Lomatia tinctoria, R. Br.

Deans, Bianca J., Tedone, Laura, Bissember, Alex C. & Smith, Jason A., 2018, Phytochemical profile of the rare, ancient clone Lomatia tasmanica and comparison to other endemic Tasmanian species L. tinctoria and L. polymorpha, Phytochemistry 153, pp. 74-78 : 77-78

4.5. Extraction of Lomatia tinctoria

Diethyl ether maceration. Fresh leaves including petiole of L. tinctoria (19.0 g) (extracted within 1 h of obtaining plant material to reduce the likelihood of decomposition) were cut at the petiole into ∼ 2.5cm pieces (to form an even layer of material in the extraction vessel). The plant material was then completely submerged in diethyl ether (250 mL) and the vessel was sealed and maintained at room temperature. After 5 h, the leaves were observed to change to a black color, while the solution displayed a vibrant orange color. The mixture was filtered, dried (Na 2 SO 4), filtered, and evaporated under reduced pressure to provide an orange residue (207 mg). This mixture was purified by automated flash chromatography to deliver juglone (111 mg, 0.58% w/w) as red/orange crystalline solid and a colorless semi-solid (25 mg, 0.13% w/w) containing nonacosane predominantly (as judged by GC-MS; see: Supporting Information).

PHWE. Immediately following the above-mentioned diethyl ether maceration, residual solvent was allowed to evaporate from the leaf material that remained (∼10 min). The ensuing L. tinctoria leaves (14.5 g) were then coarsely ground in a spice grinder, mixed with sand (2 g), and extracted using the identical PHWE method detailed previously. The ensuing extract was then concentrated under reduced pressure on a rotary evaporator (50 ̊C bath temperature) to remove EtOH. The ensuing aqueous extract was then extracted with EtOAc (3 × 60 mL), with the organic extracts combined, dried over Na 2 SO 4, filtered and evaporated under reduced pressured to provide a solid dark yellow residue (59 mg).

A portion of the total 100 mL aqueous phase (30 mL) was evaporated to dryness (193 mg) and dissolved in pyridine (1.5 mL) and cooled to 0 ̊C. Acetic anhydride (1.5 mL) was added dropwise and the magnetically stirred reaction mixture was maintained at room temperature. After 21 h, the mixture was concentrated under reduced pressure to afford a brown gum (352 mg). Purification by automated flash column chromatography (0 → 100% EtOAc/hexanes; 10 min) provided α- and β- Glucose pentaacetate (91 mg).

4.6. Intraspecific studies of L. tasmanica , L. tinctoria and L. polymorpha

Diethyl ether maceration. Three specimens from each of the respective species were separately extracted (i.e., a total of nine extractions were performed). Fresh leaves including petiole of the three L. tasmanica specimens (A–C), L. tinctoria (D–F) and L. polymorpha (G–I) (10.0 g each) were completely submerged in diethyl ether (125 mL) in a sealed vessel, without stirring at room temperature. After 5 h, the leaves were observed to darken, while the solutions displayed a vibrant orange color. The mixtures were filtered, dried (Na 2 SO 4), filtered, and concentrated under reduced pressure to provide orange residue (Specimen A, 64 mg; Specimen B, 64 mg; Specimen C, 96 mg); (Specimen D, 65 mg; Specimen E, 64 mg; Specimen F, 51 mg); (Specimen G, 85 mg; Specimen H, 84 mg; Specimen I, 68 mg). The respective crude samples were then each analyzed by 1 H NMR spectroscopy (CDCl 3) and GC-MS (see: Supporting Information).

PHWE. Immediately following the above-mentioned diethyl ether maceration, residual solvent was allowed to evaporate from the remaining leaf material (∼10 min). The ensuing leaves were then coarsely ground in a spice grinder: Specimen A (7.6 g); Specimen B (7.7 g) Specimen C (7.5 g); Specimen D (7.9 g); Specimen E (7.8 g); Specimen F (7.7 g); Specimen G (8.5 g); Specimen H (7.8 g); Specimen I (7.7 g). Respective samples were mixed with sand (2 g), and extracted using standard PHWE method described previously. The ensuing extract was then concentrated under reduced pressure to remove EtOH (50 ̊C bath temperature). The ensuing aqueous extracts were then extracted with EtOAc (3 × 30 mL), with the organic extracts combined, dried over Na 2 SO 4, filtered and evaporated under reduced pressured to provide solid dark green residues: L. tasmanica (Specimen A, 26 mg; Specimen B, 32 mg; Specimen C, 21 mg); L. tinctoria (Specimen D, 11 mg; Specimen E, 24 mg; Specimen F, 21 mg); L. polymorpha (Specimen G, 76 mg; Specimen H, 21 mg; Specimen I, 18 mg). The respective crude samples were then each analyzed by 1 H NMR spectroscopy (acetone- d 6) and LC-MS (see: Supporting Information).