An intensive study was undertaken in order to isolate and identify bioactive compounds in bracken, Pteridium aquilinum (L.) Kuhn (Dennstaedtiaceae). A systematic phytochemical investigations of the underground rhizomes of this plant afforded thirty-five pterosins and pterosides along with the main carcinogen in bracken, ptaquiloside, 5-hydroxyisocalamene and 5-(β-hydroxy)ethyl-2, 2, 4, 6-tetramethyl-1, 3-indandione. By detailed analysis of one- and two-dimensional nuclear magnetic resonance spectroscopy, circular dichroism and highresolution mass spectrometric data, thirteen previously undescribed pterosins and pterosides have been identified. Interestingly, for the first time 12-O-β-D-glucopyranoside substituted pterosins, rhedynosides C and D, and the sulfate-containing pterosin, rhedynosin H, alongside the two known compounds, trans-histiopterosin A and (2S)-pteroside A2, were isolated from the rhizomes of subsp. aquilinum of bracken. In addition, the six-membered cyclic ether pterosins and pterosides rhedynosin A and rhedynoside A are the first examples of this type of pterosin-sesquiterpenoids. Additionally, the three previously reported compounds [(2S)- rhedynosin I, (2S)-2-hydroxymethylpterosin E and (2S)-12-hydroxypterosin A] were obtained for the first time from plant source as opposed to mammalian metabolic products. Single crystal X-ray diffraction analysis was applied to the previously undescribed compounds (2R)- rhedynoside B, (2R)-pteroside B and (2S)-pteroside K, yielding the first crystal structures for pterosides, and three known pterosins, (2S)-pterosin A, trans-pterosin C and cis-pterosin C. Rhedynosin C is the only example of the cyclic lactone pterosin with a keto group at position C-14. Six selected pterosins ((2S)-pterosin A, (2R)-pterosin B and trans-pterosin C) and associated glycosides ((2S)-pteroside A, (2R)-pteroside B and pteroside Z) were assessed for their anti-diabetic activity using an intestinal glucose uptake assay; all were found to be inactive at 300 μM. A simple, reproducible and rapid reverse phase high performance liquid chromatography (RP-HPLC) method was developed which was linked to the UV detection source. The method validated for quantification of pterosin B and pteroside B in different plant matrices, soil and water samples. A new solid phase extraction (SPE) method was developed and established for sample preparation based on using 500 mg Supelco-SPE cartridges. The RP-HPLC was performed with mobile phase containing 0.1% formic acid (FA) in water and 0.1% FA in acetonitrile with an appropriate gradient and a flow rate of 1 mL/min. Detection of the analyte peaks was performed at 254 nm. The method showed good linearity (correlation coefficient (r) > 0.99), and appropriate limit of detection (LOD) and limit of quantification (LOQ). The proposed RP-HPLC-UV method has a retention time of 20 min (3 samples/h). Overall, this work focuses on the application of SPE–HPLC-UV analysis to quantify pterosin B and pteroside B in different matrices including soil, water and bracken samples. The method involves pre-concentration and clean-up by SPE cartridges. Final analysis of the selected compounds was carried out by the developed HPLC-UV method. The extraction efficiency was checked by recovery experiment while the accuracy of the method was tested by relative standard deviation (% RSD). Recoveries were ranged from 90.29 to 96.23 % (pterosin B) and 93.64 to 101.03 % (pteroside B). The RSD (%) for both target analytes (pterosin B and pteroside B) was less than 2.5 % for all the analysed bracken stem samples. Preliminary results demonstrated that the present method was suitable for determination of pterosin B and pteroside B in bracken tissues, soil and water samples. The simplicity, accuracy and sensitivity of the developed SPE-HPLC-UV method, recommending that the method is also convenient and useful for quick check and detection of trace amounts of pterosin B and pteroside B in water, soil and plant samples. The quantification of pterosin B and pteroside B was applied to bracken, divided into rhizomes, stems and fronds, the top soil of the sample cores and a set of water samples. The rhizome contained the largest concentration of pteroside B through the completed bracken growth period (750-2950 mg/kg). Its concentration was approximately one order of magnitude above pterosin B (10-245 mg/kg). The above ground stems and fronds showed distinct increases in concentrations of pterosin B (ca. 500 mg/kg) and pteroside B (ca. 250 mg/kg) at the time of crosier emergence, that were about one order of magnitude higher than the other samples collected during the growth periods. The soil sampled reflected the increased mobilisation of pterosin B during the onset of above ground growth (May to June) while no pteroside B was detected. The absolute pterosin B concentrations determined were about three orders of magnitudes lower (0.02-0.3 mg/kg). Water samples that drain bracken covered areas were found to contain pterosin B between 9-47 ng/L and pteroside B in range of 16-85 ng/L. Overall, the seasonal variation of pterosin B and pteroside B showed that the compounds are stored in the rhizome, preferably as the glycoside, and are mobilised to protect the early tissue as a means of chemical defence. The stability of pterosin B was exemplified by its environmental presence in plant, soil and water.