In vitro and in vivo toxicological evaluation of extract and fractions from Baccharis trimera with anti-inflammatory activity.
BACKGROUND
Baccharis trimera (Less) DC. (Asteraceae), popularly known in Brazil as "carqueja", have been used in folk medicine to treat gastrointestinal, hepatic and renal diseases, and inflammatory processes as rheumatism.


OBJECTIVE
To evaluate the in vitro and in vivo toxicological effects of anti-inflammatory Baccharis trimera aqueous extract and fractions.


METHODS
Aqueous extract of Baccharis trimera (AEBt) was produced by infusion in boiling water. After lyophylization AEBt was extracted with 80% ethanol, originating the ethanolic supernatant fraction (EFBt) and the aqueous sediment fraction (AFBt). Anti-inflammatory properties of AEBt, EFBt or AFBt (3, 30 or 300 μg/kg b.w.) were evaluated by the carrageenan-induced mouse paw edema using indomethacin (10mg/kg) as positive control. The growth of rat hepatoma cells (HTC) and human embryo kidney epithelial cells (HEK) was determined by protein staining assay. Cytotoxicity was assayed by the tetrazolium salt (MTT) reduction. Cyclosporin was used as reference cytotoxic drug for spleen cells and doxorubicin for HTC and HEK cells. For in vivo toxicological evaluation SW male mice were daily and oral (gavage) treated with extract/fractions at 4.2mg/kg or 42 mg/kg during 15 days. After treatment liver or kidney cells were submitted to comet assay to determine the DNA damage index, and the glutathione S-transferase activity was assayed towards ETHA (class Pi) and CDNB (several classes). Mutagenicity was evaluated by the Ames test using Salmonella typhimurium strains TA97, TA98, TA100, and TA102.


RESULTS
The anti-inflammatory effects of EFBt were higher than those of AEBt or AFBt. Mice treatment (3-300 μg/kg) with AFBt reduced the paw edema (3h) at lower levels (29.2-37.3%; P<0.01), than those observed for AEBt (44.7-54.2%; P<0.001), EFBt (49.3-58.2%; P<0.001) or indomethacin (64.6%, P<0.001, 10mg/kg). The growth of kidney cells (HEK) was inhibited by AEBt (IC(50) 182.6 μg/ml), EFBt (IC(50) 78.1 μg/ml) and AFBt (IC(50) 86.2 μg/ml), with lower effects on HTC hepatic cell (IC(50) 308.8 μg/ml, 396.5 μg/ml and 167.9 μg/ml, respectively). As evaluated by MTT test, AFBt exhibited cytotoxicity for HEK cells (IC(50) 372.5 μg/ml), but none for HTC ones; by the way, AFBt stimulated spleen cells (EC(50) 2.2 μg/ml) while cyclosporine, a cytotoxic reference drug inhibited them with IC(50) of 0.42 μg/ml; the IC(50) for doxorubicin for HEK and HTC cells was 0.28 μg/ml and 14.4 μg/ml, respectively, at 96h. No mutagenic potential was observed. Mice treatment with AEBt or AFBt at 42 mg/kg for 15 days altered the kidney relative weight, but not at 4.2mg/kg. Baccharis trimera did not change liver, spleen or popliteal lymph node relative weight. DNA damage index of kidney cells was observed on mice treated with AEBt/AFBt, but not on animals treated with EFBt, while DNA lesions were detected on liver cells only after AFBt treatment. The general activities of hepatic GST and Pi GST were reduced by EFBt and AFBt treatment, respectively.


CONCLUSIONS
Baccharis trimera did not show mutagenicity, inhibited the GST activity, a hepatic detoxification enzyme, and induced in vivo (genotoxicity) and in vitro toxicological effects to kidney cells.
Departamento de Bioquímica, Instituto de Biologia Roberto Alcantara Gomes, Centro Biomédico, Universidade do Estado do Rio de Janeiro, Av. Professor Manoel de Abreu, 444, PAPC, 4o andar, CEP 20550-170, Rio de Janeiro, RJ, Brazil.
N P A
NP
Nogueira
P A
PA
Reis
G A T
GA
Laranja
A C
AC
Pinto
C A F
CA
Aiub
I
I
Felzenszwalb
M C
MC
Paes
F F
FF
Bastos
V L F C
VL
Bastos
K C C
KC
Sabino
M G P
MG
Coelho
D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology / Q000633:toxicity*; D031175:Baccharis / Q000737:chemistry*; D002478:Cells, Cultured; D020552:Comet Assay; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D010936:Plant Extracts / Q000494:pharmacology / Q000633:toxicity*; D051381:Rats; D014407:Tumor Cells, Cultured; D014867:Water
D000893:Anti-Inflammatory Agents; D010936:Plant Extracts; D014867:Water; D005982:Glutathione Transferase