Natural products isolation studies of native Australian fern species

Keywords: ferns, flavonoid, glycoside, natural products, natural products isolation, neolignan, Polystichum, Todea.

Ferns are a group of vascular plants bearing complex leaves called megaphylls. These plants do not produce flowers or seeds and reproduce via spores. There are more than 12 000 species of ferns that are widely distributed across the globe, with the greatest diversity typically found in the tropics.^[1] Ferns represent the phylogenetic bridge between the lower and higher plants in the plant kingdom. For centuries, ferns have been used in many different contexts: as food, medicines, and ornaments. The fiddleheads (or croziers) of many fern species often feature in Asian cuisine. Indeed, it is reported that 52 species feature in Chinese food and it is estimated that the actual number of edible ferns may extend to 144 species.^[2] In Japan, ostrich (Matteuccia struthiopteris (L.) Tod.), bracken (Pteridium aquilinum (L.) Kuhn), and royal ferns (Osmunda japonica Thunb.) are the most popular edible ferns that are harvested.^[3] In Australia, the sporocarps of the small freshwater fern, nardoo (Marselia drummondii A. Braun) are consumed as baked cakes by first nations people following proper and extensive preparation.^[4] In addition to their use in cuisine, many fern species feature in traditional pharmacopoeias and are used to treat an array of ailments.^[5–8] In this context, relative to other species of vascular plants, ferns and lycophytes are poorly represented.^[9]

Ferns and lycophytes, like angiosperms, are a rich source of phytochemicals with interesting biological properties. Natural products isolation studies reveal that they contain flavonoids, terpenoids (including steroids), and polyphenols (Fig. 1).^[5,10] They also contain more distinctive alkaloid secondary metabolites. For example, lycopodium alkaloids such as lycopodine (1), lycodine (2), fawcettimine (3), and phlegmarine (4) skeletons have been isolated from Lycopodiaceae and Huperziaceae.^[10–12] Flavonoids are commonly isolated from numerous fern species. For example, species of the genus Pteris are rich in flavonoids with mainly α- and β-glucosides, galactosides, rhamnosides or arabinosides present.^[13,14] Distinctive flavonoids that have been isolated include neoflavonoids, calomelanols A–J (5–14) from farinose of Pityrogramma calomelanos (L.) Link,^[15,16] bioflavonoids such as hinokiflavone (15), 7″-O-methylhinokiflavone (16) amentoflavone (17) and 7,7″-di-O-methylamentoflavone (18) from Selaginella tamariscina (P.Beauv.) Spring,^[17] involvenflavones A–F (19–24) from S. involven (Sw.) Spring^[18] and prenylated flavonoids (25–28) from Helminthostachys zeylanica (L.) Hook.^[19] Many sesquiterpenoid compounds with indane or cadinene skeletons are found in ferns.^[10] Sesquiterpenyl indanones, known as pterosins, and their glycosides (pterosides) have been isolated from bracken fern species and polypodiaceous ferns.^[20] For example, multifidosides A–C (29–31) have been isolated from Pteris multifida Poir.^[14] and the carcinogenic pteroside, ptaquiloside (32) has been isolated from Pteridium aquilinium (L.) Kuhn.^[21] The terpenoids obtained from ferns are typically ent-kaurane-, ent-atisane- and ent-primarane-type diterpenoids, which are present in Pteris species.^[22] Labdane- and clerodane-type diterpenoids, diterpenoid glycosides and triterpenoids are major constituents in the Gleicheniaceae family,^[23–26] and ecdysteroids have been isolated from species of genera Microsorum and Diplopterygium in the family Polypodiaceae.^[27–31] Phenolic compounds are another class of secondary metabolites widely distributed in ferns. Commonly isolated molecules of this type include caffeic (33), chlorogenic (34) and vanillic (35) acids.^[10,32,33] The glycosylated phenolic acid, 7-O-caffeoylhydroxymaltol-3-β-d-glucopyranoside (36) was isolated from Pteris ensiformis Burm.,^[33–35] in addition to a chalcone derivative, licoagrochalcone D (37) from Pteris multifida Poir.^[36]

Fig. 1.  Examples of secondary metabolites isolated from ferns and lycophytes: alkaloids (1–4), complex flavonoids (5–28), sesquiterpenoids (29–32) and miscellaneous natural products (33–37); *stereogenic centres were not assigned in these molecules

Natural products research concerning ferns native to Australia are mainly restricted to toxicity studies. For example, the sporocarps of the freshwater fern nardoo, Marsilea drummondii A. Braun, which are used for food by Australian Aborigines,^[4] are reportedly toxic to humans, cattle, and sheep.^[37] Studies on this waterfern has revealed that its toxicity derives from high levels of the enzyme thiaminase which breaks down thiamine (vitamin B1).^[38] Similarly, bracken ferns Pteridium aquillinum (L.) Kuhn and P. esculentum (G.Forst.) Nakai have been the subject of many phytochemical and pharmacological studies in order to elucidate the mechanism of toxicity involving the carcinogenic norsesquiterpene glucoside, ptaquiloside (32).^[39–42] Consequently, many sesquiterpenoid compounds have been isolated from P. aquillinum (L.) Kuhn.^[20,43–45] The presence of ptaquiloside has been reported in fern species from the genera Pteris, Microlepia, Hypolepis and Chelianthes.^[43,44,46] Beyond their toxicity, very limited information regarding the phytochemistry of ferns found in Australia exists. Nevertheless, due to their wide geographic distribution, many native Australian ferns are also found in Asia and South America and natural products isolation studies of species found in these locations have been undertaken. For example, Helminthostachys zeylanica (L.) Hook which is found widely distributed in tropical parts of Asia, the Pacific region, and Australia,^[1] contains prenylated flavonoids, ugonins,^[47,48] cyclised geranyl stilbenes, and ugonstiblenes.^[49] Similarly, Salvinia species have a global distribution and the species, S. auriculata Aubl. and a hybrid, S.xmolesta D.S.Mitch. are found in Australia.^[1] Bioactivity-guided phytochemical investigation of these two Australian species allowed for the isolation of more than sixty different secondary metabolites that include diterpenes, polyphenols, fatty acids, triterpene, apocarotenoids, acyclic sesquiterpenoids, monoterpenes, jasmonates, steroids and coumarins.^[50–52] However, a large number of native Australian ferns, particularly endemic species, have not been the subject of natural products isolation studies.

In this report, a total of 16 Australian native fern species formed the basis of natural products isolation studies. Specifically, our research concerned Todea barbara (L.) T. Moore, Alsophila australis (R.Br.) Domin, Dicksonia antarctica Labill., Calochlaena dubia (R.Br.) M. D. Turner & R. A. White, Polystichum proliferum (R.Br.) C. Presl, P. vestitum (G. Forst.) C. Presl, Pellaea falcata (R.Br.) Fée, Lecanopteris pustulata subsp. pustulata (G. Forst.) Testo & A. R. Field, Oceaniopteris cartilaginea (Sw.) Gasper & Salino, Lomaria nuda (Labill.) Willd., Doodia australis (Parris) Parris, Austroblechnum penna-marina (Poir.) Gasper & V. A. O. Dittrich subsp. alpina (R.Br.) S. Jess. & L. Lehm., Parablechnum wattsii (Tindale) Gasper & Salino, Gleichenia alpina R.Br., Histiopteris incisa (Thunb.) J.Sm. and Pteridium esculentum (G. Forst.) Nakai subsp. esculentum. Two of these species, Polystichum vestitum (G. Forst.) C. Presl and Gleichenia alpina R.Br., were previously thought to be endemic to Tasmania,^[53] however, these species were recently found in New Zealand.^[1] We isolated a total of 14 different compounds (38–51) from 12 native Australian fern species, including γ- and δ-lactones; flavonoid glycosides, a dihydrobenzofuran neolignan, in addition to hydroxycinnamate/caffeic acid esters (Fig. 2).

Fig. 2.  Secondary metabolites 38–51 isolated from 12 of the 16 native Australian fern species investigated in this study.

For each of the 16 native Australian fern species that we investigated, PHWE of leaf material, followed by liquid–liquid extraction of the aqueous PHWE extract with ethyl acetate provided a crude organic extract after concentration under reduced pressure. In each case, the remaining aqueous phase was concentrated under reduced pressure to afford a crude aqueous extract. The combined yield (% w/w) of the respective crude extracts thus obtained is shown in Table 1. With the exception of P. vestitum (0.38% w/w), yields of crude extracts were >0.5% w/w in all cases. The crude extracts were then subjected to various standard flash column chromatography and preparative thin layer chromatography procedures.

Table 1.  Yields (% w/w) of the respective crude extracts obtained following PHWE of 16 native Australian fern species in this study.

Dicksonia antarctica and Pteridium esculentum are the only 2 of these 16 fern species that have been the subject of previous natural products isolation studies. Previously, the phenolic compounds (5S,6S,9S,10S)-15-hydroxycadina-3,11-dien-2-one and p-hydroxystyrene β-vicianoside, in addition to p-hydroxystyrene β-d-glucoside, kaempferol 3-O-β-d-glucoside, kaempferol 3-O-(2-O-β-d-xylosyl)-β-d-glucoside, kaempferol 3-O-(6-p-coumaroyl)-β-d-glucoside and chlorogenic acid have been isolated from P. esculentum fronds (1.2 kg of dry plant material).^[54] 4-O-Caffeoylshikimic acid and 4-O-(p-coumaroyl) shikimic acid were isolated from croziers of D. antarctica (2.4 kg of fresh plant material) in 1997.^[55,56] In this present study, secondary metabolites were not isolated from D. antarctica, Doodia australis, Oceaniopteris cartilaginea and Histiopteris species. However, 14 different compounds were isolated from the remaining 12 fern species we investigated. A combination of standard ¹H, ¹³C and 2D (COSY, HMBC and HSQC) NMR spectroscopic techniques were employed to elucidate the structures of these compounds. In each case, these data were consistent with equivalent data reported in the literature. Specifically, we obtained 5,6-dihydro-5-hydroxy-6-methyl-2H-pyran-2-one (38), 5-(1-hydroxyethyl)-2(5H)-furanone (39), osmundalin (40), astragalin (41), 4β-carboxymethyl-(−)-epicatechin (42), (2R)-eriodictyol-7-O-β-d-glucopyranoside (43), naringin (44), (−)-trans-blechnic acid (45), (p-hydroxybenzyl)malonic acid (46), quercitrin (47), quercetin 3-O-(6″-acetyl-β-d-glucopyranoside) (48), 5-O-caffeoylshikimic acid (49), rutin (50), and tiliroside (51) (Fig. 2).

5,6-Dihydro-5-hydroxy-6-methyl-2H-pyran-2-one (38) and 5-(1-hydroxyethyl)-2(5H)-furanone (39) were obtained as a mixture from Todea barbara in a ~2:1 ratio, as judged by NMR spectroscopic and GC-MS analysis (see Supplementary Material). Osmundalin (40) was also isolated from T. barbara. Lactones 38–40 have been isolated from Osmunda japonica, a common Japanese fern species, and are reported to exhibit antifeedant properties against the larvae of yellow butterfly, Eurema hecabe mandarina.^[57,58] In addition, all three natural products have also been isolated from Angiopteris caudatiformis, a fern species used in Chinese folk medicine for the treatment of a broad range of ailments.^[59] Natural products 38 and 39 have been isolated from the fern species A. esculenta^[60] and angiopteroside, an epimer of osmundalin (40), was isolated from Angiopteris evecta.^[61]

4β-Carboxymethyl-(−)-epicatechin (42),^[62] was isolated from Polystichum vestitum. This secondary metabolite has been isolated from Davallia divaricata,^[62] D. solida^[63] and Dryopteris crassirhizoma.^[64] We also obtained molecule 42 from Polystichum proliferum and Lecanopteris pustulata subsp. pustulata. All of these fern species are members in the order Polypodiales. Dihydrobenzofuran neolignane, (−)-trans-blechnic acid (45) was isolated from Austroblechnum penna-marina subsp. alpina; a species formerly classified within the genus Blechnum. (−)-trans-Blechnic acid (45) and its epimer, epiblechnic acid, represent characteristic constituents of the family Blechnaceae.^[65] Blechnic acid has been isolated from various fern species, including Blechnopsis orientalis, Spicantopsis amabilis, S. niponica, Woodwardia orientalis, W. prolifera, Brainea insignis^[65] and Struthiopteris spicant.^[66] We isolated 5-O-caffeoylshikimic acid (49) (0.5% w/w yield) from Parablechnum wattsii (also formerly in the genus Blechnum). Compound 49 is a major secondary metabolite present in this fern and a known enzymatic browning agent present in dates, Phoenix dactylifera.^[55,67] It is a major phytochemical and an anti-thiamine factor isolated from Pteridium aquillinum var. latiusculum and reportedly causes depression of leucocytes and thrombocytes in calves.^[68] 5-O-Caffeoylshikimic acid is found widely distributed in Equisetaceae family and in ferns from the families Adiantaceae, Dryopteridaceae, Athyriaceae, Dennstaedtiaceae, Osmundaceae and Thelypteridaceae.^[69] Interestingly, we isolated p-hydroxybenzylmalonic acid (46) and 4β-carboxymethyl-(–)-epicatechin (42) from Lecanopteris pustulata subsp. pustulata. Natural product 46 has been isolated from liquorice previously.^[70] Liquorice primarily derives from three species, Glycyrrhiza glabra, Glycyrrhiza uralensis and Glycyrrhiza inflata and the presence of p-hydroxybenzylmalonic acid (46) has been reported from all three.^[71]

Flavonoid glycosides 41, 43, 44, 47, 48, 50, and 51 were also isolated in our study. Specifically, we obtained the common flavonoid glucoside astragalin (41) from Alsophila australis, Calochalaena dubia and Pteridium esculentum. It has been isolated from many plant species including from the bracken fern P. aquilinum.^[72–76] We isolated (2R)-eriodictyol-7-O-β-d-glucopyranoside (43) from P. vestitum. This natural product has been isolated from a wide range of flowering plants.^[77–85] In ferns, its presence has been identified in species of Pyrrosia.^[86] Molecule 43 is a reported Nrf2 activator and confers protection against cisplatin-induced toxicity and cerebral ischemic injury.^[84,87] Naringin (44) and rutin (50) were isolated from Pellaea falcata and Gleichenia alpina, respectively. Both are commonly reported flavonoid glycosides found in citrus, and exhibit a broad range of pharmacological activity.^[88–95] Compounds 44 and 50 have also been found in fern species.^[96–99] Trace amounts of quercitrin (47) and quercetin 3-O-(6″-acetyl)-glucoside (48) were isolated from Lomaria nuda. Both molecules are present in an array of plant species and possess wide ranging biological properties. Tiliroside (51), a kaempferol flavonoid glucoside with a coumaroyl moiety, was isolated from Pteridium esculentum. Its presence has been identified in various plant species, including P. aquilinum; and compound 51 is a reported carcinogen found in bracken fern.^[100–103] However, tiliroside also exhibits profound anti-hyperglycemic, anti-hyperlipidemic and antioxidant effects, and has potential therapeutic applications for the treatment of diabetes.^{[100,104–106]}

Among the 14 secondary metabolites isolated in this study, osmundalin (40), 4β-carboxymethyl-(−)-epicatechin (42) and trans-blechnic acid (45) have only been isolated from fern sources to date (Table 2). This reveals that various Australian fern species investigated in our study contain secondary metabolites that are consistent with other members in the genera, families or orders that are found distributed beyond Australia. For example, Todea barbara is exclusively a southern hemisphere species and both Osmunda japonica and Angiopteris caudatiformis are species found in eastern Asia regions. trans-Blechnic acid (45) has only been obtained from fern species of the family Blechnaceae. The remaining natural products are regularly isolated from angiosperms, including various fern species. 4β-Carboxymethyl-(−)-epicatechin (42) is a particularly rare natural product and its isolation solely from ferns suggests that its formation might derive from a biosynthetic pathway unique to certain fern species (Scheme 1).

Table 2.  Distribution of isolated secondary metabolites 38–51 in ferns species.

Scheme 1.  Overview of possible biosynthetic pathway leading to 4β-carboxymethyl-(−)-epicatechin (42).

Our natural products isolation studies provide further evidence that fern species represent a source of structurally diverse phytochemicals with interesting biological properties. Phytochemical screening of 16 native Australian ferns enabled the isolation of 14 previously reported compounds. The structures of these compounds were primarily elucidated via 1D (¹H and ¹³C) and 2D (COSY, HSQC and HMBC) NMR spectroscopy. In each case, the characterisation data were consistent with equivalent data reported in the literature. These isolated compounds included flavonoid glycosides, caffeic acid esters, lactones, and a dihydrobenzofuran neolignan. Lactone molecules, 5,6-dihydro-5-hydroxy-6-methyl-2H-pyran-2-one, 5-(1-hydroxyethyl)-2(5H)-furanone and osmundalin were isolated from Todea barbara. 4β-Carboxymethyl-(−)-epicatechin was isolated from both species of Polystichum investigated in this study. Our isolation of (−)-trans-blechnic acid from Austroblechnum penna-marina subsp. alpina is consistent with this natural product representing a characteristic compound in the family Blechnaceae.

Leaf material from 13 ferns Todea barbara, Alsophila australis, Dicksonia antarctica, Calochlaena dubia, Polystichum proliferum, P. vestitum, Pellaea falcata, Lecanopteris pustulata subsp. pustulata, Oceaniopteris cartilaginea, Lomaria nuda, Doodia australis, Austroblechnum penna-marina subp. alpina and Parablechnum wattsii were collected from the Royal Botanical Gardens of Tasmania in Hobart during February 2020. Aerial parts of Gleichenia alpina was collected from Wombat Moor at Mt Field National Park (42.6829°S, 146.6174°E; 1073 m above sea level) in February 2021. Histiopteris incisa was collected from Mt Field National Park (42.7806°S, 146.5844°E; 461 m above sea level) in February 2021. Aerial parts of Pteridium esculentum were collected from five healthy plants growing at Marion Bay (42.8218°S, 147.8671°E) in October 2021. Voucher specimens have been provided to the Tasmanian Herbarium, Tasmanian Museum and Art Gallery (no. HO607603–HO607618). With the exception of Pteridium esculentum, all plant material was air-dried for 2 weeks and stored prior to extraction. P. esculentum plant material was dried in an oven at 45°C for 3 days prior to extraction.

Solvents used in all experiments were of analytical grade or purified by standard laboratory procedures. Plant material was ground using a Sunbeam spice grinder. Pressurised hot water extraction (PHWE) was performed using Breville Expresso Machine Model 800ES. This PHWE method is a well-established natural products extraction technique.^[107–109] The extracted organic solvents were dried using anhydrous MgSO₄ and Na₂SO₄. Solvents were removed under reduced pressure on a rotary evaporator. Flash column chromatography was performed using flash grade silica gel (Kieselgel 60). Automated flash chromatography was performed using a Grave Reveleris X2 flash column chromatography system or a Büchi Flash Pure system with 40 µm silica gel cartridges. TLC analysis was performed using Merck silica gel 60-F₂₅₄ plates. NMR spectroscopy was performed on a Bruker Avance III NMR spectrometer operating at 400 MHz (¹H) and 100 MHz (¹³C) or Bruker Ascend^TM 600 NMR spectrometer operating at 600 MHz for (¹H) and 150 MHz (¹³C). The deuterated solvents used were D₂O, CDCl₃, acetone-d₆, CD₃OD and DMSO-d₆. Spectra were calibrated by assignment of the residual solvent peak to δ_H 7.26 and δ_C 77.16 for CDCl₃; δ_H 2.50 and δ_C 39.52 for DMSO-d₆; δ_H 3.31 and δ_C 49.00 for CD₃OD; δ_H 4.79 for D₂O; and δ_H 2.05 and δ_C 29.84 for acetone-d₆.^[110] Infrared spectroscopy was performed using a Shimadzu FTIR 8400s spectrometer, with samples prepared as thin films on NaCl plates. Gas chromatography mass spectrometry (GC-MS) experiments were performed on Agilent 6850 GC and Agilent 5975C mass spectrometers. HRESIMS analyses were conducted on a Thermo LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific).

T. barbara dried leaflets (29 g) were finely ground using a spice grinder, mixed with sand (~4 g), and extracted via PHWE (35% EtOH/H₂O). This provided a hot extract (200 mL) that was cooled immediately in an ice bath. This process was repeated a further two times to provide a combined extract (600 mL). The extract was concentrated under reduced pressure (35°C) to remove EtOH and extracted with EtOAc (150 mL and 2 × 100 mL). The combined organic phase was dried (MgSO₄), filtered, and concentrated under reduced pressure to provide extract A (646 mg). The remaining aqueous phase was concentrated under reduced pressure (50°C) to yield a dark brown extract B (10 g). Extract A: Extract A (646 mg) was redissolved in EtOAc, adsorbed onto silica/Celite^® (1:1 mixture by mass), and subjected to flash column chromatography {silica; hexanes (50 mL), 10% acetone/hexanes (50 mL), 20% acetone/hexanes (100 mL), 30% acetone/hexanes (150 mL), 40% acetone/hexanes (100 mL), 50% acetone/hexanes (50 mL), 60% acetone/hexanes (50 mL) and acetone (150 mL)} to provide a mixture of compounds 38 and 39 (23 mg, 0.8% w/w yield in a ~2:1 ratio) as colourless crystalline solids. This mixture was analysed by GC-MS. Extract B: Extract B (10 g) was soaked in MeOH (~200 mL) for 1 h, repeated five times, combined, and concentrated to provide extract B.1 (4 g). Extract B.1 was then soaked in acetone (~200 mL) for 1 h, repeated 5 times, combined, and concentrated to provide extract B.2 (~2 g). Extract B.2 (~1 g) was redissolved in acetone, absorbed onto silica/Celite^® (1:1 mixture by mass), and subjected to flash column chromatography {silica; hexanes (50 mL), 10% acetone/hexanes (100 mL), 50% acetone/hexanes (50 mL), acetone (50 mL) and 50% MeOH/acetone (50 mL)}. Following TLC and ¹H NMR spectroscopic analysis, the resulting fractions were combined to afford six fractions, F.1–6. Fraction F.5 (709 mg) was again adsorbed onto silica/Celite^® (1:1 mixture by mass) and subjected to flash column chromatography {silica; hexanes (50 mL), 10% acetone/hexanes (50 mL), 30% acetone/hexanes (200 mL), 50% acetone/hexanes (200 mL), and acetone (30 mL)}, which provided compound 40 (570 mg, 2.0% w/w yield) as an off-white solid. Extract B.1 (1.5 g) was absorbed onto silica/Celite^® (1:1 mixture by mass) and subjected to flash column chromatography {silica; CH₂Cl₂ (75 mL), 13% MeOH/CH₂Cl₂ (150 mL), 27% MeOH/CH₂Cl₂ (75 mL), 53% MeOH/CH₂Cl₂ (150 mL) and MeOH (100 mL)}. Following TLC and ¹H NMR spectroscopic analysis the resulting fractions were combined to afford four fractions, F.1–4. Fraction F.3 (641 mg) was redissolved in MeOH, absorbed onto silica/Celite^® (1:1 mixture by mass) and subjected to flash column chromatography {silica; CH₂Cl₂ (50 mL), 25% MeOH/CH₂Cl₂ (100 mL), 66% MeOH/CH₂Cl₂ (75 mL) and MeOH (50 mL)}, which provided compound 40 (30 mg, 2.0% w/w yield) as an off-white solid.

Osmundalactone (38).^[57] Colourless crystalline solid. ¹H NMR (600 MHz, CDCl₃) δ 6.78 (1H, dd, J = 9.9 and 2.3 Hz; H-4), 5.92 (1H, dd, J = 9.9 and 1.9 Hz; H-3), 4.31 (1H, m; H-6), 4.18 (1H, d, J = 8.7 Hz; H-5), 1.42 (3H, d, J = 6.4 Hz; 7-CH₃) ppm; ¹³C NMR (150 MHz, CDCl₃) δ 163.2 (C-2), 148.5 (C-4), 120.7 (C-3), 79.0 (C-6), 67.7 (C-5), 18.2 (7-CH₃) ppm.

5-(1-Hydroxyethyl)-2(5H)-furanone (39).^[57,58] Colourless crystalline solid. ¹H NMR (600 MHz, CDCl₃) δ 4.35 (1H, m; H-5), 4.08 (1H, m; H-6), 2.53 (1H, m; H-3a), 2.48 (1H, m; H-3b), 2.19 (1H, m; H-4a), 2.12 (1H, m; H-4b), 1.13 (3H, d, J = 6.5 Hz; 7-CH₃) ppm; ¹³C NMR (150 MHz, CDCl₃) δ 177.5 (C-2), 83.5 (C-5), 67.4 (C-6), 28.6 (C-3), 20.9 (C-4), 17.7 (7-CH₃) ppm.

Osmundalin (40) (CAS# 54835-71-1).^[57] Off-white solid. [α] –65.1° (c 0.075, MeOH), lit. [α] –107° (c 1.0, MeOH).^{[58] 1}H NMR 600 MHz, CD₃OD) δ 7.04 (1H, dd, J = 9.9 and 2.9 Hz; H-3), 5.98 (1H, dd, J = 9.9 and 1.5 Hz; H-2), 4.48 (1H, quintet, J = 7.4 Hz; H-5), 4.45 (1H, d, J = 7.8 Hz; H-1′), 4.43 (1H, ddd, J = 7.6, 2.8 and 1.5 Hz; H-4), 3.85 (1H, dd, J = 11.8 and 5.6 Hz; H-6′b), 3.79 (1H, dd, J = 11.8 and 1.9 Hz; H-6′a), 3.33 (1H, t, J = 8.9 Hz; H-3′), 3.26 (2H, m; H-4′,5′), 3.16 (1H, t, J = 9.1 Hz; H-2′), 1.42 (3H, d, J = 6.5 Hz; 6-CH₃) ppm; ¹³C NMR (150 MHz, CD₃OD) δ 165.14 (C-1), 147.71 (C-3), 121.55 (C-2), 102.81 (C-1′), 79.29 (C-5), 78.16 (C-5′), 77.95 (C-3′), 74.82 (C-2′), 73.36 (C-4), 71.49 (C-4′), 62.72 (C-6′), 18.57 (C-6) ppm.

A. australis dried leaflets (30 g) were finely ground using a spice grinder, mixed with sand (~6 g), and extracted via PHWE (35% EtOH/H₂O). This provided a hot extract (200 mL) that was cooled immediately in an ice bath. This process was repeated a further two times to provide a combined extract (600 mL). The extract was concentrated under reduced pressure (35°C) to remove EtOH and extracted with EtOAc (2 × 200 mL and 150 mL). The combined organic phase was dried (MgSO₄), filtered, and concentrated under reduced pressure to provide extract A (268 mg). The remaining aqueous phase was concentrated under reduced pressure (50°C) to yield a dark brown extract B (7 g). Extract A: Extract A (268 mg) was redissolved in EtOAc and MeOH, absorbed onto silica/Celite^® (1:1 mixture by mass), and subjected to flash column chromatography {silica; hexanes (100 mL), 50% EtOAc/hexanes (100 mL), EtOAc (100 mL), 10% MeOH/EtOAc (100 mL) and 20% MeOH/EtOAc (100 mL)}. Following TLC and ¹H NMR spectroscopic analysis, the resulting fractions were combined to afford five fractions, F.1–5. Fraction F.3 (146 mg) was redissolved in EtOAc and MeOH, adsorbed onto absorbed onto silica/Celite^® (1:1 mixture by mass), and subjected to flash column chromatography {silica; CH₂Cl₂ (150 mL), 20% EtOAc/CH₂Cl₂ (100 mL), 40% EtOAc/CH₂Cl₂ (100 mL), 60% EtOAc/CH₂Cl₂ (100 mL), 80% EtOAc/CH₂Cl₂ (100 mL), EtOAc (100 mL) and 20% MeOH/EtOAc (100 mL)}, which afforded compound 41 (12 mg, 0.04% w/w) as a pale-yellow solid.

Astragalin (41) (CAS# 480-10-4).^[73,75] Pale-yellow solid. ¹H NMR (600 MHz, CD₃OD) δ 8.06 (2H, d, J = 8.9 Hz; H-2′,6′), 6.90 (2H, d, J = 8.9 and 1.9 Hz; H-3′, 5′), 6.41 (1H, d, J = 2.1 Hz; H-8), 6.21 (1H, d, J = 2.1 Hz; H-6), 5.26 (1H, d, J = 7.4 Hz; H-1″), 3.69 (1H, dd, J = 11.9 and 2.3 Hz; H-6a″), 3.53 (1H, dd, J = 11.9 and 5.6 Hz; H-6b″), 3.40–3.46 (2H, m; H-2″,3″), 3.32 (1H, m; H-4″), 3.21 (1H, m; H-5″) ppm; ¹³C NMR (150 MHz, CD₃OD) δ 179.54 (C-4), 165.98 (C-7), 163.10 (C-5), 161.58 (C-4′), 159.09 (C-2), 158.52 (C-9), 135.45 (C-3), 132.28 (C-2′,6′), 122.80 (C-1′), 116.07 (C-3′,5′), 105.75 (C-10), 104.04 (C-1″), 99.87 (C-6), 94.73 (C-8), 78.43 (C-5″), 78.04 (C-3″), 75.73 (C-2″), 71.36 (C-4″), 62.62 (C-6″) ppm.

C. dubia leaflets (30 g) were finely ground using a spice grinder, mixed with sand (~6 g), and extracted via PHWE (35% EtOH/H₂O). This provided a hot extract (200 mL) that was cooled immediately in an ice bath. This process was repeated a further two times to provide a combined extract (600 mL). The extract was concentrated under reduced pressure (35°C) to remove EtOH and extracted with EtOAc (3 × 200 mL). The combined organic phase was dried (MgSO₄), filtered, and concentrated under reduced pressure to provide dark green extract A (575 mg). The remaining aqueous phase was concentrated under reduced pressure (50°C) to yield a dark brown extract B (10 g). Extract A: Extract A (315 mg) was redissolved in MeOH and EtOAc, absorbed onto silica/Celite^® (1:1 mixture by mass), and subjected to automated flash chromatography {silica cartridge (24 g); 0–100% EtOAc/hexanes and 0–50% MeOH/EtOAc, for 12 min with flow rate of 28 mL/min}. Following TLC and ¹H NMR spectroscopic analysis, the resulting fractions were combined to give nine fractions, F.1–9. Fraction F.8 (41 mg) was subjected to flash column chromatography {silica; CH₂Cl₂ (50 mL), 10% MeOH/CH₂Cl₂ (200 mL) and 50% MeOH/CH₂Cl₂ (20 mL)}, which provided compound 41 (4 mg, 0.014% w/w).

P. vestitum dried leaflets (30 g) were finely ground using a spice grinder, mixed with sand (~6 g), and extracted via PHWE (35% EtOH/H₂O). This provided a hot extract (200 mL) that was cooled immediately in an ice bath. This process was repeated a further two times to provide a combined extract (600 mL). The extract was concentrated under reduced pressure (35°C) to remove EtOH and extracted with EtOAc (150 mL and 2 × 100 mL). The combined organic phase was dried (Na₂SO₄), filtered, and concentrated under reduced pressure to provide extract A (114 mg). The remaining aqueous phase was concentrated under reduced pressure (50°C) to yield a dark brown extract B (7 g). Extract A: Extract A (114 mg) was redissolved in EtOAc and MeOH, adsorbed onto silica/Celite^® (1:1 mixture by mass), and subjected to automated flash column chromatography {silica cartridge (4 g); 0–100% EtOAc/hexanes and 0–50% MeOH/EtOAc, for 26 min with flow rate 12 mL/min)} and afforded compound 42 (30 mg, 0.1% w/w) as a light brown solid. Following TLC and ¹H NMR spectroscopic analysis, the fractions were combined to give six fractions, F.1–6. Fractions F.3–5 (27 mg) were combined and subjected to flash column chromatography {silica; CH₂Cl₂ (50 mL), 5% MeOH/CH₂Cl₂ (50 mL), 15% MeOH/CH₂Cl₂ (50 mL) and 40% MeOH/CH₂Cl₂ (30 mL)} to provide compound 43 (4 mg) as a yellow solid. Extract B: Extract B (4 g) was redissolved in MeOH and water, adsorbed onto silica/Celite^® (1:1 mixture by mass), and fractionated through a silica plug (150 mL of EtOAc, 10% MeOH/EtOAc, 20% MeOH/EtOAc, 30% MeOH/EtOAc, 40% MeOH/EtOAc and 50% MeOH/EtOAc) which afforded six fractions, F.1–6. Following ¹H NMR spectroscopic analysis, fractions F.1 and 2 (145 mg) were recombined, absorbed onto silica and subjected to flash column chromatography {silica; CH₂Cl₂ (100 mL), 2.5% MeOH/CH₂Cl₂ (100 mL), 5% MeOH/CH₂Cl₂ (100 mL), 10% MeOH/CH₂Cl₂ (100 mL), 15% MeOH/CH₂Cl₂ (100 mL), 20% MeOH/CH₂Cl₂ (100 mL) and 35% MeOH/CH₂Cl₂ (100 mL)} to provide compound 42 (10 mg, 0.035% w/w).

4β-Carboxymethyl-(–)-epicatechin (42) (CAS# 146805-53-0).^[62] Light-brown solid. ¹H NMR (600 MHz, acetone-d₆) δ 7.08 (1H, d, J = 1.7 Hz; H-2′), 6.87 (1H, dd, J = 8.2 and 1.8 Hz; H-6′), 6.81(1H, d, J = 2.2 Hz; H-5′), 6.04 (1H, d, J = 2.2 Hz; H-8), 5.94 (1H, d, J = 2.2 Hz; H-6), 4.93 (1H, s; H-2), 4.01 (1H, s; H-3), 3.46 (1H, d, J = 6.2 Hz; H-4), 3.05 (1H, dd, J = 16.4 and 3.5 Hz; H-1b″), 2.45 (1H, dd, J = 16.4 and 11.2 Hz; H-1a″) ppm. ¹³C NMR (150 MHz, acetone-d₆) δ 173.9 (2″-COOH), 157.9 (C-7,9), 156.3 (C-5), 145.5 (C-4′), 145.3 (C-3′), 132.2 (C-1′), 119.3 (C-6′), 115.6 (C-5′), 115.3 (C-2′), 102.7 (C-10), 96.6 (C-8), 95.8 (C-6), 75.4 (C-2), 70.2 (C-3), 39.1 (C-1″), 36.0 (C-4) ppm.

(2R)-Eriodictyol-7-O-β-d-glucopyranoside (43) (CAS# 38965-51-4).^[80] Yellow solid. ¹H NMR (600 MHz, CD₃OD) δ 6.82 (1H, s, H-2′), 6.69 (2H, t, J = 10.9 Hz, H-5′,6′), 6.11 (1H, s, H-6), 6.09 (1H, s, H-8), 5.23 (1H, d, J = 12.5 Hz, H-2), 4.87 (1H, t, J = 6.6 Hz, H-1″), 3.88 (1H, d, J = 12.1 Hz, H-6a″), 3.69 (1H, m, H-6b″), 3.42–3.47 (3H, m, H-2″,3″,5″), 3.39 (1H, m, H-4″), 3.13 (1H, dd, J = 17.5 Hz, H-3a), 2.75 (1H, d, J = 17.0 Hz, H-3b) ppm. ¹³C NMR (150 MHz, CD₃OD) δ 198.5 (C-4), 167.0 (C-7), 164.9 (C-9), 164.6 (C-5), 146.9 (C-3′), 146.5 (C-4′), 131.5 (C-1′), 119.3 (C-6′), 116.3 (C-5′), 114.8 (C-2′), 104.9 (C-10), 101.2 (C-1″), 97.9 (C-8), 96.9 (C-6), 80.7 (C-2), 78.3 (C-3″), 77.8 (C-5″), 74.7 (C-2″), 71.2 (C-4″), 62.3 (C-6″), 44.1 (C-3) ppm.

P. proliferum leaves (30 g) were finely ground using a spice grinder, mixed with sand (7 g), and extracted via PHWE (35% EtOH/H₂O). This provided a hot extract (200 mL) that was cooled immediately in an ice bath. This process was repeated a further two times to provide a combined extract (600 mL). The extract was concentrated under reduced pressure (35°C) to remove EtOH and extracted with EtOAc (150 mL and 2 × 100 mL). The combined organic phase was dried (Na₂SO₄), filtered, and concentrated under reduced pressure to provide extract A (240 mg). The remaining aqueous phase was concentrated under reduced pressure (50°C) to afford a dark brown extract B (4 g). Extract A: Extract A (240 mg) was redissolved in CH₂Cl₂ and MeOH, adsorbed onto silica/Celite^® (1:1 mixture by mass), and subjected to flash column chromatography {silica; CH₂Cl₂ (200 mL), 10% MeOH/CH₂Cl₂ (100 mL), 20% MeOH/CH₂Cl₂ (100 mL) and 40% MeOH/CH₂Cl₂ (200 mL)} which provided compound 42 (16 mg, 0.06% w/w).

P. falcata dried leaflets (30 g) were finely ground using a spice grinder, mixed with sand (~8 g), and extracted via PHWE (35% EtOH/H₂O). This provided a hot extract (200 mL) that was cooled immediately in an ice bath. This process was repeated a further two times to provide a combined extract (600 mL). The extract was concentrated under reduced pressure (35°C) to remove EtOH and extracted with EtOAc (150 mL and 3 × 100 mL). The combined organic phase was dried (MgSO₄), filtered, and concentrated under reduced pressure to provide extract A (1.0 g). The remaining aqueous phase was concentrated under reduced pressure (50°C) to yield a dark brown extract B (8 g). Extract A: Extract A (1.0 g) was redissolved in EtOAc and MeOH, adsorbed onto silica/Celite^® (1:1 mixture by mass) and subjected to automated flash chromatography {silica cartridge (12 g); (0–100% EtOAc/hexanes and 0–50% MeOH/EtOAc for 21 min with a flow rate of 28 mL/min)}. Following TLC and ¹H NMR spectroscopic analysis, the resulting fractions were combined to afford seven larger fractions, F.1–7. Fraction F.5 was redissolved in MeOH, adsorbed onto silica/Celite^® (1:1 mixture by mass) subjected to flash column chromatography {silica; CH₂Cl₂ (50 mL), 10% MeOH/CH₂Cl₂ (100 mL), 20% MeOH/CH₂Cl₂ (50 mL) and 40% MeOH/CH₂Cl₂ (50 mL)} which provided compound 44 (21 mg, 0.7% w/w) as a yellow solid.

Naringin (44) (CAS# 10236-47-2).^[111,112] Yellow solid. ¹H NMR (600 MHz, CD₃OD) δ 7.32 (2H, d, J = 8.5 Hz; H-2′,6′), 6.82 (2H, d, J = 8.5 Hz; H-3′,5′), 6.18 (1H, d, J = 2.0 Hz; H-8), 6.16 (1H, d, J = 2.1 Hz; H-6), 5.37 (1H, dd, J = 12.9 and 2.7 Hz; H-2), 5.25 (1H, s; H-1″), 5.09 (1H, d, J = 7.6 Hz; H-1″), 3.93 (1H, s; H-2″), 3.85–3.90 (2H, m; H-6a″,5‴), 3.62–3.69 (2H, m; H-3‴,6b″), 3.57-3.60 (2H, m; H-2″,5″), 3.43–3.46 (1H, m; H-3″), 3.39 (2H, t, J = 9.5 Hz; H-4″,4‴), 3.16 (1H, dd, J = 17.2 and 12.9 Hz; H-3a), 2.75 (1H, dd, J = 17.2 and 2.8 Hz; H-3b), 1.28 (3H, d, J = 6.2 Hz; 6‴-CH₃) ppm. ¹³C NMR (150 MHz, CD₃OD) δ 198.5 (C-4), 166.6 (C-7), 164.9 (C-5), 164.6 (C-9), 159.1 (C-4′), 130.8 (C-1′), 129.1 (C-2′,6′), 116.3 (C-3′,5′), 104.9 (C-10), 102.5 (C-1‴), 99.4 (C-1″), 97.8 (C-6), 96.7 (C-8), 80.7 (C-2), 79.0 (C-2″), 78.9 (C-3″), 78.1 (C-5″), 73.9 (C-4″‘), 72.2 (C-3‴), 71.2 (C-2‴), 71.2 (C-4″), 69.9 (C-5‴), 62.3 (C-6″), 44.1 (C-3), 18.2 (6‴-CH₃) ppm.

A. penna-marina subsp. alpina dried leaflets (29 g) were finely ground using a spice grinder, mixed with sand (~7.5 g), and extracted via PHWE (35% EtOH/H₂O). This provided a hot extract (200 mL) that was cooled immediately in an ice bath. This process was repeated a further three times to provide a combined extract (800 mL). The extract was concentrated under reduced pressure (35°C) to remove EtOH and extracted with EtOAc (150 mL and 2 × 100 mL). The combined organic phase was dried (MgSO₄), filtered, and concentrated under reduced pressure to provide extract A (409 mg). The remaining aqueous phase was concentrated under reduced pressure (50°C) to yield a dark brown extract B (6 g). Extract A. Extract A (400 mg) was redissolved in MeOH and EtOAc, adsorbed onto silica/Celite^® (1:1 mixture by mass) and subjected to flash column chromatography {silica; hexanes (100 mL), 20% EtOAc/hexanes (200 mL), 40% EtOAc/hexanes (100 mL), 60% EtOAc/hexanes (100 mL), 80% EtOAc/hexanes (100 mL), EtOAc (50 mL), 10% MeOH/EtOAc (100 mL) and MeOH/EtOAc (100 mL)} that provided compound 45 (8.9 mg, 0.3% w/w) as a pale-green solid.

Blechnic acid (45) (CAS# 146805-53-0).^[113] Pale-green solid. [α] –23.2° (c 0.0345, MeOH), lit. [α] –28° (c = 1.0, MeOH).^{[65] 1}H NMR (600 MHz, CD₃OD) δ 7.47 (d, J = 15.9 Hz; H-7′), 7.03 (d, J = 8.5 Hz; H-6′), 6.86 (d, J = 2.0 Hz; H-2), 6.74 (dd, J = 8.2, 1.9 Hz; H-6), 6.71 (d, J = 8.4 Hz; H-5), 6.65 (d, J = 8.2 Hz; H-5′), 6.16 (d, J = 15.9 Hz; H-8′), 5.83 (d, J = 9.3 Hz; H-7), 4.50 (d, J = 9.3 Hz; H-8) ppm. ¹³C NMR (150 MHz, CD₃OD) δ 173.6 (C-9), 170.6 (C-9′), 149.6 (C-3′), 146.5 (C-4), 146.0 (C-3), 145.1 (C-4′), 143.3 (C-7′), 129.3 (C-1), 129.1 (C-2′), 124.5 (C-1′), 122.6 (C-6′), 119.7 (C-6), 118.0 (C-5′), 117.8 (C-8′), 115.9 (C-5), 115.1 (C-2), 88.4 (C-7), 55.3 (C-8) ppm. HRESIMS m/z calcd for C₁₈H₁₄O₈Na [M + Na]⁺ 381.0586; found 381.0581.

L. pustulata subsp. pustulata dried leaflets (30 g) were finely ground using a spice grinder, mixed with sand (~7 g), and extracted via PHWE (35% EtOH/H₂O). This provided a hot extract (200 mL) that was cooled immediately in an ice bath. This process was repeated a further two times to provide a combined extract (600 mL) and extracted with EtOAc (150 mL and 2 × 100 mL). The combined organic phase was dried (MgSO₄), filtered, and concentrated under reduced pressure to provide extract A (154 mg). The remaining aqueous phase was concentrated under reduced pressure (50°C) to yield a dark brown extract B (5.6 g). Extract A. Extract A (118 mg) was redissolved in MeOH, adsorbed onto silica/Celite^® (1:1 mixture by mass) and subjected to flash column chromatography {silica; hexanes (30 mL), 30% EtOAc/hexanes (50 mL), 60% EtOAc/hexanes (100 mL), 80% EtOAc/hexanes (50 mL), EtOAc (50 mL), 20% MeOH/EtOAc (50 mL) and 50% MeOH/EtOAc (25 mL)}. Following TLC and ¹H NMR spectroscopic analysis, the resulting fractions were combined to afford six larger fractions, F.1–6. Fractions F.2 (19.5 mg) was redissolved in CH₂Cl₂ and MeOH, adsorbed onto silica/Celite^® (1:1 mixture by mass) and subjected to flash column chromatography {silica; CH₂Cl₂ (25 mL), 5% MeOH/CH₂Cl₂ (25 mL) and 20% MeOH/CH₂Cl₂ (25 mL)}. Following TLC analysis, the resulting fractions were combined to afford three fractions F.1–3; and F.2 provided compound 42 (12 mg, 0.04% w/w). Extract B. Extract B (5.6 g) was redissolved in MeOH and water, adsorbed onto silica/Celite^® (1:1 mixture by mass) and subjected to flash chromatography {silica; EtOAc (200 mL), 10% MeOH/EtOAc (200 mL), 20% MeOH/EtOAc (200 mL) and 30% MeOH/EtOAc (200 mL)} that provided five fractions, F.1–5. Fraction F.1 (127 mg) was redissolved in MeOH and CH₂Cl₂, adsorbed onto silica/Celite^® (1:1 mixture by mass) and subjected to flash column chromatography {silica; CH₂Cl₂ (50 mL), 5% MeOH/CH₂Cl₂ (100 mL), 10% MeOH/CH₂Cl₂ (50 mL), 30% MeOH/CH₂Cl₂ (100 mL) and MeOH (30 mL)} that provided compound 46 (4.8 mg, 0.016% w/w) as an off-white solid.

(p-Hydroxybenzyl)malonic acid (46).^[70] Off-white solid. ¹H NMR (600 MHz, acetone-d₆) δ 7.05 (2H, d, J = 8.3 Hz; H-2′,6′), 6.68 (2H, d, J = 8.3 Hz; H-3′,5′), 3.53 (1H, t, J = 7.6 Hz; H-2), 3.06 (2H, d, J = 7.6 Hz; H-3); ¹H NMR (600 MHz, DMSO-d₆) δ 6.99 (2H, d, J = 8.4 Hz; H-2′,6′), 6.63 (2H, d, J = 8.4 Hz; H-3′,5′), 3.39 (1H, m, H-2), 2.92 (2H, d, J = 7.5 Hz; H-3) ppm. ¹³C NMR (150 MHz, acetone-d₆) δ 172.9 (C-1), 157.1 (C-4′), 130.9 (C-2′,6′), 130.9 (C-1′), 116.2 (C-3′,5′), 55.4 (C-2), 35.2 (C-3); ¹³C NMR (150 MHz, DMSO-d₆) δ 170.7 (C-1), 155.7 (C-4′), 129.6 (C-2′,6′), 128.7(C-1′), 114.9 (C-3′,5′), 53.5 (C-2), 33.5 (C-3) ppm. HRESIMS m/z calcd for C₁₀H₁₀O₅Na [M + Na]⁺ 233.0426; found 233.0423.

L. nuda fronds (30 g) were finely ground using a spice grinder, mixed with sand (~6 g), and extracted via PHWE (35% EtOH/H₂O). This provided a hot extract (200 mL) that was cooled immediately in an ice bath. This process was repeated a further two times to provide a combined extract (600 mL). The extract was concentrated under reduced pressure (35°C) to remove EtOH and extracted with EtOAc (150 mL and 2 × 100 mL). The combined organic phase was dried (MgSO₄), filtered, and concentrated under reduced pressure to provide extract A (254 mg). The remaining aqueous phase was concentrated under reduced pressure (50°C) to yield a dark brown extract B (5 g). Extract A. Extract A (254 mg) was redissolved in MeOH, adsorbed onto silica/Celite^® (1:1 mixture by mass) and subjected to automated flash chromatography {silica cartridge (4 g); 0–40% MeOH/EtOAc for 17 min}. Following TLC and ¹H NMR spectroscopic analysis, the resulting fractions were combined into seven large fractions, F.1–7. Fraction F.3 (14.5 mg) was redissolved in MeOH, adsorbed onto silica/Celite^® (1:1 mixture by mass) and subjected to flash column chromatography {silica; CH₂Cl₂ (50 mL), 5% MeOH/CH₂Cl₂ (50 mL), 10% MeOH/CH₂Cl₂ (50 mL), 20% MeOH/CH₂Cl₂ (50 mL) and MeOH (20 mL)} which provided compound 47 (2.5 mg, 0.01% w/w) as a yellow solid and compound 48 (5 mg, 0.016% w/w) as a yellow solid.

Quercetin-3-O-(6′-O-acetyl)glucoside (47) (CAS# 54542-51-7).^[114] Yellow solid. ¹H NMR (600 MHz, CD₃OD) δ 7.81 (1H, d, J = 2.2 Hz; H-6′), 7.63 (1H, dd, J = 8.5 and 2.2 Hz; H-5′), 6.88 (1H, d, J = 8.5 Hz; H-2′), 6.44 (1H, d, J = 2.0 Hz; H-8), 6.23 (1H, d, J = 2.0 Hz; H-6), 5.08 (1H, d, J = 7.9 Hz; H-1″), 4.18 (1H, dd, J = 11.4 and 7.8 Hz; H-6a″), 4.07 (1H, dd, J = 11.4 and 4.5 Hz; H-6b″), 3.83 (1H, dd, J = 9.6 and 7.4 Hz; H-2″), 3.80 (1H, d, J = 3.3 Hz; H-3″), 3.70 (1H, dd, J = 7.7 and 4.6 Hz; H-5″), 3.58 (1H, dd, J = 9.4 and 3.1 Hz; H-4″), 1.83 (3H, s; 2‴-COCH₃) ppm. ¹³C NMR (150 MHz, CD₃OD) δ 179.4 (C-4), 172.5 (1‴-CO), 167.3 (C-7), 162.9 (C-5), 158.6 (C-9), 157.8 (C-2), 150.0 (C-4′), 145.8 (C-3′), 135.7 (C-3), 123.1 (C-6′), 122.8 (C-1′), 117.6 (C-5′), 116.0 (C-2′), 105.8 (C-1″), 105.1 (C-10), 100.4 (C-6), 95.1 (C-8), 74.9 (C-3″), 74.5 (C-5″), 72.9 (C-2″), 70.2 (C-4″), 64.5 (C-6″), 20.4 (2‴-COCH₃) ppm.

Quercitrin (48) (CAS# 522-12-3).^[115] Yellow solid. ¹H NMR (600 MHz, CD₃OD) δ 7.33 (1H, d, J = 2.1 Hz; H-2′), 7.30 (1H, dd, J = 8.3 and 2.1 Hz; H-6′), 6.91 (1H, d, J = 8.3 Hz; H-5′), 6.34 (1H, d, J = 2.1 Hz; H-6), 6.18 (1H, d, J = 2.1 Hz; H-8), 5.35 (1H, s; H-1″), 4.22 (1H, m; H-2″), 3.75 (1H, m; H-3″), 3.42 (1H, m; H-5″), 3.35 (1H, m; H-4″), 0.95 (3H, d, J = 6.2 Hz; 6″-CH₃) ppm. ¹³C NMR (150 MHz, CD₃OD) δ 179.5 (C-4), 167.3 (C-7), 163.1 (C-5), 159.1 (C-2), 158.6 (C-9), 149.9 (C-4′), 146.5 (C-3′), 136.1 (C-3), 122.9 (C-6′), 122.8 (C-1′), 116.9 (C-5′), 116.4 (C-2′), 105.5 (C-10), 103.5 (C-1″), 100.3 (C-6), 95.0 (C-8), 73.3 (C-4″), 72.1 (C-3″), 72.0 (C-5″), 71.9 (C-2′), 17.6 (6′-CH₃) ppm.

P. wattsii dried leaf material (30 g) was finely ground using a spice grinder, mixed with sand (~6 g), and extracted via PHWE (35% EtOH/H₂O). This provided a hot extract (200 mL) that was cooled immediately in an ice bath. This process was repeated further two times to provide a combined extract (600 mL). The extract was concentrated under reduced pressure (35°C) to remove EtOH and extracted with EtOAc (150 mL and 2 × 120 mL). The combined organic phase was dried (MgSO₄), filtered, and concentrated under reduced pressure to provide extract A (523 mg). The remaining aqueous phase was concentrated under reduced pressure (50°C) to yield a dark-brown extract B (7 g). Extract A. Extract A (523 mg) was redissolved in MeOH, adsorbed onto silica and subjected to automated flash chromatography {silica cartridge (12 g); 0–100% EtOAc/hexanes and 0–40% MeOH/CH₂Cl₂ for 13 min at a flow rate of 25 mL/min} which provided compound 49 (143 mg, 0.5% w/w) as a dark-brown solid. Approximately 60 mg of compound 49 was absorbed onto silica and further purified by flash column chromatography {silica, 20% MeOH/CH₂Cl₂ (150 mL) then 40% MeOH/CH₂Cl₂ (100 mL)} which provided compound 49 as a pale-yellow/-green solid (20 mg).

5-O-Caffeoylshikimic acid (49) (CAS# 73263-62-4).^[55] Pale-yellow/-green solid. ¹H NMR (600 MHz, CD₃OD) δ 7.56 (1H, d, J = 15.9 Hz; H-7′), 7.04 (1H, d, J = 1.7 Hz; H-2′), 6.95 (1H, dd, J = 8.2 and 1.6 Hz; H-6′), 6.83 (br s; H-2), 6.78 (1H, d, J = 8.2 Hz; H-5′), 6.28 (1H, dd, J = 15.9 Hz; H-8′), 5.25 (1H, dd, J = 13.2 and 5.6 Hz; H-5), 4.40 (1H, s; H-3), 3.90 (1H, dd, J = 7.9 and 4.1 Hz; H-4), 2.87 (1H, dd, J = 18.8 and 4.9 Hz; H-6a), 2.32 (1H, dd, J = 18.4 and 5.1 Hz; H-6b) ppm. ¹³C NMR (150 MHz, CD₃OD) δ 170.3 (C-7), 168.6 (C-9′), 149.6 (C-4′), 147.2 (C-7′), 146.8 (C-3′), 138.2 (C-2), 131.1 (C-1), 127.7 (C-1′), 123.0 (C-6′), 116.5 (C-5′), 115.2 (C-2′), 115.1 (C-8′), 71.4 (C-5), 70.1 (C-4), 67.4 (C-3), 29.4 (C-6) ppm. HRESIMS m/z calcd for C₁₆H₁₆O₈Na [M + Na]⁺ 359.0845; found 359.0738.

G. alpina dried aerial parts (29 g) were finely ground using a spice grinder, mixed with sand (~4 g), and extracted via PHWE (35% EtOH/H₂O). This provided a hot extract (200 mL) that was cooled immediately in an ice bath. This process was repeated further two times to provide a combined extract (600 mL). The extract was concentrated under reduced pressure (35°C) to remove EtOH and extracted with EtOAc (200 mL and 2 × 150 mL). The combined organic phase was dried (MgSO₄), filtered, and concentrated under reduced pressure to provide extract A (646 mg). The remaining aqueous phase was concentrated under reduced pressure (50°C) to yield a dark-brown extract B (10 g). Extract A. Extract A (793 mg) was adsorbed onto silica/Celite^® (1:1 mixture by mass) and subjected to flash column chromatography {silica; EtOAc (300 mL), 10% MeOH/EtOAc (150 mL), 20% MeOH/EtOAc (150 mL), 30% MeOH/EtOAc (150 mL), 40% MeOH/EtOAc (150 mL) and MeOH (100 mL)}, to afford six fractions, F.1–6. Fraction F.3 (202 mg) was and adsorbed onto silica/Celite^® (1:1 mixture by mass) and subjected to flash column chromatography {silica; CH₂Cl₂ (50 mL), 10% MeOH/CH₂Cl₂ (50 mL), 20% MeOH/CH₂Cl₂ (100 mL), 20% MeOH/CH₂Cl₂ (150 mL) and MeOH (50 mL)} which provided compound 50 (6.9 mg, 0.02% w/w) as a yellow solid that contained minor impurities.

Rutin (50) (CAS# 153-18-4).^[116] Yellow solid. ¹H NMR (600 MHz, CD₃OD) δ 7.67 (1H, d, J = 2.2 Hz; H-2′), 7.63 (1H, dd, J = 8.4 and 2.2 Hz; H-6′), 6.88 (1H, d, J = 8.4 Hz; H-5′), 6.41 (1H, d, J = 2.1 Hz; H-8), 6.21 (1H, d, J = 2.1 Hz; H-6), 5.11 (1H, d, J = 7.7 Hz; H-1″), 4.52 (1H, d, J = 1.4 Hz; H-1‴), 3.80 (1H, dd, J = 11.2 and 1.5 Hz; H-6a″), 3.63 (1H, dd, J = 3.3 and 1.6 Hz; H-2″‘), 3.54 (1H, dd, J = 9.5 and 3.5 Hz; H-3‴), 3.38–3.49 (4H, m; H-2″,3″,5‴,6b″), 3.25–3.34 (3H, m; H-4″,5″,4‴), 1.12 (3H, d, J = 6.2 Hz; 6‴-CH₃) ppm. ¹³C NMR (150 MHz, CD₃OD) δ 179.4 (C-4), 166.1 (C-7), 162.9 (C-5), 159.3 (C-2), 158.5 (9), 149.8 (C-4′), 145.8 (3′), 135.6 (C-3), 123.5 (C-6′), 123.1 (C-1′), 117.7 (C-2′), 116.1 (C-5′), 105.6 (C-10), 104.7 (C-1″), 102.4 (C-1‴), 99.9 (C-6), 94.9 (C-8), 78.2 (C-3″), 77.2 (C-5″), 75.7 (C-2″), 73.9 (C-4‴), 72.2 (C-3‴), 72.1 (C-2‴), 71.4 (C-4″), 69.7 (C-5‴), 68.5 (C-6″), 17.9 (C-6‴) ppm.

D. antarctica dried leaflets (30.5 g) were finely ground using a spice grinder, mixed with sand (~7 g), and extracted via PHWE (35% EtOH/H₂O). This provided a hot extract (200 mL) that was cooled immediately in an ice bath. This process was repeated a further three times to provide a combined extract (800 mL). The extract was concentrated under reduced pressure (35°C) to remove EtOH and extracted with EtOAc (150 mL and 2 × 100 mL). The combined organic phase was dried (MgSO₄), filtered, and concentrated under reduced pressure to provide extract A (298 g). The remaining aqueous phase was concentrated under reduced pressure (50°C) to yield a dark-brown extract B (5 g). No compounds could be isolated by flash column chromatography.

D. australis dried leaflets (31 g) were finely ground using a spice grinder, mixed with sand (~7 g), and extracted via PHWE (35% EtOH/H₂O). This provided a hot extract (200 mL) that was cooled immediately in an ice bath. This process was repeated a further two times to provide a combined extract (600 mL). The aqueous extract was extracted with EtOAc (150 and 2 × 100 mL). The combined organic phase was dried (MgSO₄), filtered, and concentrated under reduced pressure to provide extract A (617 mg). The remaining aqueous phase was concentrated under reduced pressure (50°C) to yield a dark-brown extract B (3.5 g). No compounds could be isolated by flash column chromatography.

O. cartilaginea dried leaflets (33 g) were finely ground using a spice grinder, mixed with sand (~8 g), and extracted via PHWE (35% EtOH/H₂O). This provided a hot extract (200 mL) that was cooled immediately in an ice bath. This process was repeated a further two times to provide a combined extract (600 mL) and extracted with EtOAc (400 mL and 2 × 200 mL). The combined organic phase was dried (MgSO₄), filtered, and concentrated under reduced pressure to provide extract A (1.5 g). The remaining aqueous phase was concentrated under reduced pressure (50°C) to yield a dark-brown extract B (~2 g). No compounds could be isolated by flash column chromatography.

H. incisa dried leaflets (35 g) were finely ground using a spice grinder, mixed with sand (~16 g), and extracted via PHWE (35% EtOH/H₂O). This provided a hot extract (200 mL) that was cooled immediately in an ice bath. This process was repeated a further three times to provide a combined extract (800 mL) and extracted with EtOAc (3 × 250 mL). The combined organic phase was dried (MgSO₄), filtered, and concentrated under reduced pressure to provide extract A (877 mg). The remaining aqueous phase was concentrated under reduced pressure (50°C) to yield a dark-brown extract B (3 g). No compounds could be isolated by flash column chromatography.

Aerial parts of P. esculentum (150 g) were finely ground using a spice grinder, mixed with sand (~75 g), and extracted via PHWE (35% EtOH/H₂O). This provided a hot extract (200 mL) that was cooled immediately in an ice bath. This process was repeated a further nine times to provide a combined extract (2 L). The extract was concentrated under reduced pressure (35°C) to remove EtOH and extracted with EtOAc (3 × 500 mL). The combined organic phase was dried (MgSO₄), filtered, and concentrated under reduced pressure to provide extract A (2.7 g). The remaining aqueous phase was concentrated under reduced pressure (50°C) to afford brown extract B (32 g). Extract A. Extract A (1.5 g) was redissolved in MeOH, adsorbed onto silica/Celite^® (1:1 mixture by mass) and subjected to flash column chromatography {silica; CH₂Cl₂ (250 mL), 10% MeOH/CH₂Cl₂ (250 mL), 20% MeOH/CH₂Cl₂ (250 mL), 30% MeOH/CH₂Cl₂ (250 mL), 40% MeOH/CH₂Cl₂ (250 mL) and MeOH (200 mL)}. Following TLC and ¹H NMR spectroscopic analysis, the resulting fractions were combined to provide seven fractions, F.1–7. Fraction F.4 (150 mg) was redissolved in 20% MeCN/H₂O solution and subjected to automated flash chromatography (12 g C18 cartridge; 0–100% MeCN/H₂O for 17 min with a flow rate of 28 mL/min). Following TLC and ¹H NMR spectroscopic analysis, the resulting fractions were combined to afford four fractions, F.1–4. Fraction F.2 (19 mg) was redissolved in MeOH/CH₂Cl₂ solution and subjected to flash column chromatography {silica; 10% MeOH/CH₂Cl₂ (10 mL)} to provide compound 41 (12 mg, 0.006% w/w) as a pale-yellow solid. The fraction F.4 (42 mg) was subjected to preparative TLC which provided compound 51 (25 mg, 0.02% w/w) as a yellow solid.

Tiliroside (51) (CAS# 20316-62-5).^[117,118] Yellow solid. ¹H NMR (600 MHz, CD₃OD) δ 7.98 (2H, d, J = 8.8 Hz; H-2′,6′), 7.40 (1H, d, J = 15.9 Hz; H-7‴), 7.30 (2H, d, J = 8.5 Hz; H-2‴,6‴), 6.82 (2H, d, J = 8.8 Hz; H-3′,5′), 6.79 (2H, d, J = 8.6 Hz; H-3‴,5‴), 6.30 (1H, d, J = 1.9 Hz; H-8), 6.13 (1H, d, J = 2.0 Hz; H-6), 6.07 (1H, d, J = 15.9 Hz; H-8‴), 5.23 (1H, d, J = 7.3 Hz; H-1″), 4.31 (1H, dd, J = 11.8 and 1.9 Hz; H-6a″), 4.20 (1H, m; H-6b″), 3.41–3.50 (3H, m; H-2″,3″,5″), 3.31 (1H, m; H-4″) ppm. ¹³C NMR (150 MHz, CD₃OD) δ 179.4 (C-4), 168.6 (9‴-CO), 166.3 (C-7), 162.9 (C-5), 161.5 (C-4′), 161.2 (C-4‴), 159.3 (C-2), 158.5 (C-9), 146.6 (C-7‴), 135.2 (C-3), 132.2 (C-2′,6′), 131.2 (C-2‴,6‴), 127.1 (C-1‴), 122.7 (C-1′), 116.8 (C-3‴,5‴), 116.1 (C-3′,5′), 114.7 (C-8‴), 105.5 (C-10), 104.1 (C-1″), 100.1 (C-6), 94.9 (C-8), 78.0 (C-3″), 75.7 (C-2″,5″), 71.7 (C-4″), 64.3 (C-6″) ppm.

The data that support this study are available in the article and accompanying online supplementary material.

The authors declare no conflicts of interest.

We acknowledge the University of Tasmania (UTAS) School of Natural Sciences – Chemistry for funding. TG thanks the UTAS for a Tasmania Graduate Research Scholarship. ACB’s contributions were supported by an ARC Future Fellowship (FT200100049).

Supplementary material is available online.