| Summary: | Phyllanthin and related lignans were found to be responsible, at least in part, for most of the activity of <i>Phyllanthus</i> species. This observation encouraged the authors to develop methods for the preparation of an extract rich in phyllanthin and related lignans from the aerial parts of <i>P. niruri</i> L. Direct extraction with solvents produced extracts with variable yields and contents of lignans. Lignans were identified by LC-ESI-MS analysis as phyllanthin (used as marker substance), hypophyllanthin, phylltetralin, nirtetralin, and niranthin. Extraction with boiling water produced 18.10 g% (<i>w</i>/<i>w</i>) extract with a trace amount of lignans (phyllanthin content of 0.33 ± 0.10 mg/g extract), while extraction with MeOH gave 3.6 g% <i>w</i>/<i>w</i> extract with a low phyllanthin content (3.1 mg/g extract), as determined by HPLC. However, Soxhlet extraction with hexane, CH<sub>2</sub>Cl<sub>2</sub>, or acetone gave extracts with low yields (0.82, 1.12, and 3.40 g% <i>w</i>/<i>w</i>, respectively) and a higher phyllanthin contents (36.2 ± 2.6, 11.7 ± 1.68, and 11.7 ± 1.10 mg/g extract, respectively). Extraction quality and efficiency were optimized by adopting the following three different approaches: (1) Alkaline digestion of the plant material with 30% potassium hydroxide yielded 3.1 g% <i>w</i>/<i>w</i> of purified extract with high phyllanthin content (22.34 ± 0.13 mg/g); (2) microwave-assisted extraction using 80% MeOH gave an extract with a better yield (8.13 g% <i>w</i>/<i>w</i>) and phyllanthin content (21.2 ± 1.30 mg/g) (after filtration through a Diaion HP-20 column); and (3) treatment of the ground plant material at 50 °C with two hydrolytic enzymes, cellulase (9 U/g for 12 h) and then, protease (4 U/g up to 72 h) optimized the yield of extract (13.92 g% <i>w</i>/<i>w</i>) and phyllanthin content (25.9 mg/g extract and total lignans content of 85.87 mg/g extract). In conclusion, the nonconventional methods presented here are superior for optimizing the yield of extract and its lignan contents from the aerial parts of <i>P. niruri</i>.
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