Flavonoids from Artemisia copa with Anti-Inflammatory Activity

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

Bioactivity-guided fractionation of the dichloromethane and ethanol extracts from the aerial parts of Artemisia copa led to the isolation of the flavonoids spinacetin, jaceosidin, axillarin, penduletin, tricin and chrysoeriol. These compounds were studied for possible inhibitory activity on the generation of inflammatory mediators in a cell line of mouse macrophages (RAW 264.7) stimulated with lipopolysaccharide. Spinacetin and jaceosidin weakly inhibited nitric oxide production whereas all flavonoids reduced prostaglandin E₂ levels to different extents. The most active flavonoid was jaceosidin that inhibited cyclooxygenase-2 activity in a concentration-dependent manner with an IC₅₀ value of 2.8 μM. In addition, the other flavonoids partially inhibited synovial phospholipase A₂ activity. These mechanisms may provide a basis for explaining the anti-inflammatory activity of this plant.

Artemisia copa Phil. (Compositae), commonly known as ”copa-copa”, is a small bush with a height of 30 - 60 cm that grows in the Northwest of Argentina and in the North of Chile. This plant is used in popular medicine as an antitussive, a digestive, for lowering fever as well as for treatment of pulmonary diseases and hypertension as its aqueous extract [1]. The leaves, macerated in alcohol, are used for local treatment of rheumatic pains [2]. Four flavonoids have been isolated previously from this plant (jaceidin, jaceidein 7-methyl ether, luteolin and kaempferol 6-methyl ether 3-rhamnoglucoside) [3]. In previous studies, we found that the dichloromethane and the ethanol extracts of A. copa showed anti-inflammatory activity in the ear edema test [4].

Cyclooxygenase (COX) activity is an important target for anti-inflammatory drugs since this enzyme catalyzes the rate-limiting step in prostaglandin (PG) synthesis. Constitutive COX-1 and inducible COX-2 isozymes play an important role in inflammation, pain and fever [5] and are also involved in different pathologies such as cancer [6]. Inhibition of COX-1 is achieved with non-steroidal anti-inflammatory drugs in the pathogenesis of gastrointestinal damage. In this class of pharmacological agents, COX-2 inhibitors selectivity reduce the occurrence of digestive side-effects [7] although a risk for cardiovascular events has been reported [8]. Stimulation of cells with proinflammatory agents such as cytokines or lipopolysaccharide (LPS) results in the induction of COX-2 and inducible nitric oxide synthase (iNOS). The activity of these enzymes leads to the overproduction of PGs and NO, which play a key role in the pathophysiology of arthritis and other inflammatory conditions [5]. The aim of our study was to identify the anti-inflammatory compounds present in the dichloromethane and ethanol extracts of A. copa.

The flavonoids spinacetin (1), jaceosidin (2), axillarin (3), penduletin (4), tricin (5) and chrysoeriol (6) (Fig. [1]) were isolated from the dichloromethane and ethanol extracts of the aerial parts of A. copa (4), which are active in vivo, by activity-monitored fractionation using the assay of PGE₂ production in RAW 264.7 macrophages stimulated with LPS for 24 h. Flavonoids were not cytotoxic in cells at the concentrations used in the present study, either in the presence or absence of LPS, as assayed with the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. Cell viability relative to the vehicle-treated control culture was over 90 % (data not shown). As shown in Table [1], incubation of RAW 264.7 macrophages with spinacetin or jaceosidin in the presence of LPS for 24 h partially reduced the generation of NO measured as nitrite. The percentages of inhibition were 31.3 and 41.7 %, respectively, at 10 μM. As expected, the selective iNOS inhibitor 1400W used as positive control strongly reduced nitrite levels. Under the same incubation conditions, all of the tested flavonoids inhibited PGE₂ production. The percentages of inhibition ranged from 37.3 (spinacetin) to 65.8 % (jaceosidin). The latter exhibited a concentration-dependent inhibition of PGE₂ generation as shown in Fig. [2] with an IC₅₀ value and 95 % confidence limits of 5.1 (3.3 - 8.4) μM. To assess if this inhibitory effect of jaceosidin was due to inhibition of COX-2 enzyme activity, we performed the COX-2 activity assay in intact cells. Fig. [3] shows the concentration-dependent effect of jaceosidin on COX-2 activity with an IC₅₀ value and 95 % confidence limits of 2.8 (2.0 - 3.8) μM. Additional experiments were performed to determine if A. copa flavonoids inhibited COX-1 activity (Table [2]). As expected, it was strongly inhibited by the dual COX-1/COX-2 inhibitor indomethacin whereas no significant effect was observed upon incubation with the flavonoids at 10 μM. We have also tested these compounds for inhibition of other enzymatic activities relevant in the synthesis of lipid inflammatory mediators, sPLA₂. Our results indicate that all flavonoids except jaceosidin weakly inhibited sPLA₂. Inhibition percentages ranged from 16.2 (spinacetin) to 36.5 % (chrysoeriol) at 10 μM (Table [2]).

Activated macrophages express COX-2 and iNOS leading to the production of excessive amounts of PGs and NO, which are mediators of inflammatory responses and carcinogenesis. We have shown that the flavonoids present in A. copa inhibit the production of mediators relevant to inflammatory responses, which could provide a basis for the medical use of this plant. In particular, jaceosidin exhibits topical anti-inflammatory activity [9], and our results indicate for the first time that its mechanism of action could be based on inhibition of COX-2 activity. Our data also suggest that in this series of flavonoids, the presence of a hydroxy group at C-3 or a methoxy at C-3′ would be detrimental for COX-2 inhibitory activity. In addition to our findings, some of the flavonoids isolated from A. copa in the present work, like chrysoeriol [10], tricin [11] and axillarin [12], possess antioxidant properties that could contribute to the activity of the plant extracts. On the other hand, since inhibition of COX-2 suppresses angiogenesis and tumour growth [6], flavonoids like jaceosidin offer an attractive approach for cancer chemoprevention. This notion is supported by recent data on the potential use of this flavonoid in the treatment of cervical cancers associated with the human papillomavirus (13).

Materials and Methods

The aerial parts of A. copa were collected in Antofagasta de la Sierra, Catamarca province, Argentina. A voucher specimen has been deposited at the Museo Juan Dominguez, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Argentina, under BAF 10 313. The plant material was dried under flow of air in an oven between 40 and 50 °C and then powdered mechanically. Five hundred grams of powdered plant material were extracted by maceration with dichloromethane for 24 h (2 × 5000 mL) at room temperature. The marc of the dichloromethane extraction was dried at room temperature and macerated with ethanol 50 % (2 × 5000 mL). Both extracts were dried under reduced pressure. The yield of the dichloromethane and the ethanol extracts were 8.0 % and 9.4 %, respectively. A fraction (500 mg) of the dichloromethane extract was subjected to column chromatography over Sephadex LH 20. The elution with dichloromethane yielded fractions 1 - 44, and the elution with dichloromethane-methanol (9 : 1) yielded fractions 45 - 100. Fractions 11 - 14, 43, 49 and 59 were purified by paper chromatography and yielded 3.2 mg of compound (1), 2.5 mg of compound (2), 2.8 mg of compound (3), 3.0 mg of compound (4) and 2.5 mg of compound (5). The ethanol extract (300 mg) was chromatographed over a Sephadex LH 20 column. Sixty fractions were eluted using dichloromethane-methanol (1 : 1). Fractions 18 to 38 were purified by preparative paper chromatography and yielded 4.0 mg of compound (6). The isolated compounds were identified by direct comparison with authentic samples and UV spectral analysis with diagnostic shift reagents and MS analysis. Their purity was confirmed by HPLC.

The mouse macrophage RAW 264.7 cell line was cultured in DMEM medium. Cell viability was assessed using the mitochondrial-dependent reduction of MTT to formazan. Macrophages were incubated with Escherichia coli [serotype 0111:B4] LPS (1 μg/mL) at 37 °C for 24 h in the presence of test compounds or vehicle (methanol, 1 %, v/v). Nitrite levels were determined in culture supernatants by a fluorometric method [14]. PGE₂ levels were determined with a radioimmunoassay. For COX-2 activity, 24 h LPS-stimulated macrophages were washed, and fresh medium supplemented with arachidonic acid (10 μM) was added for a 2 h incubation with the test compounds. PGE₂ accumulation during this 2 h incubation period was measured in supernatants. For COX-1 activity, macrophages were incubated with test compounds and 1 μM A23187 for 30 min. PGE₂ levels were measured in supernatants as above. Human synovial phospholipase A₂ (sPLA₂) was assayed by using [³H]oleate labeled membranes of Escherichia coli as previously described [15]. The results are presented as mean ± S.E.M. IC₅₀ values were calculated from four concentrations (n = 6 - 9). The level of statistical significance was determined by analysis of variance (ANOVA) followed by Dunnett’s t-test for multiple comparisons.

Acknowledgements

This work was supported by grants Grupos03/201 Generalitat Valenciana, UBACYT 046/04 and Project X.6 (CYTED).

References

• 1 Ratera E L, Ratera M O. Plantas de la Flora Argentina empleadas en Medicina Popular. Buenos Aires; Editorial Hemisferio Sur 1980: p. 108
  Search in Google Scholar
• 2 Giberti G. Herbal folk medicine in Northwestern Argentina: Compositae.  J Ethnopharmacol. 1983;  7 321-41
  CrossrefPubMedSearch in Google Scholar
• 3 Quarenghi de Riera M V, Catalán C N, Israilev L RA, Seeligmann P. Flavonoides foliares de Artemisia copa (Compositae).  Bol Soc Argent Bot. 1991;  27 253-5
  PubMedSearch in Google Scholar
• 4 Miño J, Moscatelli V, Hnatyszyn O, Gorzalczany S, Acevedo C, Ferraro G. Antinociceptive and anti-inflammatory activities of Artemisia copa extracts.  Pharmacol Res. 2004;  50 59-63
  CrossrefPubMedSearch in Google Scholar
• 5 Vane J R, Mitchell J A, Appleton I, Tomlison A, Bishop-Bailey D, Croxtall J. et al . Inducible isoforms of cyclooxygenase and nitric-oxide synthase in inflammation.  Proc Natl Acad Sci USA. 1994;  91 2046-50
  PubMedSearch in Google Scholar
• 6 Liu X H, Kirschenbaum A, Yao S, Lee R, Holland J F, Levine A C. Inhibition of cyclooxygenase-2 suppresses angiogenesis and the growth of prostate cancer in vivo .  J Urol. 2000;  164 820-5
  PubMedSearch in Google Scholar
• 7 Warner T D, Giuliano F, Vojnovic I, Bukasa A, Mitchell J A, Vane J R. Nonsteroid drug selectivities for cyclo-oxygenase-1 rather than cyclo-oxygenase-2 are associated with human gastrointestinal toxicity: a full in vitro analysis.  Proc Natl Acad Sci USA. 1999;  96 7563-8
  CrossrefPubMedSearch in Google Scholar
• 8 Justice E, Carruthers D M. Cardiovascular risk and COX-2 inhibition in rheumatological practice.  J Hum Hypertens. 2005;  19 1-5
  CrossrefPubMedSearch in Google Scholar
• 9 Schinella G R, Giner R M, Recio M C, Mordujovich D B, Rios J L, Manez S. Anti-inflammatory effects of South American Tanacetum vulgare .  J Pharm Pharmacol. 1998;  50 1069-74
  CrossrefPubMedSearch in Google Scholar
• 10 Mishra B, Priyadarsini K I, Kumar M S, Unnikrishnan M K, Mohan H. Effect of O-glycosylation on the antioxidant activity and free radical reactions of a plant flavonoid, chrysoeriol.  Bioorg Med Chem. 2003;  11 2677-85
  PubMedSearch in Google Scholar
• 11 Kwon Y S, Kim C M. Antioxidant constituents from the stem of Sorghum bicolor .  Arch Pharm Res. 2003;  26 535-9
  PubMedSearch in Google Scholar
• 12 Kim S R, Park M J, Lee M K, Sung S H, Park E J, Kim J. et al . Flavonoids of Inula britannica protect cultured cortical cells from necrotic cell death induced by glutamate.  Free Radic Biol Med. 2002;  32 596-604
  CrossrefPubMedSearch in Google Scholar
• 13 Lee H G, Yu K A, Oh W K, Baeg T W, Oh H C, Ahn J S. et al . Inhibitory effect of jaceosidin isolated from Artemisia argyi on the function of E6 and E7 oncoproteins of HPV 16.  J Ethnopharmacol. 2005;  98 339-43
  CrossrefPubMedSearch in Google Scholar
• 14 Misko T P, Schilling R J, Salvemini D, Moore W M, Currie M G. A fluorometric assay for the measurement of nitrite in biological samples.  Anal Biochem. 1993;  214 11-6
  CrossrefPubMedSearch in Google Scholar
• 15 García Pastor P, De Rosa S, De Giulio A, Payá M, Alcaraz M J. Modulation of acute and chronic inflammatory processes by cacospongionolide B, a novel inhibitor of human synovial phospholipase A2.  Br J Pharmacol. 1999;  126 301-11
  CrossrefPubMedSearch in Google Scholar

Dr. Valeria Moscatelli

Department of Pharmacology

Faculty of Pharmacy

Junin 956

Buenos Aires 1113

Argentina

Phone: +54-11-4508-3642

Fax: +54-11-4508-3642

Email: valmosca@ffyb.uba.ar

References

• 1 Ratera E L, Ratera M O. Plantas de la Flora Argentina empleadas en Medicina Popular. Buenos Aires; Editorial Hemisferio Sur 1980: p. 108
  Search in Google Scholar
• 2 Giberti G. Herbal folk medicine in Northwestern Argentina: Compositae.  J Ethnopharmacol. 1983;  7 321-41
  CrossrefPubMedSearch in Google Scholar
• 3 Quarenghi de Riera M V, Catalán C N, Israilev L RA, Seeligmann P. Flavonoides foliares de Artemisia copa (Compositae).  Bol Soc Argent Bot. 1991;  27 253-5
  PubMedSearch in Google Scholar
• 4 Miño J, Moscatelli V, Hnatyszyn O, Gorzalczany S, Acevedo C, Ferraro G. Antinociceptive and anti-inflammatory activities of Artemisia copa extracts.  Pharmacol Res. 2004;  50 59-63
  CrossrefPubMedSearch in Google Scholar
• 5 Vane J R, Mitchell J A, Appleton I, Tomlison A, Bishop-Bailey D, Croxtall J. et al . Inducible isoforms of cyclooxygenase and nitric-oxide synthase in inflammation.  Proc Natl Acad Sci USA. 1994;  91 2046-50
  PubMedSearch in Google Scholar
• 6 Liu X H, Kirschenbaum A, Yao S, Lee R, Holland J F, Levine A C. Inhibition of cyclooxygenase-2 suppresses angiogenesis and the growth of prostate cancer in vivo .  J Urol. 2000;  164 820-5
  PubMedSearch in Google Scholar
• 7 Warner T D, Giuliano F, Vojnovic I, Bukasa A, Mitchell J A, Vane J R. Nonsteroid drug selectivities for cyclo-oxygenase-1 rather than cyclo-oxygenase-2 are associated with human gastrointestinal toxicity: a full in vitro analysis.  Proc Natl Acad Sci USA. 1999;  96 7563-8
  CrossrefPubMedSearch in Google Scholar
• 8 Justice E, Carruthers D M. Cardiovascular risk and COX-2 inhibition in rheumatological practice.  J Hum Hypertens. 2005;  19 1-5
  CrossrefPubMedSearch in Google Scholar
• 9 Schinella G R, Giner R M, Recio M C, Mordujovich D B, Rios J L, Manez S. Anti-inflammatory effects of South American Tanacetum vulgare .  J Pharm Pharmacol. 1998;  50 1069-74
  CrossrefPubMedSearch in Google Scholar
• 10 Mishra B, Priyadarsini K I, Kumar M S, Unnikrishnan M K, Mohan H. Effect of O-glycosylation on the antioxidant activity and free radical reactions of a plant flavonoid, chrysoeriol.  Bioorg Med Chem. 2003;  11 2677-85
  PubMedSearch in Google Scholar
• 11 Kwon Y S, Kim C M. Antioxidant constituents from the stem of Sorghum bicolor .  Arch Pharm Res. 2003;  26 535-9
  PubMedSearch in Google Scholar
• 12 Kim S R, Park M J, Lee M K, Sung S H, Park E J, Kim J. et al . Flavonoids of Inula britannica protect cultured cortical cells from necrotic cell death induced by glutamate.  Free Radic Biol Med. 2002;  32 596-604
  CrossrefPubMedSearch in Google Scholar
• 13 Lee H G, Yu K A, Oh W K, Baeg T W, Oh H C, Ahn J S. et al . Inhibitory effect of jaceosidin isolated from Artemisia argyi on the function of E6 and E7 oncoproteins of HPV 16.  J Ethnopharmacol. 2005;  98 339-43
  CrossrefPubMedSearch in Google Scholar
• 14 Misko T P, Schilling R J, Salvemini D, Moore W M, Currie M G. A fluorometric assay for the measurement of nitrite in biological samples.  Anal Biochem. 1993;  214 11-6
  CrossrefPubMedSearch in Google Scholar
• 15 García Pastor P, De Rosa S, De Giulio A, Payá M, Alcaraz M J. Modulation of acute and chronic inflammatory processes by cacospongionolide B, a novel inhibitor of human synovial phospholipase A2.  Br J Pharmacol. 1999;  126 301-11
  CrossrefPubMedSearch in Google Scholar

Dr. Valeria Moscatelli

Department of Pharmacology

Faculty of Pharmacy

Junin 956

Buenos Aires 1113

Argentina

Phone: +54-11-4508-3642

Fax: +54-11-4508-3642

Email: valmosca@ffyb.uba.ar

• Supplementary Material