Vernonia patens Kunth, an Asteraceae species with phototoxic and pharmacological activity

1 Facultad de Ciencias, Departamento de Biología. 2 Instituto de Química. Universidad Nacional Autónoma de México. Ciudad Universitaria. 04510. México, D.F. México. Address Correspondence to: Dr. Maria Cristina Pérez-Amador; Facultad de Ciencias. UNAM. Circuito Exterior s/n Ciudad Universitaria. C.P. 04510. México D.F.; e-mail: perez_amador@yahoo.com, mcpa@hp.fciencias.unam.mx. Recibido/Received 20.V.2008. Aceptado/Accepted 12.VIII.2008. Abstract. The presence of phototoxic compounds in stems and leaves of young plants of Vernonia patens Kunth was confirmed by TLC. These compounds were in smaller amounts in younger than in adult plants of this species. Only the stems presented specific activity of these compounds from the two study plant organs. It included characteristic UV bands at 200 and 300nm, and phototoxic activity against Bacillus subtilis (ATCC-6633). Stems and leaves of Vernonia patens also showed anti-inflammatory activity and bactericide potential.


INTRODUCTION
In our systematic study of species of the Asteraceae family, searching for phototoxic compounds which could be taxonomic markers, we analyzed Vernonia patens plants.
Since this species have a confirmed pharmacological activity, we also determined the biological activity of the extracts: anti-inflammatory and bactericidal potentials.

MATERIALS AND METHODS
P Pl la an nt t m ma at te er ri ia al l. .Plants were collected in Nauzontla, Puebla, Mexico, on 13 March 2006.This is the time when this species starts flowering.Harvested plants had a very small root system.From these plants, we studied the stems and the leaves.Voucher specimens were deposited in the National Herbarium, Instituto de Biología, UNAM (MEXU).P Pr re ep pa ar ra at ti io on n o of f e ex xt tr ra ac ct ts s. .Plants were divided in stems and leaves.Extracts were prepared from these plant parts using hexane, ethyl acetate and methanol; the solvents were eliminated at reduced pressure.The dry extracts were used for the different tests.P Ph ho ot to ot to ox xi ic c c co om mp po ou un nd ds s. .Presence of polyacetylenes and thiophenes was evident from the hexane extracts by TLC.This was confirmed by (1) the UV spectrum of the extract, with characteristic bands, and (2) by its bactericidal potential, after being irradiated with UV-light (Daniels, 1965).
A An nt ti ib ba ac ct te er ri ia al l p po ot te en nt ti ia al l. .From the three extracts of stems and leaves, the antibacterial activity was measured against Bacillus subtilis (ATCC-6633) and Escherichia coli (ATCC-6051).
The bactericidal test was performed by the paper disc diffusion method (Cavalieri, 2005).Petri dishes with agar containing a bacterial concentration of 10 6 UFC were used with this purpose.Hexane, ethyl acetate das características al UV entre 200 y 300 nm, y actividad fototóxica frente a Bacillus subtilis (ATCC-6633).Los tallos y hojas de Vernonia patens también mostraron actividad antiinflamatoria y potencial bactericida.and methanol were essayed each at concentrations of 0.25, 0.5, 1, 2, or 4 mg/ml.Petri dishes were incubated at 37 ºC during 24h.Anhydrous ampicilline (0.05 mg) (Sigma) was used as a positive control.Each assay was repeated three times.
A An nt ti i--i in nf fl la am mm ma at to or ry y t te es st t. .The bioassay was conducted by the mouse ear edema test induced with TPA (12-O-tetradecanoyl-phorbol-13-acetate) (De Young et al., 1989).For each determination, three male CDI mice (25-30 g) were used; 10 μL of an ethanolic TPA solution (Table 3) were applied on the surface of the right ear.The same amount of ethanol (control) was applied on the left ear.Ten minutes after application of the TPA, 20 μL of the extracts were applied topically (1 mg dissolved in ethanol).After 4h, mice were sacrificed and an ear section (7 mm) was cut off and weighed.Increases in weight of the right compared to the left ears indicated swelling.Percentage swelling inhibition was calculated by comparison with the control (left ear).Indometacine (0.046, 0.085, 0.15 mg/ear) was used as a drug reference.
S St ta at ti is st ti ic ca al l a an na al ly ys si is s. .When corresponding, results were analyzed using the Student's t test (Steel & Torrie, 1985).Differences were considered either significant or highly significant with respect to the control when p ≤ 0.05 or p ≤ 0.01, respectively.

RESULTS
P Ph ho ot to ot to ox xi ic c c co om mp po ou un nd ds s. .The hexane extracts contained the phototoxic compounds, which were confirmed by TLC and developed with ceric sulphate.The UV spectrum gave the characteristic bands of these compounds at 200 and 300 nm (Table 1), and the phototoxic activity showed an inhibition halo at different concentrations.Only the stem extract was active, giving an inhibition halo of (1) 7.9 mm (39.5%) at a concentration of 0.25 mg/ml, and (2) 8.9 mm (44.5%) at a concentration of 1 mg/ml, compared to ampicilline (control).It gave an halo of 20 mm (100%) at a concentration of 0.05 mg/ml.
A An nt ti ib ba ac ct te er ri ia al l p po ot te en nt ti ia al l. .It was performed with hexane, ethyl acetate and methanol extracts against Bacillus subtilis (ATCC-6633) and Escherichia coli (ATCC-6051) (inoculation 10 6 UFC).Ampicilline was used as a control at a concentration of 0.05 mg/ml.
In the hexane extract, leaves showed no activity against Bacillus subtilis at concentrations of 0.25, 0.5, 1.0, 2.0 and 4.0 mg/ml.Stems had activity at 0.25 and 1 mg/ml, with an inhibition halo of 10.8 mm (54%) and 15 mm (75%), respectively, while the inhibition halo was 20 mm in the control.Stems and leaves did not present activity against Escherichia coli (Table 2).Positive control: Anhydrous ampicilline (D[-]-α-Aminobenzylpenicillin)(Sigma).Zone of inhibition, including the diameter of the filter paper disc (5 mm); values are the mean of three replicates.
Pérez-Amador MC et al., ΦYTON 77 (2008) A An nt ti i--i in nf fl la am mm ma at to or ry y a ac ct ti iv vi it ty y. .The three study extracts (hexane, ethyl acetate and methanol) were active.Extracts from the stem were the most active, showing a higher ear edema inhibition than indometacine (0.15 mg/ear) (drug reference).Leaf ethyl acetate and methanol extracts were more active than indometacine (0.085 mg/ear) (Table 3).

CONCLUSION
The high inhibitory values of the hexane, ethyl acetate and methanol extracts from the stems of Vernonia patens clearly demonstrate that this plant has a good pharmacological activity.In the near future, it could be used as a medicinal plant for the treatment of inflammatory processes.
Bandas de absorción en el rango de 200 a 300 nm son características de poliacetilenos.

Table 2 .
Antibacterial activity of leaf and stem extracts of V. patens.