The Hemolytic Activity of Bracken Extracts in Guinea Pigs.

Abstract

ABSTRACT. This study was conducted to elucidate the hemolytic activity of a new toxic substance in bracken fern. A crude extract (CE) was prepared from the methanol extracts of bracken by the column chromatography. When the CE was injected subcutaneously in guinea pigs, the hemoglobinuria and hemolysis were observed within 6 hr, and 3 days later edema and hemorrhages in the urinary bladder were observed. The CE was then fractionated by high performance liquid chromatography (HPLC), and three (HF, BF and CF) of the fractions showed the toxic activities in guinea pigs. The HF caused the hemolysis, whereas both the BF and the CF caused the hemorrhagic cystitis without any hemolytic activities. The HF was further fractionated by the HPLC, resulting of the 3 fractions (HF-I, II and III). The hemolysis was caused only with the HF-II, and HF-II as well as HF did not cause the hemorrhagic cystitis. HPLC analysis revealed that both BF and CF contains braxin B and braxin C, respectively, and both HF and HF-II do not contain braxin A, B or C. These facts suggest that bracken fern contains a new toxic substance (hemolysin) which induces the acute hemolysis in guinea pigs.-KEY WORDS: bracken fern, guinea pig, hemolysin, hemolytic effect.J. Vet. Med. Sci. 61(2): 129-133, 1999 prefecture in Japan. The fronds were immediately freezed at 20°C, followed by a freeze-drying. The dried materials were stored at 20°C. The freeze-dried bracken fern was hashed in ice-cold methanol containing 0.5% acetic acid with a mixer and Polytron (PT10/35, Kinematica, Luzern, Switzerland), and the mixture was filtered with suction. The methanol extract was washed repeatedly with hexane and ether, and was applied to column chromatography with RP-18 (Lobar, Merck, Darmstadt, Germany) eluted with methanol and 0.1% acetic acid to obtain CE. The CE was then separated into fractions using the high performance liquid chromatography (HPLC) with a preparative column of ASAHIPAK HIKARISIL-C18 (20 × 250 mm, Asahi