Phytochemistry, Chemotaxonomy, and Biological Activities of the Araucariaceae Family—A Review

In this review article, the phytochemistry of the species belonging to the Araucariaceae family is explored. Among these, in particular, it is given a wide overview on the phytochemical profile of Wollemia genus, for the first time. In addition to this, the ethnopharmacology and the general biological activities associated to the Araucariaceae species are singularly described. Lastly, the chemotaxonomy at the genus and family levels is described and detailed.


Introduction
Araucariaceae Henkel and W. Hochstetter is a family of coniferous trees, classified under the order Pinales, the class Pinopsoda, the division Pinophyta, and the Clade Tracheophytes [1].
It is a very ancient family since its maximum diversity was achieved during the Jurassic and Cretaceous periods with a worldwide distribution. Yet, during the extinction events occurred in the transition from Cretaceous to Paleogene, these species totally vanished from the Northern Hemisphere whereas they remained in the Southern Hemisphere apart for a very few exceptions. In particular, Araucariaceae species are well present in South America, Australia, New Zealand, New Guinea, New Caledonia, and other South Pacific islands while Malaysia represents the exception [2].
From the taxonomic point of view, the family comprises four genera: Agathis Salisb., Araucaria Juss., Columbea Salisb., and Wollemia W.G.Jones, K.D.Hill and J.M.Allen. Yet, Columbea and Wollemia genera are formed by one only species each i.e., Columbea brasiliensis (A. Rich.) Carrière and Wollemia nobilis W.G.Jones, K.D.Hill and J.M.Allen. On the other hand, Agathis genus is formed by 18 accepted species and 4 unresolved species whilst Araucaria genus is formed by 19 accepted species [3].
From the phylogenetic standpoint derived from molecular data, Araucariaceae family belongs to a major subdivision that includes the Podocarpaceae, Sciadopityaceae, Cupressaceae, Cephalotaxaceae, and Taxaceae families. In particular, Araucariaceae family belongs to the same clade as Podocarpaceae and represents the least evolved family of the subdivision. Within the family itself, the phylogeny tree forecasts Wollemia genus as the least evolved one followed by Agathis and Araucaria genera. Within the Araucaria genus, the situation is more complex, with several existing sub-clades [1].
The species belonging to this genus are usually monoecious. They are characterized by a large and very robust trunk with no branching in the inferior part when they are mature trees. Indeed, when they are young, they are generally conical and have more irregular crowns. The bark is smooth and grey-brownish colored, usually with a peeling that form irregular flakes that become thicker and thicker as the age of the tree proceeds. The branches are often horizontal, or ascending when they are too large. The lower ones often leave circular branch scars when they detach from the lower trunk. The juvenile leaves are larger than the adult ones and are more or less acute, with an ovate to lanceolate shape. They are often coppery-red colored also. Indeed, the adult leaves are opposite, from linear to elliptical, quite thick and very leathery. They produce two cones: male (pollen) and female (seed). The male ones appear only on the largest trees. The female ones usually develop on short lateral branchlets and get mature after two years. They are generally oval or globe shaped ( Figure 1) [4].

Distribution
Agathis is a quite widespread genus of the family. In fact, their species can be found in New Zealand, the Philippines, New Guinea, Melanesia, and Australia, but also in Malaysia, beyond the Equator line. They grow on diverse substrates including podzolized sands, ultramafics, carbonates, and silicates. They occur from near sea level to the altitude of about 2500 m. They mainly prefer sites that never see frost, and that receive between five and ten meters of rain per year [5]. Table 1 shows data of all the compounds identified in the genus divided according to the species.

Species Collection Site Organs Studied Compound Study Methods References
A. dammara (Lamb.) Rich. and A.Rich.

Ethnopharmacology
The ethnopharmacological uses of species belonging to the Agathis genus are quite limited. In fact, only a couple of works have dealt with this argument. In particular, it was reported that, in Borneo, A. borneensis is used to treat fever by boiling the bark in water and drinking it as an herbal tea [35]. Moreover, still in Borneo, the powdered woods of A. borneensis, A. philippinensis and A. dammara are employed to treat headache and myalgia [36].

Extracts
The biological activities associated with the essential oil or the extracts of Agathis species are quite numerous.
A. atropurpurea resin extract has shown to possess medium antifungal properties against Aspergillus niger and Rhizopus stolonifer with MIC values equal to 625 and 1250 µg/mL, respectively [37]. It also showed strong antileishmanial activities against L. amazonensis promastigotes and amastigotes with IC 50 values equal to < 12.5 and 19.3 µg/mL, respectively, as well as weak cytotoxic effects against BALB/c mouse macrophage cells with a CC 50 value equal to 118.4 µg/mL [38].
A. borneensis leaf methanol extract is able to exert strong antiplasmodial properties against Plasmodium falciparum D10 strain (sensitive strain) with an IC 50 value equal to 11.00 µg/mL [36].
The essential oil of A. dammara exerts good antibacterial effects against several bacterial strains (Staphylococcus aureus, Bacillus subtilis, Pneumonia aeruginosa, and Escherichia coli) with inhibition zones in the range of 14.5-23.7 mm and with MIC values ranging from 1.25 to 2.5 mg/mL [12]. In addition to this, the methanolic extract obtained from its leaves was found to be active also against Proteus vulgaris with an inhibition zone in the range 19-21 mm [13]. Moreover, the n-hexane, methanol, and ethyl acetate extracts of its resin display very low antioxidant effects (IC 50 values equal to 438.55, 313.51, 245.99 mg/mL, respectively) [39].
The hydroalcoholic extract of A. robusta from England at the concentration of 400 µg/mL has shown good anti-inflammatory activities according to the HRBC (HumanRedBloodCell) membrane stabilization and heat-induced hemolytic methods with percentages of denaturation inhibition equal to 76.84% and 77.12%, which are comparable to those observed for diclofenac (79.25%) and aspirin (83.78%) for the respective models [23]. Moreover, the resin essential oil has shown interesting antibacterial effects against several bacterial strains (Staphylococcus spp., Klebsiella pneumoniae, Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa) with MIC and MBC values ranging from 250 to 500 and from 500 to more than 1000 µg/mL, respectively [24].

Phytochemicals
In literature there are also some works about the biological activities associated with specific compounds isolated from Agathis species.

Other Facts
In literature some interesting curiosities about Agathis species are also reported. In particular, these curiosities regard other uses of these species in the past.
Agathis spp. have been widely used for their timber in order to make panels, cabinets, joinery, turnery, moldings, patterns making, battery separators, piano parts, and artificial limbs. This is because the timber is straight-grained, strong, knot-free, with a silky and lustrous surface and it can be easily worked. In addition, the resin has been used to make the so-called Manila copal to be a component of varnishes, mainly. This resin derives from their living inner barks, is translucent or clear white, and slowly hardens on exposure to air with age, eventually becoming brittle. It is soluble in alcohol to a varying degree and has a melting point between 115-135 • C. Actually, this copal is a complex mixture of monoterpenes, sesquiterpenes, and diterpenes [40].
The young gum of A. australis has been used for many centuries as chewing gum by Maori people [41].
The Borneo aborigens consider A. boorneensis as a magical plant, capable to exert special powers including protection against bad spirits.

Botany
The species belonging to this genus are mostly dioecious trees even if monoecious trees can also be found. Moreover, some trees are even able to change sex with time. These species are characterized by a massive and erect stem. They can reach up to 80 m high. The branches are gathered in whorls and grow horizontally. They are covered with leathery or needled leaves, with no branching in the inferior part when they are mature trees. In some species, the leaves are narrow and lanceolate, barely overlapping each other, whilst in others they are very broad, flat, and widely overlapped with each other. They produce two cones: male (pollen) and female (seed). The female cones are globose and can be very variable size according to the species. They are found only on the top of the tree and they contain many large edible seeds. Indeed, the male cones are smaller in size and present a broad cylindrical shape ( Figure 2) [42].
overlapping each other, whilst in others they are very broad, flat, and widely overlapped with each other. They produce two cones: male (pollen) and female (seed). The female cones are globose and can be very variable size according to the species. They are found only on the top of the tree and they contain many large edible seeds. Indeed, the male cones are smaller in size and present a broad cylindrical shape ( Figure 2) [42].

Distribution
Araucaria is widespread only in the Southern Hemisphere even if at different meridians. In fact, their species can be found in New Guinea, Australia, but also in Chile, Argentina, Brazil, New Caledonia, and Norfolk Island. In addition to this, there is a naturalized population of A. columnaris(G.Forst.) Hook. on the island of Lanai in the Hawaii. The greatest biodiversity of the genus is in New Caledonia. They prefer ultrabasic schistose and calcareous soils [43]. Table 2 shows data of all the compounds identified in the genus divided according to the species.

Ethnopharmacology
The ethnopharmacological uses of species belonging to the Araucaria genus are quite numerous. In particular, in Brazil, A. angustifolia leaves are used as emollient, antiseptic, and to treat respiratory infections and rheumatisms. Their dyes are also used for the treatment of wounds and herpes eruptions [90]. In addition, the tinctures derived from the nodes are employed to treat rheumatism. The infusions of the nodes are used orally for the treatment of kidney diseases and sexually transmitted diseases. The infusions of the bark are used topically to treat muscular tensions and varicose veins. The syrup produced of the resin is used for the treatment of respiratory infections [91].
A. araucana resin has been used by Amerindian Mapuche tribes located in Southern Chile and Argentina to treat contusions, ulcers, as well as to help cicatrization of skin wounds [60,61].
A. bidwillii bark is employed in South Africa against amenorrhoea and as a body and steam wash [92]. Moreover, it is employed in the Lahu tribes of Northern Thailand to treat insomnia [93].
A. cunninghamii bark is used by the Yali tribe in West Papua for thatching and in several rituals [94].
A. heterophylla aerial parts are used in Peru for toothache and to extract teeth [95].

Extracts
The biological activities associated with the essential oil or the extracts of Araucaria species are also quite numerous.
The biflavonoid rich fraction derived from the fresh needles of A. angustifolia has shown to be a potent UV-A UV-B radiation protector [53] as well as to protect liposomes against peroxidative degradation caused by UV-irradiation [52]. The ethyl acetate and n-butanol fractions derived from the whole plant of A. angustifolia showed strong antiviral effects against HSV-1 with IC 50 values equal to 8.19 and 11.04 µg/mL, respectively [56]. The ethanol and water extracts of its seeds showed good antioxidant properties [55]. The hydroalcoholic and ethyl acetate extracts of its dead bark showed high antioxidant properties in the DPPH assay with IC 50 values equal to 1 and 0.9 µg/mL, respectively. Moreover, the same extracts showed medium activity in the lipid peroxidation assay induced by UV, ascorbyl, and hydroxyl free radicals with IC 50 values equal to 36 and 25 µg/mL, respectively, for the former case, 18 and 17 µg/mL, respectively, for the second case and 12 and 22 µg/mL, respectively, for the latter case [46]. Indeed, the water extract of its female strobili also exerts a time-dependent antiproliferative activity against HEp-2 (human laryngeal cancer) cell lines. In particular, at the concentrations of 250 and 500 µg/mL, it was able to inhibit tumor growth by about 50% after 24 h from the subministration whereas, after 48 h, the percentage of inhibition was about 65 and 70%, respectively, and after 72 h, for both, the percentage of inhibition was 80%, approximately [48]. The same extract showed good DPPH and SOD activities with IC 50 values equal to 10.0 and 14.7 µM, respectively, as well as good antimutagenic effects against H 2 O 2 in three different loci i.e., Lys, His, and Hom at the concentrations of 0.05, 0.1, and 0.15% with a percentage of survivals of 100% [49]. The water extract of its bracts at the concentration of 50 µg/mL is also able to completely avoid, in human lung fibroblast cells, cell mortality, protein damage, and SOD and CAT depletions induced by H 2 O 2 [45].
The crude A. araucana resin possesses dose-dependent gastroprotective effects on ethanol-HCl-induced gastric lesions in mice [60]. In addition, the methanol extract of its wood showed moderate antibacterial activity against Citrobacter pilifera, Bacillus subtilis, Micrococcus luteus, and Staphylococcus aureus with growth inhibition percentages around 20%, which are values much lower than gentamicin used as control. Indeed, the same extract showed moderate antifungal activities against Mucor miehei, Paecilomyces variotii, Ceratocystis pilifera, and Trametes versicolor with growth inhibition percentages from 28.7% for the second one to 57.1% for the latter one. The relative IC 50 values were in the range 1250-2000 µg/mL [62].
The petroleum ether and methanolic extracts of the leaves and oleoresin of A. bidwillii, at the doses 300 mg/kg for the former ones and 100 mg/kg for the latter ones, possess strong anti-insomnia, analgesic, and anti-inflammatory activities [96]. In addition to this, the ethanol extract of its leaves, at the dose of 5 mg/Kg, exerts strong anti-inflammatory activity with percentages of inhibition similar to those of indomethacin i.e., 68.51% vs. 63.28%, respectively [97]. The same extract demonstrated high antinociceptive effects at the concentration of 300 mg/Kg in four different tests: the hot plate test, the acetic acid-induced writhing test, the carrageenan-induced edema test, and the serotonin-induced rat paw oedema test. The associated percentages of inhibition were equal to 81.69%, 54.64%, 45.64%, and 40.75%, respectively. All these values are comparable with those reported for the standard compounds in the relative tests [97]. In addition, its methanolic and ethyl acetate extracts derived from the leaves are able to exert good antitumor effects against HL-60 and K-562 (chronic myelogenic leukemia) cancer cell lines with IC 50 values equal to 33.11 and 39.81 µg/mL, respectively, for the former and 28.18 and 34.64 µg/mL, respectively, for the latter [98]. The leaf methanol extracts at the concentration of 100 µg/mL showed also strong anti-inflammatory activity acting as an inhibitory agent on the levels of IL-1β, TNF-α by reducing them by 58.4% and 56.4%. Indeed, for what concerns IL-6, the effect was observed to be concentration-dependent. Additionally, the n-butanol polyphenolic rich extract at the concentration of 10 µg/mL showed these effects but in minor extent i.e., 44.8% inhibition on the levels of IL-1β and 33.6% inhibition on the levels of TNF-α. Indeed, for what concerns IL-6, also this effect was concentration-dependent. All these values were quite similar to those observed for indomethacin [68]. Its oleoresin possesses good antipyretic activity on female albino rats at the dose of 100 mg/Kg showing the maximum decrease in the rectal temperature after 60 min (−1.35 • C) [71].
The ethanol extract of the branches of A. columnaris showed good antioxidant and antiradical activities in absolute with values equal to 93.14 and 74.12% for the DRSC (DPPH radical scavenging activity) and NOSC (nitric oxide scavenging capacity) assays, respectively. Moreover, it also showed a good ferric reducing antioxidant power with a value equal to 113.05 mg Fe(II)E/g FS, a good cupric ion reducing capacity with a value equal to 128.34 mg TE/g FS. Indeed, its dichloromethane extract showed good TAC and MCA activities with values equal to 93.26 mg AAE/g FS and 81.50 mg EDTAE/g FS, respectively [75]. The 70% aqueous methanol extract of the needles of A. columnaris showed moderate antioxidant effects with a SC 50 value equal to 73.0 µg/mL which is, anyway, much higher than ascorbic acid (SC 50 = 8.0 µg/mL) [73]. Different extracts of its leaves showed to possess also medium antioxidant properties and good α-amylase inhibitory and antibacterial effects against Pseudomonas and Klebsiella spp. and Escherichia coli [76]. These results were also confirmed by another study by Zaffar et al. [99]. Indeed, the study performed by Joshi et al. [100] demonstrated that these extracts were also active against Xanthomonas phaseoli and Erwinia chrysanthemi with the best MIC values i.e., 62.5 µg/mL for the methanol and n-hexane extracts for both bacterial strains.
The methanolic extract of A. columnaris bark exerts strong antibacterial effects against Staphylococcus aureus, Escherichia coli and Bacillus subtillis with maximum inhibition zones equal to 20, 18 and 15 mm, respectively. The same extract was also found to be quite cytotoxic against HEK (human kidney) cancer cell line, having an IC 50 value equal to 95.0 µg/mL [74].
The extracts of A. cunninghamii leaves in different solvents (n-hexane, chloroform, ethanol, methanol) possess good antifungal activities against Alternaria alternata, Colletotrichum falcatum, Fusarium oxysporum, Pyricularia oryzae, Sclerotinia rolfsii, Sclerotinia sclerotiorum, and Tillatia indica with inhibition percentages from 39% for the chloroform extract against A. alternata to 57% of the n-hexane extract against A. alternata itself. All the extracts were active except the n-hexane and chloroform ones against Fusarium oxysporum. Most of the extracts showed percentages of inhibition similar or better than clotrimazol used as reference [101]. The methanolic extract of its leaves also showed good DPPH radical scavenging activities with an IC 50 value equal to of 181.9 µg/mL as well as a little reducing power (IC 50 = 1384.42 µg/mL) and a moderate prevention effect of nitric oxide radical (IC 50 = 1026.51 µg/mL) [82]. Moreover, the extracts of A. cunninghamii stem bark resin in different solvents showed different biological activities. In particular, the methanol extract showed high α-glucosidase inhibition effects with a percentage equal to 48.48% which is very close to that of acarbose i.e., 48.69. The n-hexane and dichloromethane effects were lower i.e., 24.2% and 26.58%, respectively. The dichloromethane showed strong cytotoxic effects against in Chang liver cells with an IC 50 value equal to 92.9 µg/mL. The n-hexane and methanol extracts showed minor effect with IC 50 value equal to 386 and above 500 µg/mL, respectively [84]. In addition to this, its essential oil derived from the foliage showed moderate antibacterial activity against Salmonella typhimurium, Staphylococcus aureus, and Staphylococcus epidermidis with inhibition zones equal to 9, 6, and 5 mm, respectively, whereas it was low against Staphylococcus aureus and Bacillus subtilis with inhibition zones both equal to 4 mm. Indeed, its essential oil derived from the resin was moderately active only against Staphylococcus aureus with an inhibition zone equal to 5 mm. The relative MIC values were in the range 250 and 500 µg/mL and the minimum bactericidal concentrations were 1000 µg/mL or more [81].
The resin extract of A. heterophylla stems showed strong cytotoxic effects in vitro against colon (HCT116) and breast (MCF7) human cancer cell lines with IC 50 values equal to 0.54 and 0.94 µg/mL, respectively, which are quite similar to those observed for doxorubicin (0.70 and 0.96 µg/mL, respectively) [86]. The extracts of its leaves in different solvents showed strong to weak anticancer activity against HEPG-2 (hepatocellular carcinoma), MCF-7, PC-3 (human prostate cancer), and Hela (epitheliod carcinoma) cell lines. In particular, the n-butanol extract was one of the most effective with IC 50 values equal to 12.06, 9.13, 17.42, and 7.69 µg/mL, respectively. These values are lower than doxorubicin but absolutely comparable. The ethyl acetate extract was the most efficient one against MCF-7 and Hela cancer cell lines with IC 50 values equal to 7.64 and 6.72 µg/mL, respectively. The water extract was more effective against Hela cell lines with an IC 50 value to 9.84 µg/mL. The petroleum ether and dichloromethane extracts showed quite moderate activities against all the studied cancer cell lines with IC 50 values above 20 µg/mL, except for the petroleum ether extract against Hela whose IC 50 value was 19.34 µg/mL [85].

Phytochemicals
In literature there are also some works about the biological activities associated with specific compounds isolated from Araucaria species.
Protocatechuic acid, quercetin, (-)-epiafzelechin protocatechuate, and (-)-epiafzelechin p-hydroxybenzoate extracted from A. angustifolia dead bark from Brazil showed all antioxidant effects in the DPPH assay with IC 50 values ranging from 0.6 µM of quercetin to 11 µM of (-)-epiafzelechin p-hydroxybenzoate. Moreover, only quercetin and (-)-epiafzelechin protocatechuate showed activity in the lipid peroxidation assay and only in that induced by UV and ascorbyl free radicals with IC 50 values equal to 9 and 21 µM, respectively, for the former case and 30 and 35 µM, respectively, for the second case [46]. Imbricatolic acid, 15-hydroxy-imbricatolal, and 15-acetoxy-imbricatolic acid isolated from A. araucana resin from Chile showed dose-dependent gastroprotective effects on ethanol-HCl-induced gastric lesions in mice with maximum activity at doses up to 100 mg/Kg [60]. Moreover, at the dose of 100 mg/kg, 15-hydroxy-imbricatolal, 15-acetoxy-imbricatolic acid, and 15-acetoxylabd-8(17)-en-19-ol were also seen to have effects similar to those of lansoprazole, a standard a proton pump inhibitor drug, reducing the lesions by 78, 69, and 73%, respectively [61]. In addition to this, 15,19-diacetoxylabd-8(17)-en, again isolated from A. araucana resin, was observed to possess a good cytotoxic activity against AGS cells (human gastric epithelial) and fibroblasts after treatment for 24 h with IC 50 values equal to 52 and 72 µM, respectively. These values are much better than those observed for lansoprazole (IC 50 values equal to 162 and 306 µM, respectively) [61]. Additionally, seco-isolariciresinol, pinoresinol, eudesmin, lariciresinol extracted from the wood of this species from Chile showed weak antibacterial activities against Citrobacter pilifera, Bacillus subtilis, Micrococcus luteus, and Staphylococcus aureus with growth inhibition percentages below 20%, which are, again, values much lower than gentamicin used as control. Indeed, the same compounds showed moderate antifungal activities against Mucor miehei, Paecilomyces variotii, Ceratocystis pilifera, and Trametes versicolor with growth inhibition percentages above 20.0% but below 50%. Actually, pinoresinol was the only compound not active against Paecilomyces variotii whereas the seco-isolariciresinol was the most active in all the cases with growth inhibition percentages equal to 29.7%, 21.9%, 41.5%, and 45.1%, respectively [62].
The two compounds extracted from the A. bidwillii leaves from Egypt, 7-hydroxy-labda-8 (17) The biflavonoid rich fraction from A. bidwillii, both at the concentration of 100 and 200 mg/Kg, was also observed to be a strong protective agent against ischemia-induced oxidative stress in rats by inhibiting free radicals generation, by scavenging reactive oxygen species and by modulating the intracellular antioxidants against ischemia/reperfusion-induced decreases [102]. Anyway, most of these data are worse than those observed for ampicillin [80].

Other Facts
In literature, one interesting curiosity about the Araucaria species is also reported. In particular, the edible part of the seeds of A. angustifolia are eaten by animals and people for their high nutritional value [47].

Genus Columbea
The overall data of this genus including its morphological description, its distribution, its phytochemistry, its ethnopharmacological uses, and its pharmacological activities are associated to the data of its only existing species i.e., Columbea brasiliensis (A. Rich.) Carrière. Yet, besides its distribution which is endemic to Brazil [103], there are no data reported in literature for what concerns the other arguments.

Botany
The overall data of this genus including, its morphological description, its distribution, its phytochemistry, its ethnopharmacological uses, and its pharmacological activities are associated to the data of its only existing species i.e., Wollemia nobilis W.G.Jones, K.D.Hill and J.M.Allen.
This species is a monoecious tree which can reach up to 40 m tall. The bark is brown. The stem is multiple with a complex root system. The branching is vertical and lateral. The leaves are flattened, and arranged spirally on the shoots but twisted at the base to form two or four ranks flattened at their own time and they open in November or December depending on the location of the tree. There are two different cones. The male ones (the pollen) are conic and large with a brown-reddish color. Indeed, the female ones (the seeds) are lighter in color and narrower. These cones are disposed in lower positions than the male cones ( Figure 3) [104,105].

Botany
The overall data of this genus including, its morphological description, its distribution, its phytochemistry, its ethnopharmacological uses, and its pharmacological activities are associated to the data of its only existing species i.e., Wollemia nobilis W.G.Jones, K.D.Hill and J.M.Allen.
This species is a monoecious tree which can reach up to 40 m tall. The bark is brown. The stem is multiple with a complex root system. The branching is vertical and lateral. The leaves are flattened, and arranged spirally on the shoots but twisted at the base to form two or four ranks flattened at their own time and they open in November or December depending on the location of the tree. There are two different cones. The male ones (the pollen) are conic and large with a brown-reddish color. Indeed, the female ones (the seeds) are lighter in color and narrower. These cones are disposed in lower positions than the male cones ( Figure 3) [104,105].

Distribution
This species is native to Australia and is very rare. In fact, it grows wild only in three different localities within the Wollemi National Park, NSW of Australia where 20 large trees (up to 40 m in height) and 20 juvenile trees are present. Additionally, it was considered to be extinct until 1994. Given these elements, it has been subjected to several protection and conservation programs with the aims to keep its growth habitat secret, monitor them against illegal visits, and develop a plan that favors its cultivation and marketing all around the world. Right because of this, nowadays several W. nobilis exemplars are now hosted in a few Botanical Gardens outside Australia (including Italy) as well as in thousands of Australian home gardens. The species prefers sandy soils with good drainage and watering [104,105]. Table 3 shows data of all the compounds identified in W. nobilis.

Distribution
This species is native to Australia and is very rare. In fact, it grows wild only in three different localities within the Wollemi National Park, NSW of Australia where 20 large trees (up to 40 m in height) and 20 juvenile trees are present. Additionally, it was considered to be extinct until 1994. Given these elements, it has been subjected to several protection and conservation programs with the aims to keep its growth habitat secret, monitor them against illegal visits, and develop a plan that favors its cultivation and marketing all around the world. Right because of this, nowadays several W. nobilis exemplars are now hosted in a few Botanical Gardens outside Australia (including Italy) as well as in thousands of Australian home gardens. The species prefers sandy soils with good drainage and watering [104,105]. Table 3 shows data of all the compounds identified in W. nobilis.

Collection site Organs Studied Compounds Study Methods References
Italy (Botanical garden of Rome) Leaves pheophorbide a, isocupressic acid, acetyl-isocupressic acid, sandaracopimaric acid, agathic acid, 7,4 ,4 "-tri-O-methyl-agathisflavone, 7,4 ,7 ',4 "-tetra-O-methyl-agathisflavone, caffeic acid, shikimic acid SE, CC, NMR, MS [32] Half-matured female cones acetyl-isocupressic acid, methyl-(E)-communate, sandaracopimaric acid, wollemol, As Table 3 clearly shows, most of the phytochemical works reported in the literature about this species regard exemplars collected in Italy [28][29][30][31][32][33]. Yet, this fact is not so unusual since, as already mentioned, Italy is one of the main places where this species has been introduced in order to favor its survival. One only work has regarded the phytochemistry of an exemplar from Australia but only for the essential oil composition [8]. In addition, there are two works regarding the essential oil content of a sample collected in Belgium [106] and one regarding the polar fraction metabolites of an exemplar purchased from a company in Poland [107]. The studied organs were mainly the leaves [8,32,106] but also other organs were taken into considerations i.e., twigs [106,107], half-matured female cones [33], male reproduction organs [29], unripe female cones [31], and male cones for two different studies in the time distance of one year [28,30]. For what concerns the identified compounds, also W. nobilis is known to biosynthesize both components of the essential oil and polar fraction metabolites. For none of the studied samples, both essential oil and polar fraction composition were studied. In particular, only the essential oil composition was analyzed for the exemplar from Australia [8] and for the leaves and twigs of the exemplar coming from Belgium [106], whereas only the polar fraction composition was analyzed in all the other cases [28][29][30][31][32][33]107]. Among the essential oil metabolites, only β-pinene and germacrene D are present in all the studied samples [8,106]. Yet, it is still not possible to draw a general conclusion on this matter since the exemplars studied until now are still too few. Anyhow, they cannot be considered as chemotaxonomic markers since they represent very common compounds of the essential oils of different plants. Among the polar fraction metabolites, none have been evidenced in all the studied samples. Yet, biflavonoids and diterpenes have been generally evidenced in all the studied samples [28][29][30][31][32][33]107], but they are mostly considered as chemotaxonomic markers at the family level. For what concerns the methodology, in one case, the essential oil was obtained through solvent distillation [8] whereas in two cases, it was obtained through hydrodistillation [106]. In one case, GLC was also used for the separation of the essential oil metabolites [8] whilst in all the other cases GC-MS was used for the separation and identification of the essential oil metabolites [8,106]. In one case [8], [α] D and NMR analyses accompanied the GC ones. Indeed, for the analysis of the polar fraction metabolites, SE was the only method for the extraction of these compounds [28][29][30][31][32][33]107]. CC was the method for the separation procedure of the compounds in most cases [28][29][30][31][32][33] whereas HPLC and TLC were used in one case [107]. Lastly, NMR and MS techniques were used for the identification procedure of the compounds in all the cases [28][29][30][31][32][33]107]. In one case, UHPLC-HRMS was used for the specific separation and identification of one compound [30].

Chemotaxonomy of the Araucariaceae Family
As Tables 4 and 5 clearly show, the phytochemistry of the Araucariaceae family is quite complex. Several metabolites belonging to different classes of natural compounds have been evidenced within it. Yet, none of them have an occurrence spread in all of it even if some compounds have been isolated in many different species. Conversely, other compounds have been isolated in specific species, if not specific exemplars and this fact is not atypical since the qualitative and quantitative content in secondary metabolites of plants is much affected by environmental and genetic factors [26].
Plants 2020, 9, x FOR PEER REVIEW 68 of 75 As Tables 4 and 5 clearly show, the phytochemistry of the Araucariaceae family is quite complex. Several metabolites belonging to different classes of natural compounds have been evidenced within it. Yet, none of them have an occurrence spread in all of it even if some compounds have been isolated in many different species. Conversely, other compounds have been isolated in specific species, if not specific exemplars and this fact is not atypical since the qualitative and quantitative content in secondary metabolites of plants is much affected by environmental and genetic factors [26].
A particular speech regards the presence of alkyl chains. These were evidenced in several species of the family [8] even if not always the exact compounds were identified, but only the general molecular formula. By the way, these compounds are also quite widespread [26,27,[110][111][112][113][114]. Yet, none of these compounds can be actually considered as chemotaxonomic markers since they all are widespread compounds in the plant kingdom [26,27,[110][111][112][113][114].
A particular speech regards the presence of alkyl chains. These were evidenced in several species of the family [8] even if not always the exact compounds were identified, but only the general molecular formula. By the way, these compounds are also quite widespread [26,27,[110][111][112][113][114].
A particular speech regards the presence of alkyl chains. These were evidenced in several species of the family [8] even if not always the exact compounds were identified, but only the general molecular formula. By the way, these compounds are also quite widespread [26,27,[110][111][112][113][114].
Indeed, among the polar fraction metabolites, the most common compounds were found to be:  As previously mentioned, the derivatives of agathisflavone can be really considered as chemotaxonomic markers of the whole Araucariaceae family [22,[28][29][30][31][32][33] whereas cupressuflavone and its derivatives have now been started to be evidenced also in other families [115] and so they can no longer be considered as chemotaxonomic markers of the family.
For what concerns the diterpenes, even if no specific compound of this class has been evidenced in all the species reported in literature, as previously mentioned, they are also known to be used as chemotaxonomic markers of the family. In particular, this concerns labdane diterpenes which are, anyway, a very big sub-class of natural compounds and so, they are not so specific also because this kind of compound has been isolated from other families also [116].

Conclusions
This review article has clearly showed the huge importance of Araucaricaeae species both under different standpoints: phytochemistry, chemotaxonomy, ethnobotany, and pharmacology.
In fact, as for the first point, many compounds, components of the essential oil, and of the polar fraction have been reported in them, including several new ones. For what concerns chemotaxonomy, some compounds could be eventually used as chemotaxonomic markers even if some other studied on this point are necessary in order to develop this concept better. Several species of the Araucariaceae family are also used for ethnobotanical and ethnopharmacological purposes, especially the species belonging to the Araucaria genus. Lastly, several extracts derived from Araucariaceae species as well several compounds isolated from them have been found to possess interesting and amazing pharmacological activities, ranging from the mere antioxidant to the greater cytotoxic effects.
Nevertheless, the studies in these fields about this family are quite limited in several senses, not to say absent for the genus Columbea, and this review article wants to be a first of its kind but also an incentive to continue the phytochemical, chemotaxonomic, ethnobotanical, and pharmacological studies on the Araucariaceae family in a specific as well as generic manner.