Indigenous Uses, Phytochemical Analysis, and Anti-Inflammatory Properties of Australian Tropical Medicinal Plants

Australian tropical plants have been a rich source of food (bush food) and medicine to the first Australians (Aboriginal people), who are believed to have lived for more than 50,000 years. Plants such as spreading sneezeweed (Centipeda minima), goat’s foot (Ipomoea pes-caprae), and hop bush (Dodonaea viscosa and D. polyandra) are a few popular Aboriginal medicinal plants. Thus far, more than 900 medicinal plants have been recorded in the tropical region alone, and many of them are associated with diverse ethnomedicinal uses that belong to the traditional owners of Aboriginal people. In our effort to find anti-inflammatory lead compounds in collaboration with Aboriginal communities from their medicinal plants, we reviewed 78 medicinal plants used against various inflammation and inflammatory-related conditions by Aboriginal people. Out of those 78 species, we have included only 45 species whose crude extracts or isolated pure compounds showed anti-inflammatory properties. Upon investigating compounds isolated from 40 species (for five species, only crude extracts were studied), 83 compounds were associated with various anti-inflammatory properties. Alphitolic acid, Betulinic acid, Malabaric acid, and Hispidulin reduced proinflammatory cytokines and cyclooxygenase enzymes (COX-1 and 2) with IC50 values ranging from 11.5 to 46.9 uM. Other promising anti-inflammatory compounds are Brevilin A (from Centipeda minima), Eupalestin, and 5′-methoxy nobiletin (from Ageratum conyzoides), Calophyllolide (from Calophyllum inophyllum), and Brusatol (from Brucea javanica). D. polyandra is one example of an Aboriginal medicinal plant from which a novel anti-inflammatory benzoyl ester clerodane diterpenoid compound was obtained (compound name not disclosed), and it is in the development of topical medicines for inflammatory skin diseases. Medicinal plants in the tropics and those associated with indigenous knowledge of Aboriginal people could be a potential alternative source of novel anti-inflammatory therapeutics.


Introduction
Since time immemorial, plants have been a vital source of food, shelter, clothing, tools, and weapons for humankind. Before modern allopathic medicines, early civilizations dealt with illnesses and diseases mostly with natural products from native plants and fungi, and they were taken either in raw or partially processed form. Moreover, these plants have been one of the vital sources of modern drugs, and medicinal plants still play a significant role in the biodiscovery of chemical leads for developing novel therapeutics. Of 52,885 medicinal plants identified globally [1], the phytochemical profile of only about 15% of these species has been reported thus far [2]. The World Health Organization (WHO) estimated that about 80% of the population in developing countries still rely on medicinal plants for their primary healthcare [3,4]. Many indigenous communities globally, including Australian indigenous people-popularly known as Aboriginal people, still use plants for preventive and curative purposes, despite having adequate access to advanced modern healthcare systems.
The Australian Aboriginal people are known to have occupied the country more than 50,000 years ago, and currently, they constitute 3.3% of the total Australian population [5,6]. Aboriginal people have developed a profound connection with their native flora and fauna. Their longstanding survival could have resulted from the prolonged use of medicinal plants in their diet and home remedies [7], and they still use medicinal plants in their day-to-day life. However, as they have already merged with mainstream modern society, it has become crucial to properly document their vast indigenous knowledge for their future generation [8]. The Northern Territory government, in collaboration with the Commonwealth in the 1980s, compiled "Traditional Bush Medicines," an Aboriginal pharmacopeia of the Northern Territory [9], a first-ever initiative to record dying Aboriginal medicinal lore. Since then, more collaborations have occurred between Aboriginal communities and scientists from various universities across Australia to explore Aboriginal medicinal plants [10,11]. Exploring indigenous food and medicinal plants may give a wealth of potential candidates for novel therapeutics, and Australian native plants could be an intriguing source. The geographic isolation of Australia from Gondwana and other parts of the world for over 65 million years [12,13] has become home to unique and complex flora, where approximately 85% of its vascular plants are endemic species [10]. Plants growing in the tropics produce more phenolics, flavonoids, and terpenoids during adaptation to its extreme vegetative and climatic conditions [14]. Phenolics and flavonoids are antioxidative and anti-inflammatory [15][16][17], and thus tropical plants may yield novel drug leads for treating infectious and non-infectious diseases, including chronic inflammatory conditions [18]. More than 900 medicinal plant species have been recorded in the Tropical region of Australia (shaded green in Figure 1) [19]. Aboriginal people have used these medicinal plants for treating various diseases, including inflammations and inflammatory-related diseases.  [20]. Inflammation is usually characterized by swelling and redness, pain, high body temperature (or heat), and loss of tissue function triggered by pathogens, toxic compounds, and damaged cells, whereby our immune system usually responds to initiate the healing process [21]. Anti-inflammatory drugs are used to manage inflammation (redness and swelling) and pain when conditions become severe. Currently, only FDA (United States Food and Drug Administration)-approved nonsteroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen, indomethacin, naproxen, cyclooxygenase-2 enzyme (COX-2) (categorized as non-selective NSAIDs), and celecoxib (selective NSAIDs), are available to manage inflammations and inflammatory processes [22]. These NSAIDs are associated with adverse effects on gastrointestinal, cardiovascular, nervous, hepatic, respiratory, renal, and hematologic systems [23]. Therefore, there is a need for safer alternative plant-based anti-inflammatory drugs. This study comprehensively reviewed 45 medicinal plants of tropical Australia used by Aboriginal people for inflammation and inflammatory-related diseases and explored their potential for biodiscovery of promising drug candidates to develop anti-inflammatory therapeutics. These medicinal plants also grow in Australia's sub-tropical and rainforest eco-regions.

Plant Selection and Literature Review Methods
The anti-inflammatory medicinal plants included in this review were selected following the schematic method outlined in Figure 2. Initially, we have freely listed medicinal plants which are used by Aboriginal people of Australia, referring to various books on Australian medicinal plants [8,[24][25][26][27][28], bush food [29][30][31], and bush medicines [27,32-34], followed by further verification by the senior Aboriginal Ethnobotanist at Traditional Indigenous Ethnobotany Centre, Australian Tropical Herbarium, James Cook University. Subsequently, we selected those medicinal plants found in the tropical regions of Australia (175 species) by cross-checking their distribution on the Australian virtual herbarium site [35], as medicinal plant use records are known to be more prevalent in the northern regions of Australia, where traditional knowledge is much preserved [36][37][38]. After that, we selected only those medicinal plants (78 species) primarily associated with the treatments of inflammation and inflammatory-related diseases through data mining using the online database (GoogleScholar and PubMed). We used keywords such as 'anti-inflammatory', 'anti-inflammatory uses', 'inflammation,' 'inflammatory', 'pro-inflammatory cytokines', 'inhibition', 'anti-inflammatory compound', and the name of each medicinal plant to search for the relevant literature and the information. Once the list of anti-inflammatory medicinal plants was confirmed, we reviewed their phytochemical and pharmacological properties (crude extracts and pure isolated compounds) and selected 45 species by searching the published literature indexed in PubMed using similar search keywords mentioned above. We have included only those research articles that were indexed in PubMed and Scopus databases and excluded those not indexed in these databases since the quality of the information was questionable and unreliable. Most medicinal plants included in this review are also reported to grow in the tropics of other countries, which are widely studied. However, there are limited phytochemical and pharmacological studies on medicinal plants endemic to tropical Australia. All chemical structures included in this review are referred from PubChem [39] and ChemSpider [40].

Ethnomedical Uses of Selected Medicinal Plants
Out of 78 tropical medicinal plants used by the Aboriginal people of Australia for treating inflammation and inflammatory-related diseases, 45 species were selected for this review ( Table 1). Most of these medicinal plants were trees (18 plant species), followed by herbs (16 species) and shrubs (10 species), and one grass ( Figure 3a). All parts of the plants were indicated for at least one or more inflammatory conditions (Table 1). Aerial parts such as leaves and bark were used maximum, followed by roots and wood. Medicinal plant exudates such as gums, latex, and sap were indicated least used. Fruits are known to be rich in antioxidant compounds such as phenolics and flavonoids [41], but only fruits of two medicinal plants were found to be used Figure 3b). A paste made from different parts of medicinal plants is applied to cuts, wounds, and sores to promote healing and relieve body and joint pains. A few medicinal plants are used in combination with other plants and products. For example, whole plant part of Centipeda minima is mixed with C. cunninghamii and C. thespidioides, to prepare decoction or infusion to wash inflamed eyes due to the conjunctiva and purulent ophthalmia [42,43]. Powdered leaves of Centella asiatica are mixed with lime to treat sores in babies [42]. Some of these medicinal plants are also used in other countries for treating similar types of diseases. For example, Aboriginal people use the mashed plant of Ageratum conyzoides to enhance wound healing [32,42], and Nigerians use it for treating wounds [32]. The powdered nut kernel of Calophyllum inophyllum is mixed with red pigment and water, which is then rubbed on the affected body parts to ease body pain [42]. This plant is used for treating rheumatism in India [31,32].
The whole meshed plant is applied externally to relieve rheumatism, swellings, headaches, colds, ulcers, and open-sores; seeds are eaten to relieve fever and diarrhoea [8,42].

Manihot esculenta Crantz (Euphorbiaceae)
The plant is known to be effective against belly aches and diarrhoea [196].
The plant is known to be effective against headache, sinusitis, cough and colds, and skin sores [32,42].
The whole plant is chewed or soaked in the water before applying it to the sores [131].
Nauclea orientalis (L.) L. (Rubiaceae) Aqueous bark infusion is used for sore belly; it is also applied externally to relieve rheumatic pains; the wood infusion is used for relieving fevers [32,43].
Milky juice from leaves is used against diarrhoea [31].
The plant is used to relieve fevers [230]. The plant is used to treat sore throat [234,235].
The plant is indicated for skin sores and eye complaints [237].
The plant is indicated for sore throat [196].
A decoction made from the whole plant is applied externally to overcome fever and rheumatic pain [31,42,246].

Overview of the Anti-Inflammatory Mechanism of Action/Pathways
Bioactive crude extracts or isolated compounds from medicinal plants used by Aboriginal people attenuate inflammatory conditions through three major mechanisms, namely, (i) inhibition of nuclear factor kappa B (NF-κB) pathway, (ii) inhibition of iNOS (inducible nitric oxide synthase), and cyclooxygenase (COX-1 and COX-2) protein expression/activity, and (iii) inhibiting or downregulating NO production and various pro-inflammatory cytokines (such as interleukins, IL-1β, IL-6, interferon, IFN-γ, and tumour necrosis factor, TNF-α) or upregulating anti-inflammatory cytokines (such as IL-10 and transforming growth factor, TGF-β) (Figure 4).
Briefly, NF-κB is a key to inducing gene expression of many pro-inflammatory cytokines (including IL1b, IL-6, IL-12p40, TNF-α, and COX-2) in innate and adaptive immune cells [250]. NF-κB proteins consist of five members, including NF-κB1 (or p50), NF-κB2 (or p52), ReIA (or p65), ReIB, and c-ReI, and NF-κB activation occurs in two signalling pathways, canonical and alternative non-canonical pathways [251], where both pathways are involved in regulating immune and inflammatory responses. Innate immune cells (e.g., macrophages, dendritic cells, and neutrophils) are central to innate immunity and inflammation. Innate immune cells have PRRs (pattern recognition receptors) that can detect microbial products, including pathogen-associated molecular patterns (PAMPS) and also damage-associated molecular patterns (DAMPS)-released by damaged cells and tissues [252]. Five types of PRRs expressed by mammalian cells are toll-like receptors (TLRs), RIG-I-like receptors, NOD-like receptors (NLRs), C-type lectin-like receptors, and cytosolic DNA sensors [251], each with distinct structures to bind with different PAMPs and DAMPs. All PRRs share a similar signalling pathway by activating the canonical NF-κB pathway, and this pathway either directly induces inflammation by expressing pro-inflammatory cytokines and chemokines or indirectly promoting inflammatory T cells differentiation [253]. In response to different PAMPs and DAMPs, macrophages are activated and subsequently differentiate into M1 or M2, followed by the secretion of an array of cytokines and chemokines [250]. Pro-inflammatory cytokines such as IL-1, IL-6, IL-12, and TNF-α are characteristic of M1 macrophages. TLR signals play a vital role in regulating macrophage polarization, and for instance, TLR4 ligand lipopolysaccharide (LPS) promotes the formation of M1 phenotypes (Figure 4a) [250,254]. LPS is a bacterial-derived lipopolysaccharide that can induce inflammation in macrophage cells in in vitro assays. Activated M1 macrophages can also promote other inflammatory T cells, including Th1 and Th17 cells, and these cells, in turn, also mediate inflammation (Figure 4a).
Inhibiting NO production is another anti-inflammatory mechanism shown by many compounds isolated from selected medicinal plants. In mammalian cells, NO is mainly produced from the L-arginine:NO metabolic pathway by the enzyme called nitric oxide synthase (NOS), which has three isoforms of NOS-eNOS (endothelial NOS), nNOS (neuronal NOS), and iNOS [255]. eNOS and nNOS produce a controlled amount of NO in endothelial cells and neurons, respectively, under the Ca 2+ /calmodulin system [256] (Figure 4c), while iNOS produces NO only upon activation by specific cytokines (e.g., IFN-γ) or microbial products (e.g., LPS). Sustained NO production enhances the formation of reactive nitrogen oxide species (RNOs), and prolonged exposure to such free radicals is harmful to healthy body cells [257], whereby iNOS must be regulated. Cyclooxygenase enzymes break down arachidonic acid and form prostaglandin H 2 (PGH 2 ), subsequently catalysed into PGE 2 and prostacyclin (PGI 2 ) (Figure 4c) [260], and they mediate inflammations. COX-1 activity (constitutive) is responsible for maintaining vital physiological functions, while COX-2 activity (inducible) is usually activated by adequate stimuli coming from any damaged cells of inflammatory conditions [261].

Anti-Inflammatory Crude Extracts
Out of 45 anti-inflammatory medicinal plants included in this review, crude extracts from 30 species were already tested for anti-inflammatory activities in both in vitro and in vivo assays (Table 1). Pure compounds from 15 species have also been isolated and tested for their anti-inflammatory activities to validate their ethnopharmacological uses. Among 30 species, crude extracts of Acalypha wilkesiana, Brucea javanica, Centipeda minima, Euphorbia hirta, Melaleuca leucadendra, and Terminalia catappa are most widely studied against different inflammatory conditions ( Table 2). Most of the studies on crude extracts have shown that they inhibit NO, PGE 2 , iNOS productions, and COX-2 expression in murine macrophage cells (e.g., RAW 264.7 cells) stimulated with bacterial LPS. Moreover, they also inhibit the production of pro-inflammatory cytokines, mainly TNF-α, IL-1β, and IL-6.
For example, when LPS-induced RAW 264.7 cells were treated with an aqueous crude extract of C. minima, there was a significant decrease in NO production at a 100 µg/mL concentration and also reduced inflammatory cytokines levels (TNF-α and IL-1β) significantly [262]. Moreover, the aqueous extract also inhibited the expression of iNOS and COX-2 proteins by 80.2% and 71.2%, respectively, when incubated with LPS-activated RAW 264.7 cells for 24 h. The extract also significantly inhibits the expression of iNOS and COX-2 proteins in carrageenin-induced mice paw oedema [262]. Chan et al. [263] also observed a significant decrease in the expression of monocyte chemokine attractants, particularly CCL8, in LPS-stimulated RAW 264.7 cells by the crude extract of C. minima, which could have contributed to the inhibition of monocyte chemotaxis and macrophage infiltration in DSS (dextran sodium sulphate)-induced acute colitis in C57BL/6J mice. The crude extract also inhibits the LPS-induced production of TNF-α and IL-1β, protecting HT22 (immortalized mouse hippocampal cell line) neuronal cells from inflammatory damage [264]. Crude extracts from A. wilkesiana, E. hirta, and M. leucadendra also showed a similar anti-inflammatory activity ( Table 2).
When Huang et al. (2017) studied the effect of oil emulsion from B. javanica in DSS (Dextran Sodium Sulphate)-induced acute colitis mouse model (0.5, 1, and 2 g/kg), oil emulsion improved disease activity index, including colon length, and body weight [265]. Additionally, when they analysed cytokines production, B. javanica oil emulsion at higher concentrations (1 and 2 g/kg) has significantly (p < 0.01) lowered the levels of six inflammatory cytokines (IL-1β, IL-6, IL-8, IL-17, IFN-γ, and TNF-α) in the colon tissues when compared to positive controls (sulfasalazine and azathioprine) [265]. Crude extracts from T. catappa bark, which Aboriginal people use to treat a sore throat, were anti-inflammatory. When Daram et al. (2021) compared ethanol and water extracts from T. catappa bark, ethanol extract was better in inhibiting 50% of protein denaturation in the in vitro egg-albumin denaturation assay, and diclofenac was used as a positive control [266]. Both extracts reduced carrageenan-induced paw oedema at a 500 mg/kg concentration in the rat. Leaves ethanol extract and chloroform fraction from T. catappa were tested in chronic and acute models of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear oedema. When crude extracts were applied topically at a 1 mg/kg concentration twice daily for four days, the chloroform fraction reduced oedema by 60% compared to ethanol extract (32%) [242]. In the same study, chloroform fraction yielded two pure compounds showing anti-inflammatory activities, discussed in the subsequent sections.

Anti-Inflammatory Compounds
When 45 selected Aboriginal medicinal plants were reviewed for their phytochemical compositions and pharmacological properties, 40 species were studied for their phytochemistry. For rest of the five species, only crude extracts were studied. When compounds isolated from 40 Aboriginal medicinal plants (Table 1) were further reviewed, 83 compounds have shown various anti-inflammatory activities ( Table 2) in in vitro cellular and in vivo animal models. Out of 83 anti-inflammatory compounds, majority were terpenes and terpenoids (30 compounds), followed by flavonoids (16 compounds), coumarins (10 compounds), alkaloids (6 compounds), glycosides, sterols, lignans, and carboxylic acids (3 compounds each). The rest of the compounds were phenolics, aldehydes, tannins (2 compounds each), pyrans, phenylpropanoids, and fatty acid esters (one compound each).

Terpenes and Terpenoids
Terpenes are one of the most structurally diverse secondary metabolites in plants, and five-carbon isopentyl units (2-methyl-1,3-butadiene or isoprene) form their main skeleton (additional functional groups such as ketone and hydroxyl rings are present in terpenoids), which are categorized into seven types based on a number of isoprene units they contain [14]. Among terpenoids, clerodane diterpenoid and polyandric acid A (isolated from Australian Dodonaea polyandra) ( Figure 5) have shown potent anti-inflammatory activity [133]. Simpson et al. [133] tested this compound in acute and chronic mice models of TPA-induced ear oedema. Mice with TPA-induced ear oedema, when treated with 0.9 µM/ear of polyandric acid, significantly reduced (p < 0.05) the IL-1β (78%) compared to its negative control (TPA only treated group) [133], but it did not have any effect on TNF-α. In the chronic mouse ear oedema model, treatment of TPA-induced mice with polyandric acid A (same dosage, for four days) resulted in a significant reduction (p < 0.001) in the oedema (41.1%), where positive control drug betamethasone was reduced to 54.2%. In the same study, they further evaluated the inhibition of neutrophil infiltration into the inflamed tissues of TPA-induced mice ear oedema (i.e., assessed by measuring the level of MPO-myeloperoxidase), and polyandric acid A reduced MPO level by 81.5% (p < 0.001) compared to the negative control group. Positive control (betamethasone) showed 90% reduction of MPO at the same concentration (0.9 µM/ear) [133]. The compound also significantly reduced (p < 0.05) IL-β, proIL-β, and IL-6 by THP-1 monocytes and primary neonatal human epidermal keratinocytes (nHEKs) at both high (30 µM) and low (3 µM) concentrations. Hautriwaic acid, isolated from Mexican medical plant, Dodonaea viscosa, also reduced TPA-induced ear oedema in mice by 60.2%, 70.2%, and 87.1% at 0.25, 0.5, and 1.0 mg/ear concentrations [138]. When Feng et al. (2020) evaluated the 'drug-likeliness' of 65 anti-inflammatory clerodane diterpenoids (compared with marketed drugs) using SwissADME, a web-based tool by the Swiss Institute of Bioinformatics [267], many of them fulfill parameters within SwissADME [268]. They found that a lactone ring between C-18 and C-19 is mainly responsible for anti-inflammatory activity.

Flavonoids
Flavonoids comprise seven different types, namely, anthocyanins, flavanols, flavanones, flavones, isoflavonoids, and flavan-3-ols (or catechins) [14]. Flavonoids are known to possess anti-inflammatory potential by inhibiting the expression of transcription factors and regulatory enzymes involved in the inflammation [272,273]. Sixteen flavonoids isolated from medicinal plants used by Aboriginal people of Australia have shown various anti-inflammatory activities ( Figure 6; Table 2). However, most of these compounds were isolated from medicinal plants from other parts of the world, not from the Australian tropical region. Hispidulin isolated from the leaves of Thai Clerodendrum inerme inhibited NO production in LPS-stimulated RAW 264.7 cells with an IC 50 value of 43.7 ± 4.0 µM [274]. Moreover, it also showed NF-κB pathway inhibitory effect. Agallochanin K from Excoecaria agallocha [154,167] showed a similar activity of NF-κB pathway inhibition (Table 2). Hispidulin was also reported from the Chinese C. inerme [105], but it was not tested for anti-inflammatory activity. There is no study on C. inerme from the tropical region of Australia.  [275]. When administered before pleurisy in mice (induced by carrageenin), both compounds inhibited the phosphorylation level of NF-κB p65 and p38 MAPK in the lungs compared with both negative and dexamethasone-treated positive controls [276].

Alkaloids
Alkaloids contain nitrogen atom(s), and seven types of alkaloids (based upon their amino acid precursor) are present in at least 20% of plant species [14]. Plant-derived alkaloids are promising against inflammatory bowel diseases as many of them can restore epithelial barrier functions, ameliorate colonic inflammation, and reduce oxidative stress [278]. Plant families, such as Papaveraceae, Ranunculaceae, Solanaceae, and Amaryllidaceae, are rich in alkaloids. Among 45 selected reviewed here (Table 1), Crinum pedunculatum is the only species that belongs to alkaloid-rich families (Amaryllidaceae), and Aboriginal people use this whole plant against inflammation caused by marine stings. There are no reports of its phytochemistry yet, and this plant may contain novel anti-inflammatory alkaloids. When Barbosa-Filho et al. [279] reviewed anti-inflammatory properties of 171 alkaloids, 137 of them were found to possess anti-inflammatory properties, of which isoquinoline alkaloids were reported to be mostly bioactive. A total of six alkaloids isolated from Chinese medicinal plants Ochrosia elliptica (ellipticine, 10-methoxyconolidine, apparicine, yunnanensine, and isositsirikine) and Nelumbo nucifera (neferine) were reported as anti-inflammatory ( Figure 7; Table 2). Indole alkaloid ellipticine at a concentration of 5 µM downregulated the TNF-α and IL-6 levels in LPS-stimulated RAW 264.7 cells and human peripheral blood monocytes [280]. When  assessed four alkaloids isolated from leaves of O. elliptica, 10-methoxyconolidine and apparicine showed significant inhibition of nitric oxide production in LPS-stimulated RAW 264.7 cells at IC 50 values of 2.4 ± 0.08 µM and 5.8 ± 0.13 µM, respectively. However, the other two alkaloids (yunnanensine and isositsirikine) showed only mild inhibition [226]. Aboriginal people of Australia have used the bark of this plant for treating dysentery, but there is no report of compounds isolated from the bark. Neferine, a benzylisoquinoline alkaloid from the seed embryo of Chinese Nelumbo nucifera, also showed a similar anti-inflammatory activity by inhibiting TNF-α and IL-6 in LPS-induced RAW 264.7 cells; additionally, it suppressed COX-2 and iNOS expression, and DSS-induced ulcerative colitis in the mice (at dosages of 10 and 25 mg/kg) [281]. Min et al. (2019) showed that neferine protected mice against DSS-induced colitis at concentrations of 5 and 10 mg/kg [282]. Neferine further reduced MPO activity and secretion of inflammatory cytokines (TNF-α, IL-6, and IL-1β). There are many reported cases of poisoning from the consumption of medicinal plants and herbal products, and the presence of alkaloids such as pyrrolizidine alkaloids (PAs) is responsible for the toxicity [283,284]. Plants belonging to Asteraceae, Fabaceae, and Boraginaceae are known to contain PAs. When Wang et al. (2021) analysed 386 herbal products from the Chinese Pharmacopoeia (2020) for the presence of PAs using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), 271 of their tested samples contained 0.1-25,567.4 µg/kg, out of which 20 samples had PAs above the estimated daily intake value of 0.007 µg/kg body weight/day [285]. Senkirkine  [286]. One must be aware of their detoxification processes before utilizing them in any form of medicinal preparations because, as per the World Health Organisation (WHO), daily intake of PAs above 10 µg/kg body weight can cause hepatic veno-occlusive disease [287]. A C19 norditerpenoid alkaloid aconitine from Aconitum root is another example of a toxic alkaloid and the Aconitum species are widely used in traditional medicine in Asian countries [284]. Only two plants (Ageratum conyzoides and Centipeda minima) out of 45 species reviewed here belonged to PAs containing family (Asteraceae). A. conyzoides contains PAs lycopsamine, dihydrolycopsamine, and acetyllycopsamine [288]. The toxic effect of hydroalcoholic extract of A. conyzoides leaves in Caco-2 cells (IC 50 = 44 µg/mL) is attributed to pyrrolizidine alkaloid content [289]. However, both plants are indicated for external applications for wounds and eye inflammations by Aboriginal people, which may be considered safe, but it would not be advisable for oral consumption unless processed properly, as PAs are hepatoxic and carcinogenic. Aboriginal healers have extensive knowledge of detoxifying some toxic plants before eating or using them in medical applications [46,47], but it requires adequate documentation.

Brasenia schreberi
NT Quercimeritrin Reduced the expression of iNOS and NO in LPS-stimulated RAW 264.7 cells; also prevented the overexpression of COX-2 and granulocyte macrophage-colony-stimulating factor [66].

Plant Species Crude Extracts Tested Isolated Compounds Tested Anti-Inflammatory Activities
Hibiscus tiliaceus Whole plant extract NT Whole plant extract reduced acute auricle swelling induced by dimethylbenzene in mice [316].
Whole plant extract suppressed LPS-induced NO and PGE2 production, and COX-2 expression in RAW 264.7 cells [320]. Stachyurin (or Casuarinin) reduced ethanol-induced gastric ulceration in rats by increasing mucin production and reducing acidity; it also increased glutathione and catalase levels; it suppressed the immunoexpressing of NF-κB, COX-2, and iNOS to their normal values [207].

Ocimum tenuiflorum
Leaves extract NS Leaves extract reduced the LPS-induced inflammation in RAW 264.7 cells [334].

Phragmites australis
Aerial extract NT Aerial crude extract inhibited the production of NO and ROS, and pro-inflammatory cytokines (TNF-α and IL-1β) in LPS-induced RAW 264.7 cells [335].

Sarcostemma viminale
Whole plant extract NS Whole plant extract inhibited the production of pro-inflammatory cytokines in RAW 264.7 cells [337].

Scaevola taccada
Leaves extract NT Leaves extract reduced IL-1β levels in the tissues of Sprague-Dawley rats suffering from mastitis [338].
Abbreviations: NS: compounds not isolated from the plant; NT: crude extracts not tested, or compounds isolated but not tested for anti-inflammatory activities.

Coumarins
Costatamins A-C isolated from the leaves of Australian Angophora costata inhibited the production of NO and pro-inflammatory cytokine-TNF-α in LPS-activated RAW 264.7 cells with IC 50 values in the range of 20-30 µg/mL [54]. Aboriginal people used an aqueous solution of reddish exudate from the bark/or trunk orally to cure diarrhoea [8,53], not leaves; thus, studying bark's phytochemical constituents could help further validate their use. Calophyllolide (Figure 8) is one of the promising examples of coumarins from one of the selected medicinal plants. The compound isolated from fresh seeds of Vietnamese Calophyllum inophyllum showed wound-healing potential [82]. Aboriginal people have used grounded nut kernel to rub against body pain, but in Vietnam, C. inophyllum oil is used for treating skin and rheumatic disease and burns. Calophyllolide (6 mg/animal) accelerated the wound healing process in the surgically created wound in the mice model more than iodopovidone (100 mg/animal) at 14 days post-treatment, and it was determined that the reduced MPO and increased M2-related gene expression could have promoted the wound healing in mice [82]. Calophyllolide also downregulated pro-inflammatory cytokine IL-1β, IL-6, and TNF-α, and upregulated anti-inflammatory cytokine IL-10 in LPS-activated RAW 264.7 cells [82]. Based on these promising activities, calophyllolide could be a potent lead for developing topical wound-healing agents or creams. Other parts of the C. inophyllum, particularly those used for ethnomedicinal purposes, may also contain promising antiinflammatory compounds. Scopoletin and isoceopoletin from fruits of Morinda citrifolia showed mild activity against 5-LO and 15-LO (lipoxygenase) and COX-2 enzymes, but IC 50 values were low [209]. Other coumarins, cleomiscosins A-C from dried seeds of Indian Cleome viscosa, also showed anti-inflammatory activities by inhibiting pro-inflammatory cytokines (TNF-α and IL-6) when given in a mixture at concentrations of 30-100 mg/kg body weight in female Swiss albino mice [302] (Table 2).

Biodiscovery Potential and Challenges
Drug discovery from natural sources is a challenging and resource-intensive process. It requires high throughput screening (HTS) of large extracts, followed by tedious characterization and identification of bioactive constituents. Among different natural products, medicinal plants are still one of the vital sources of promising therapeutic leads. When Fabricant and Farnsworth (2001) evaluated 122 compounds used worldwide as drugs, 80% were associated with medicinal plants used in Indigenous medicines [344]. For example, taxol and camptothecin [345]. However, there is no evidence of income amount generated by commercialising these drugs to the Indigenous peoples who own intellectual property rights. Intellectual piracy/biopiracy incidences were rampant in the olden days, whereby the researchers/pharmaceutical companies stole the traditional knowledge without any financial returns to the indigenous communities. It is fair to respect the indigenous rights and equally share the benefits arising from value-addition to their knowledge. There is an urgent need to document Aboriginal people's indigenous knowledge about medicinal plants and establish a proper database such that knowledge-ownership is secured in the hands of their younger generations.
Today, this has changed for a good cause. About two-thirds of medicinal plant species used worldwide (sourced from their natural habitats) [346] are strictly regulated by the equal benefit-sharing agreement that is highlighted in the Convention on Biological Diversity [347] and Nagoya Protocol [348], as well as local state and federal biodiversity protection acts. The plants associated with indigenous knowledge are bound by more stringent regulations, including the United Nations Declaration on the Rights of Indigenous People {Article 31(1)} [349], and local in-country biodiscovery acts and rules. All traditional knowledge-related developments, including cultural expression and biodiscovery, remain within their jurisdiction [349]. Moreover, indigenous intellectual property is their heritage, and they have the authority to approve or reject the consent to access as they must maintain secrecy [350]. In Australia, the Biodiscovery Act of Queensland 2004 [351] protects the IP and guides the biodiscovery involving Aboriginal medicinal plants, although only recognized in Queensland State.
This review has compiled 45 medicinal plants that Aboriginal people of Australia use to treat various inflammatory conditions. Some of these plant extracts and compounds showed potent anti-inflammatory activities, presenting unprecedented biodiscovery and drug development potential. In addition, most of the studied medicinal plants for their phytochemical and anti-inflammatory activities to date were reported from other countries, the majority from China, India, Japan, Taiwan, Thailand, and Vietnam. Australia reported only five species (Alphitonia petriei, Angophora costata, Corymbia terminalis, Dodonaea polyandra, and Excoecaria agallocha). Variations in the geography, habitats, and climate [14] can cause variations in plants' chemical constituents and their associated bioactivities (antioxidant and anti-inflammatory) [352][353][354]. Thus, it is worthwhile to study the Australian medicinal plants, particularly those unstudied endemic medicinal plants, in collaboration with the traditional owners.

Conclusions
Australia is home to unique biodiversity, including many native and endemic species. The Aboriginal people of Australia, who are said to have lived from 40,000 to 50,000 years ago, possess profound oral traditional knowledge about their native flora and fauna. More than 900 medicinal plants are recorded in the tropical region alone, and associated traditional medicinal knowledge belongs to traditional owners of the land. The present study found that Aboriginal people used about 78 medicinal plants against various inflammation and inflammatory-related conditions. Of the 78 species, 45 were already studied for their phytochemical and anti-inflammatory properties. However, most phytochemical, and pharmacological studies were reported from other countries, mainly China, India, Japan, Thailand, Vietnam, and Taiwan. Australia reported only five studies on Alphitonia petriei, Angophora costata, Corymbia terminalis, Dodonaea polyandra, and Excoecaria agallocha. Eightythree isolated compounds have shown various anti-inflammatory activities in vitro assays (mostly LPS-activated macrophage cells) by inhibiting the production of pro-inflammatory cytokines and chemokines and the gene expression of mediators involved in the inflammatory pathways, including the NF-κB pathway.
Furthermore, those bioactive compounds in in vitro assays showed significant protection in the various animal disease models, mostly ear and paw oedema and colitis mice models. Most isolated bioactive compounds belonged to the chemical class of terpenes and diterpenoids, and flavonoids. Many Australian medicinal species are not yet studied and studying these plants will undoubtedly help the scientific validation and documentation of their traditional knowledge. It is evident from this review that many Aboriginal medicinal plants are associated with anti-inflammatory properties that can guide the discovery of novel anti-inflammatory drug leads in collaboration with the indigenous communities of Tropical Australia.
Author Contributions: Conceptualization, P.W. and K.Y.; methodology, P.W., K.Y. and G.T.; writing-original draft preparation, K.Y. and T.J.; writing-review and editing, P.W. and G.T; supervision, P.W.; funding acquisition, P.W. All authors have read and agreed to the published version of the manuscript.