Medicinal Plants of the Australian Aboriginal Dharawal People Exhibiting Anti-Inflammatory Activity

Chronic inflammation contributes to multiple ageing-related musculoskeletal and neurodegenerative diseases, cardiovascular diseases, asthma, rheumatoid arthritis, and inflammatory bowel disease. More recently, chronic neuroinflammation has been attributed to Parkinson's and Alzheimer's disease and autism-spectrum and obsessive-compulsive disorders. To date, pharmacotherapy of inflammatory conditions is based mainly on nonsteroidal anti-inflammatory drugs which in contrast to cytokine-suppressive anti-inflammatory drugs do not influence the production of cytokines such as tumour necrosis factor-α or nitric oxide. However, their prolonged use can cause gastrointestinal toxicity and promote adverse events such as high blood pressure, congestive heart failure, and thrombosis. Hence, there is a critical need to develop novel and safer nonsteroidal anti-inflammatory drugs possessing alternate mechanism of action. In this study, plants used by the Dharawal Aboriginal people in Australia for the treatment of inflammatory conditions, for example, asthma, arthritis, rheumatism, fever, oedema, eye inflammation, and inflammation of bladder and related inflammatory diseases, were evaluated for their anti-inflammatory activity in vitro. Ethanolic extracts from 17 Eucalyptus spp. (Myrtaceae) were assessed for their capacity to inhibit nitric oxide and tumor necrosis factor-α production in RAW 264.7 macrophages. Eucalyptus benthamii showed the most potent nitric oxide inhibitory effect (IC50  5.57 ± 1.4 µg/mL), whilst E. bosistoana, E. botryoides, E. saligna, E. smithii, E. umbra, and E. viminalis exhibited nitric oxide inhibition values between 7.58 and 19.77 µg/mL.


Introduction
Inflammation is an important biological process and is essential to maintain the body's homeostasis, to fight against pathogens effectively, and to repair the damaged tissue [1]. However when uncontrolled and chronic, inflammation gives rise to a number of (often age related) diseases including asthma, rheumatoid arthritis, inflammatory bowel disease, Crohn's disease, and tendonitis. Furthermore, a chronic inflammatory response with accompanying oxidative stress is a significant force driving the progression of peripheral diseases like atherosclerosis, diabetes, and metabolic syndrome, as well as neurodegenerative diseases such as multiple sclerosis, Parkinson's disease, and Alzheimer's disease [2][3][4][5].
While some chronic/remitting neurological diseases, such as multiple sclerosis, have long been recognized as inflammatory, the term "neuroinflammation" is now applied to chronic activation of microglia and astroglia that do not reproduce the classic characteristics of inflammation in the periphery but may cause neurodegeneration [6][7][8]. Some examples of diseases characterized by neuroinflammation are Alzheimer's disease (AD) and Parkinson's disease and even autism-spectrum and obsessive-compulsive disorders [9][10][11][12]. Microglial and astroglial activation, accompanied by increased levels of proinflammatory mediators such as TNF-, IL-1 and IL-6, prostaglandins, and reactive oxygen and nitrogen species, as well as reactive carbonyl species and advanced glycation end products, is observed in the AD brain at all stages of the disease [13][14][15][16][17][18]. Genetic and pharmacoepidemiological studies also point to the importance of inflammation in AD. For example, three immune-relevant genes were shown to be associated with an increased risk of 2 Evidence-Based Complementary and Alternative Medicine AD; these are CLU (clusterin), CR1 (complement receptor 1), and TREM2 (triggering receptor expressed on myeloid cells 2) [19].
At present, both steroidal and nonsteroidal anti-inflammatory drugs (NSAIDs) are used to treat inflammation. NSAIDs in particular can cause severe side effects, most importantly gastric ulcers. NSAIDs are specifically designed as inhibitors of cyclooxygenase (COX) enzymes and, in contrast to CSAIDs, do not influence the production of proinflammatory cytokines such as TNF-or free radicals such as nitric oxide [28]. CSAIDs specifically target p38 MAPK and NF-B signalling pathways to inhibit cytokinemediated events with demonstrated efficacy in a range of animal models [29,30].
Excessive production of inflammatory cytokines and reactive radical species can damage cellular biomolecules like proteins, lipids, and carbohydrates as well as nucleic acids, leading to cellular and tissue damage, which further perpetuates the inflammatory cascade. Therefore, pharmacological compounds with the ability to attenuate the production of these inflammatory molecules may have potential for the treatment of many inflammatory diseases including AD [21,22,28,34,35].
The use of natural substances, especially those derived from plants, in order to prevent, manage, or cure diseases is a centuries-old practice which has led to the discovery of many modern pharmaceuticals. In recent years, the search for novel anti-inflammatory drugs from a wide range of medicinal plant resources has been intensified, and a variety of plant secondary metabolites including apigenin, curcumin, cinnamaldehyde, and resveratrol have already been found to suppress inflammatory responses [21,22,28].
For example, turmeric (Curcuma longa) and its main ingredient curcumin, which has long been used for treatment of rheumatic disorders, exerts both anti-inflammatory and antiatherosclerotic effects [23,36]. Ginger extract (Zingiber zerumbet) and its main active compound, 3-O-methyl kaempferol, significantly attenuated carrageenan-induced mouse paw oedema in an in vivo model and were also found to inhibit the production of nitric oxide (NO) and prostaglandin E 2 (PGE 2 ), as well as iNOS expression in a cell culture model. Aqueous and hydroalcoholic as well as ethanolic extracts from another ginger species (Zingiber officinale) demonstrated significant anti-inflammatory activity and its active constituent [6] gingerol again showed antiinflammatory activity by inhibiting the production of NO and PGE 2 [37] and was also successful in inhibiting carrageenaninduced rat paw oedema [38].
Triterpenoid saponins, from the Australian desert tree Acacia victoriae, have shown anti-inflammatory effects via inhibiting activation of NF-B, by preventing its nuclear localization and inhibiting its ability to bind to DNA [39]. Another Australian indigenous plant Tinospora smilacina is claimed to possess long chain unsaturated fatty acids which possess anti-inflammatory properties [40]. The fruits of the Australian native Kakadu plum (Terminalia ferdinandiana), Illawarra plum (Podocarpus elatus), and Native currant (Acrotriche depressa) also exhibited significant antiinflammatory activity [41].
There is large scope to investigate Australian native plants for their bioactivity and chemical constituents [42]. Traditional medicine is still practised by the many tribal Aboriginal people, particularly in Central and Northern Australia and this ethnomedicinal knowledge is recorded in some cases [43]. The "Dharawal Pharmacopeia" written by botanist and Aboriginal Elder Frances Bodkin (known as Aunty Fran) is a compilation of the Aboriginal medicinal and ceremonial uses (and corresponding taxonomic identification) of thousands of native Australian plants. Of interest to our research, a number of plant species described in the Dharawal pharmacopeia have been claimed to possess anti-inflammatory activities (Table 1) [44,45]. Plants from Eucalyptus species have special importance for the Dharawal indigenous people and are used for their anti-inflammatory activity along with other medicinal uses as well as for shelter and weapons. As stated in the Dharawal pharmacopeia, Eucalypts are mostly distributed in Blue Mountains, Southern Highlands, Woronora Plateau, and coastal area of New South Wales, Australia.
The aim of our research is to evaluate the anti-inflammatory activity of Australian native plants with ethnopharmacological importance and subsequently characterise the bioactive components. In this manuscript, dried extracts from 17 Eucalyptus spp. were evaluated for anti-inflammatory activity via the suppression of NO and TNF-production induced by lipopolysaccharide (LPS) and interferon gamma (IFN-) in RAW 264.7 cells. Cytotoxicity of the crude extracts was also examined using an Alamar blue cell viability assay.

Plant Material.
Plants known to be used by the Dharawal people (also known as Tharawal) to treat inflammation and related illnesses were selected under the guidance of botanist and Aboriginal Elder Auntie Fran (Frances Bodkin) and the Dharawal pharmacopeia. Leaf material of 17 Eucalyptus spp. was collected in the month of August, 2015 from the "Australian Botanic Gardens" at Mount Annan, NSW, Australia (Table 1).

Extraction of Plants Leaves for Biological Assays and HPLC and MS Analysis.
Approximately 40 g of fresh leaf material from each plant was extracted using absolute ethanol. The leaves were first cut into small pieces with scissors and then ground to a coarse powder using a hand blender. The coarse powder was filled into the thimbles of an accelerated solvent extraction system (Buchi B-811, Switzerland) and then extracted under standard soxhlet mode (for 2 × 15 minutes cycles). The volume of the extracts was reduced to ca. 2-4 mL using a rotary evaporator and then evaporated to dryness with nitrogen gas for biological assays. Percentage yields (g/g% fresh weight) are recorded in Table 2.

Maintenance and Preparation of RAW 264.7 Macrophages.
RAW 264.7 macrophages were grown in 175 cm 2 culture flasks on DMEM (Dulbecco's Modified Eagle's Medium) containing 5% FBS (fetal bovine serum) that was supplemented with antibiotics (1%) and glutamine (1%). The cell line was maintained in 5% CO 2 at 37 ∘ C, with media being replaced every 3-4 days. Once cells had grown to confluence in the culture flask, they were removed using a rubber policeman cell scraper, as opposed to using trypsin, which can remove membrane-bound receptors such as RAGE. The cell suspension was concentrated by centrifugation for 3 min at 900 rpm and resuspended in a small volume of fresh DMEM (with 1% antibiotics and 5% FBS). Cell densities were estimated using a Neubauer counting chamber. Cell concentration was adjusted with DMEM (with 1% antibiotics and 5% FBS) to obtain 60000 cells/100 L cell suspension. The 100 L cell suspension was then dispensed into the inner wells of 96-well plates. Plates were incubated at 37 ∘ C and 5% CO 2 for 18 h before the activation experiments were carried out.

Activation of RAW 264.7 Macrophages.
From each well, the media were removed and replaced with fresh DMEM containing 0.1% FBS. For assays with extracts, a 90 L volume of the dilutions in DMEM (with 0.1% FBS) was added an hour prior to addition of the activator. Due to the often inconsistent nature of LPS at activating cells, a combination of LPS (10 g/mL) and IFN-(10 U/mL), both in DMEM (with 0.1% FBS), was used for activation. A maximum dose of the extracts used is 900 g/mL and diluted serially by 50% up to a minimum of 10 doses (900, 450, 225, 112.5, 56.25, 28.125, 14.062, 7.031, 3.515, 1.7578, and 0.8789 g/mL in the wells, resp.). After activation, the cells were incubated for 24 h at 37 ∘ C and 5% CO 2 and then NO and TNF-inhibition and cell viability were determined. Cells with media alone were used as negative control and activated cells used as positive control.

Determination of Nitric Oxide Production by Griess Assay.
Nitric oxide was determined by Griess reagent quantification of nitrite, one of its stable reaction products. Griess reagent was freshly made up of equal volumes of 1% sulfanilamide and 0.1% naphthylethylene-diamine in 5% HCl. In the presence of nitrite this reagent forms a violet colour. From each well, 50 L of supernatant was transferred to a fresh 96well plate and mixed with 50 L of Griess reagent, and the colour produced was measured at 540 nm in a microplate 4 Evidence-Based Complementary and Alternative Medicine

Determination of Cell Viability by Alamar Blue Assay. The
Alamar Blue assay is a colorimetric assay involving the cellular reduction of resazurin to resorufin. Alamar Blue solution [100 L of 10% Alamar Blue (resazurin) in DMEM medium] was added to each well and incubated at 37 ∘ C for 1-2 h. After incubation, fluorescence was measured (excitation at 530 nm and emission at 590 nm) using a POLARstar Omega microplate reader (BMG Labtech, Mornington, Australia) and expressed as a percentage of that in control wells after background fluorescence was subtracted. .0097 ng/mL in the wells, resp.) and was used as the internal standard. TNF-was detected with a biotinylated second antibody and an avidin peroxidase conjugate with TMB as detection reagent. After ∼30 min, the reaction was stopped using 0.5 M sulfuric acid, and the absorbance was measured at 450 nm of measurement filter with a 655 nm of reference filter. The absorbance data was expressed as a percentage of that in control wells after conversion of the concentrations by using a standard curve constructed with defined concentrations of TNF-. Curve fitting of this standard curve and extrapolation of experimental data were performed using nonlinear regression analysis.

Data Presentation and Analysis.
As the experiments were done in triplicates, the results were expressed as the mean ± SEM. In addition, linear relationships and significance tests of these data sets were also conducted. GraphPad Prism version 6.01 (GraphPad Software Incorporated, USA) was used for growth curve analysis in dose-dependent experiments and to determine the IC 50 values for NO and TNF-inhibition as well as LC 50 .

Results and Discussion
In  Table 2).
The RAW 264.7 murine macrophages release NO and TNF-when exposed to bacterial LPS and IFN-and on this principle, has become an established experimental model to evaluate in vitro anti-inflammatory activity of extracts [28]. For the purpose of interpretation, the IC 50 values of NO inhibition are divided into three groups: extracts with IC 50 < 20 g/mL are considered as highly potent extracts; a value between 21 and 80 g/mL is considered as moderately potent, and an IC 50 < 80 g/mL is considered as an extract with low potency.
The highest concentration of ethanolic crude extract tested in the anti-inflammatory assay was 900 g/mL with 0.  Figure 1).
The plant extracts also showed promising TNFinhibitory activity (  Figure 1).
The use of Alamar Blue (resazurin) to measure cytotoxicity is an established technique [46]. The results of cytotoxicity (LD 50 ) of our leaf extracts are shown in Table 3 Figure 1).
In future experiments, we will purify the most potent extracts to identify the most active compounds. One major candidate for carrying the anti-inflammatory activity could be 1,8-cineole, the major monoterpene of eucalyptus oil, as it can represent between 60 and 80% of the volatile oils 6 Evidence-Based Complementary and Alternative Medicine Note. Results represent the mean ± SEM of 3 experiments in triplicate for NO production and cytotoxicity whereas for TNF-production it is 1 experiment in triplicate.
derived from eucalyptus leaves depending on the species. Therapeutic concentrations of 1,8-cineol (1.5 g/mL = 10 −5 M) inhibited significantly cytokine production in lymphocytes and monocytes [47,48]. It has to be noted that 1,8-cineol has already gained market acceptance for its anti-inflammatory properties in mouthwashes and cough suppressants or antiasthmatic medications [48,49]. The plants studied here were chosen on the basis of their traditional use to treat inflammatory conditions by the Dharawal people of the Campbelltown region (Southwest Sydney Australia). All of the plants showed antiinflammatory activity and demonstrated inhibitory effect on downregulation of NO and TNF-production with varying potencies, which supports their use in traditional Aboriginal medicine. The content of the anti-inflammatory compounds in the plants, according to traditional knowledge, is also dependent on the plant's environment. In Dharawal country, what is most important when seeking particular medicines from plants is where the plant is growing, that is, not so much the soils, but the other plants that are growing around the particular plant required. For instance, with the Eucalypts, close proximity of an Ironbark (Muggago) and a Ribbon bark (Kai'yeroo) is needed for the anti-inflammatory medicine from the Burringoa (Eucalyptus tereticornis) to be most effective. As another example, the Ironbark itself does not need other Eucalypts close by, but it does need the Einadia (one of the saltbushes) to be growing at its base. In addition, if it had been struck by lightning (and this can be confirmed by a line of interrupted bark running from the top of the tree almost to its base), then the antiinflammatory medicine would be most effective, when using the leaves of the Eucalypts as medicine, the leaves of the trees younger than 7 years were placed on a low fire and the smoke inhaled. However, when the tree is bearing the mature leaves, the leaves were collected and boiled then allowed to cool before being rubbed on the affected part of the body, depending on the species. For the present screening study, the plant material was provided by the Botanical Gardens from random trees in the garden, but for future studies, we will investigate if collection practice based on Dharawal knowledge will improve the inherent activity and/or yield of the anti-inflammatory compounds.

Conclusions
The present study suggests that most of the Eucalyptus spp. potentially possess interesting anti-inflammatory compounds with low toxicity and the in vitro activity appears to support the traditional use. Eucalyptus benthamii, E. bosistoana, E. botryoides, E. saligna, E. smithii, E. umbra, and E. viminalis leaf extracts exhibited strong anti-inflammatory activity by inhibiting NO and TNF-production in LPS and INF-stimulated RAW 264.7 macrophages. Purification and structure identification of the most these extracts are currently underway.