanism-based screening for novel inhibitors of PKC from a variety of natural sources, the CH 2 Cl 2 extract of T. microphyllum was investigated in the PMA-mouse ear model and was selected for frac- tionation. The subsequent fractionation of the extract, with parallel pharmacological studies, led to the isola- tion and identification of two new active principles of T. microphyllum. Their characterization and biological activity are reported in this paper. The CH 2 Cl 2 extract of T. microphyllum has been shown to demonstrate significant antiedema activity on carrageenan-induced paw edema in rats and mice. 9,11 This extract (and all subsequent fractions) was tested using the PMA-induced ear model in mice, to evaluate its antiinflammatory properties. The effects on swelling and other inflammatory parameters are described here. Screening of CH 2 Cl 2 extract on a PMA-induced edema in mice gave positive results. The extract significantly inhibited PMA-induced mouse ear edema in a dose- dependent manner. At the highest dosage (3 mg/ear) it was more potent than the reference agent indometha- cin, even 5 h after administration of PMA (Table 1). All fractions of the extract also reduced swelling more potently than indomethacin, fractions A, B, F, and G (3 mg/ear) being the major active fractions with inhibition around 80%. The vascular permeability response to PMA application was considerably reduced by the test materials (data not shown). The mean inhibition of PMA-induced permeability was 68% with CH 2 Cl 2 ex- tract at a dosage of 3 mg/ear, and around 40% with indomethacin at the same dose. Bioassay-guided fractionation of the crude CH 2 Cl 2 extract, using medium-pressure liquid chromatography (MPLC) and flash chromatography, yielded compounds 1 and 2. The spectral data identified compound 1 as 5,7- dihydroxy-3,6,4′-trimethoxyflavone (santin) 13,14 and com- pound 2 as 5,7-dihydroxy-3,4′-dimethoxyflavone (erma- nin). 15,16 * To whom correspondence should be addressed. Phone: (91) 394 18 71. FAX: (91) 394 17 64. X Abstract published in Advance ACS Abstracts, January 15, 1997. 142 J. Nat. Prod. 1997, 60, 142-144 S0163-3864(96)00163-2 CCC: $14.00 © 1997 American Chemical Society and American Society of Pharmacognogy + +