Phytochemical Composition and Toxicological Screening of Anise Myrtle and Lemon Myrtle Using Zebrafish Larvae.

Plants are an immense source of drugs, and 50% of modern pharmacopeia has a plant origin. With increasing life expectancy in humans, many age-related degenerative diseases converge on oxidative cellular stress pathways. This provides an opportunity to develop broad treatments by targeting the cause of common pathologic cell degeneration. Toxicological effects can be readily assessed in a live animal model system to establish potential fauna for clinical use. Here, we characterized and evaluated the antioxidant potential and toxicological effects of anise myrtle (Syzygium anisatum) and lemon myrtle (Backhousia citriodora) leaves. Using zebrafish larvae, a model for high-throughput pre-clinical in vivo toxicology screening, we identified safe levels of extract exposures for development of future therapeutics. The antioxidant capacity and toxicity were very similar in these two myrtles. The LC50-96h for anise myrtle was 284 mg/L, and for lemon myrtle, it was 270 mg/L. These measurements are comparable to ongoing studies we are performing using the same criteria in zebrafish, which allow for robust testing and prioritization of natural fauna for drug development.

Citral in lemon myrtle, lemongrass, litsea, and melissa essential oils suppress the growth and invasion of breast cancer cells.

OBJECTIVE: Although cancer therapy suppresses recurrence and prolongs life, it may be accompanied by strong side effects; thus, there is a strong demand for the development effective treatments with fewer side effects. Cancer therapy using plant-derived essential oils is attracting attention as one promising method. This study investigated the antitumor effects of essential oil volatiles on breast cancer cells and identifies four essential oils that display antitumor activity. METHODS: Breast cancer cells were cultured in a 96-well plate, then one of twenty essential oils was added dropwise to the central well. The plate was incubated at 37 °C for 48 h and the effect of the volatile components of each essential oil on the surrounding breast cancer cell growth ability was examined using an MTT assay. Gas chromatography was used to investigate the concentration of the transpiration components that may affect cancer cells. RESULTS: Of the 20 essential oils, Lemongrass, Lemon myrtle, Litsea, and Melissa displayed strong anti-tumor effects. These essential oils inhibited the growth of nearby breast cancer cells, even when diluted more than 500-fold. The transpiration component of lemon Myrtle showed the strongest antitumor effect, but was the least cytotoxic to mononuclear cells in normal peripheral blood (PBMC). Each of these essential oils contained a very large amount of citral. The IC50 against breast cancer cells when citral was volatilized from each essential oil was 1.67 µL/mL for geranial and 1.31 µL/mL for neral. Volatilized citral alone showed strong anti-proliferation and infiltration-inhibiting effects. CONCLUSION: The transpiration components of Lemongrass, Lemon myrtle, Litsea, and Melissa are thought to inhibit breast cancer cell proliferation due to their high levels of citral.

LC-ESI-QTOF-MS/MS Identification and Characterization of Phenolic Compounds from Leaves of Australian Myrtles and Their Antioxidant Activities.

Phenolic compounds, present in plants, provide substantial health advantages, such as antioxidant and anti-inflammatory properties, which enhance cardiovascular and cognitive well-being. Australia is enriched with a wide range of plants with phytopharmacological potential, which needs to be fully elucidated. In this context, we analyzed leaves of aniseed myrtle (Syzygium anisatum), lemon myrtle (Backhousia citriodora), and cinnamon myrtle (Backhousia myrtifolia) for their complex phytochemical profile and antioxidant potential. LC-ESI-QTOF-MS/MS was applied for screening and characterizing these Australian myrtles' phenolic compounds and the structure-function relation of phenolic compounds. This study identified 145 and quantified/semi-quantified 27 phenolic compounds in these Australian myrtles. Furthermore, phenolic contents (total phenolic content (TPC), total condensed tannins (TCT), and total flavonoids (TFC)) and antioxidant potential of phenolic extracts from the leaves of Australian myrtles were quantified. Aniseed myrtle was quantified with the highest TPC (52.49 ± 3.55 mg GAE/g) and total antioxidant potential than other selected myrtles. Catechin, epicatechin, isovitexin, cinnamic acid, and quercetin were quantified as Australian myrtles' most abundant phenolic compounds. Moreover, chemometric analysis further validated the results. This study provides a new insight into the novel potent bioactive phenolic compounds from Australian myrtles that could be potentially useful for functional, nutraceutical, and therapeutic applications.

Efficacy of Lemon Myrtle Essential Oil as a Bio-Fungicide in Inhibiting Citrus Green Mould.

The effectiveness of lemon myrtle (LM) (Backhousia citriodora) essential oil (EO) was investigated to combat Penicillium digitatum by in vitro agar diffusion and vapour assay and in artificially infected oranges. The main constituent of LM EO was revealed as citral when analysed in gas chromatography-mass spectrometry. Pure citral was also included in the experiment for comparison. The in vitro fungal growth was significantly inhibited by LM EO at 1, 2, 3, 4 and 5 µL per disc while complete growth inhibition by both the pure citral and LM EO occurred at 4 and 5 µL per disc. Inoculated fruits treated by dipping in 1000 µL L-1 LM EO solutions for 5, 10, 15, 30 and 120 s showed significantly lower fungal wounds compared to control. While longer dipping times led to some rind injuries, fruits with a 5 and 10 s dip were found free from any injury. The evaluation after dipping and storage confirmed that the fruits maintained the sensory attributes and were not compromised by the incorporation of the essential oil. The results of this study indicate that LM EO can be a promising alternative to synthetic fungicides for preserving the quality of citrus fruits during storage.

Essential oil of lemon myrtle (Backhousia citriodora) induces S-phase cell cycle arrest and apoptosis in HepG2 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Lemon myrtle (Backhousia citriodora F.Muell.) leaves, whether fresh or dried, are used traditionally in folk medicine to treat wounds, cancers, skin infections, and other infectious conditions. However, the targets and mechanisms related to anti-cancer effect of lemon myrtle are unavailable. In our study, we found that the essential oil of lemon myrtle (LMEO) showed anti-cancer activity in vitro, and we initially explored its mechanism of action. MATERIALS AND METHODS: We analyzed the chemical compositions of LMEO by GC-MS. We tested the cytotoxicity of LMEO on various cancer cell lines using the MTT assay. Network pharmacology was used also to analyze the targets of LMEO. Moreover, the mechanisms of LMEO were investigated through scratch assay, flow cytometry analysis, and western blot in the HepG2 liver cancer cell line. RESULTS: LMEO showed cytotoxicity on various cancer cell lines with values of IC50 40.90 ± 2.23 (liver cancer HepG2 cell line), 58.60 ± 6.76 (human neuroblastoma SH-SY5Y cell line), 68.91 ± 4.62 (human colon cancer HT-29 cell line) and 57.57 ± 7.61 µg/mL (human non-small cell lung cancer A549 cell line), respectively. The major cytotoxic chemical constituent in LMEO was identified as citrals, which accounted for 74.9% of the content. Network pharmacological analysis suggested that apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1), androgen receptor (AR), cyclin-dependent kinases 1 (CDK1), nuclear factor erythroid 2-related factor 2 (Nrf-2), fatty acid synthase (FASN), epithelial growth factor receptor (EGFR), estrogen receptor 1 (ERα) and cyclin-dependent kinases 4 (CDK4) are potential cytotoxic targets of LMEO. These targets are closely related to cell migration, cycle and apoptosis. Notley, the p53 protein had the highest confidence to co-associate with the eight common targets, which was further confirmed by scratch assay, flow cytometry analysis, and western blot in the HepG2 liver cancer cell line. LMEO significantly inhibited the migration of HepG2 cells in time-dependent and dose-dependent manner. Moreover, LMEO caused a S-phase blocking on HepG2 cells and promoted apoptosis in the meanwhile. Western blot results indicated that p53 protein, Cyclin A2 and Bax proteins were up-regulated, while Cyclin E1 and Bcl-2 proteins were down-regulated. CONCLUSION: LMEO showed cytotoxicity in various cancer cell lines in vitro. Pharmacological networks showed LMEO to have multi-component and multi-targeting effects that are related to inhibit migration of HepG2 cells, and affect cell cycle S-phase arrest and apoptosis through modulation of p53 protein.

Lemon Myrtle (Backhousia citriodora) Extract and Its Active Compound, Casuarinin, Activate Skeletal Muscle Satellite Cells In Vitro and In Vivo.

Sarcopenia is an age-related skeletal muscle atrophy. Exercise is effective in improving sarcopenia via two mechanisms: activation of skeletal muscle satellite cells (SCs) and stimulation of muscle protein synthesis. In contrast, most nutritional approaches for improving sarcopenia focus mainly on muscle protein synthesis, and little is known about SC activation. Here, we investigated the effect of lemon myrtle extract (LM) on SC activation both in vitro and in vivo. Primary SCs or myoblast cell lines were treated with LM or its derived compounds, and incorporation of 5-bromo-2'-deoxyuridine, an indicator of cell cycle progression, was detected by immunocytochemistry. We found that LM significantly activated SCs (p < 0.05), but not myoblasts. We also identified casuarinin, an ellagitannin, as the active compound in LM involved in SC activation. The structure−activity relationship analysis showed that rather than the structure of each functional group of casuarinin, its overall structure is crucial for SC activation. Furthermore, SC activation by LM and casuarinin was associated with upregulation of interleukin-6 mRNA expression, which is essential for SC activation and proliferation. Finally, oral administration of LM or casuarinin to rats showed significant activation of SCs in skeletal muscle (p < 0.05), suggesting that LM and casuarinin may serve as novel nutritional interventions for improving sarcopenia through activating SCs.

Backhousia citriodora F. Muell. (Lemon Myrtle), an Unrivalled Source of Citral.

Lemon oils are amongst the highest volume and most frequently traded of the flavor and fragrance essential oils. Citronellal and citral are considered the key components responsible for the lemon note with citral (neral + geranial) preferred. Of the myriad of sources of citral, the Australian myrtaceous tree, Lemon Myrtle, Backhousia citriodora F. Muell. (Myrtaceae), is considered superior. This review examines the history, the natural occurrence, the cultivation, the taxonomy, the chemistry, the biological activity, the toxicology, the standardisation and the commercialisation of Backhousia citriodora especially in relation to its essential oil.

Lemon myrtle extract inhibits lactate production by Streptococcus mutans.

Backhousia citriodora (lemon myrtle) extract has been found to inhibit glucansucrase activity, which plays an important role in biofilm formation by Streptococcus mutans. In addition to glucansucrase, various virulence factors in S. mutans are involved in the initiation of caries. Lactate produced by S. mutans demineralizes the tooth enamel. This study investigated whether lemon myrtle extract can inhibit S. mutans lactate production. Lemon myrtle extract reduced the glycolytic pH drop in S. mutans culture and inhibited lactate production by at least 46%. Ellagic acid, quercetin, hesperetin, and myricetin, major polyphenols in lemon myrtle, reduced the glycolytic pH drop and lactate production, but not lactate dehydrogenase activity. Furthermore, these polyphenols reduced the viable S. mutans cell count. Thus, lemon myrtle extracts may inhibit S. mutans-mediated acidification of the oral cavity, thereby preventing dental caries and tooth decay.

Antioxidant and anti-inflammatory activities of phenolic compounds extracted from lemon myrtle (Backhousia citriodora) leaves at various extraction conditions.

Lemon myrtle leaves were extracted with ethanol at different temperatures (25, 50, and 80 °C) and times (2, 4, 6, and 10 h) to examine the effect of extraction conditions on total polyphenol contents (TPC), total flavonoid contents (TFC), their antioxidant, anti-inflammatory activities, and amount of phenolic compounds. Under optimal extraction conditions (80 °C and 6 h), the values were 23.37%, 102.72 mg gallic acid equivalents (GAE/g dry basis), 23.37 mg rutin equivalents (RE/g dry basis), 83.31%, 60.13%, and 1.10% for yield, TPC, TFC, DPPH, ABTS radical scavenging activity, and reducing power, respectively. In addition, total amount of the phenolic compounds of extract was determined as 43.9 µg/g. The anti-inflammatory effect was determined in lipopolysaccharide-stimulated RAW 264.7 cells and inhibited the production of inflammatory mediators such as nitric oxide (NO). These results indicate that extracts of lemon myrtle leaves have potential as a valuable natural product with antioxidant and anti-inflammatory.

Pyrolysis of solid waste residues from Lemon Myrtle essential oils extraction for bio-oil production.

Solid waste residues from the extraction of essential oils are projected to increase and need to be treated appropriately. Valorization of waste via pyrolysis can generate value-added products, such as chemicals and energy. The characterization of lemon myrtle residues (LMR) highlights their suitability for pyrolysis, with high volatile matter and low ash content. Thermogravimetric analysis/derivative thermogravimetric revealed the maximum pyrolytic degradation of LMR at 335 °C. The pyrolysis of LMR for bio-oil production was conducted in a fixed-bed reactor within a temperature range of 350-550 °C. Gas chromatography-mass spectrometry showed that the bio-oil contained abundant amounts of acetic acid, phenol, 3-methyl-1,2-cyclopentanedione, 1,2-benzenediol, guaiacol, 2-furanmethanol, and methyl dodecanoate. An increase in pyrolysis temperature led to a decrease in organic acid and ketones from 18.09% to 8.95% and 11.99% to 8.75%, respectively. In contrast, guaiacols and anhydrosugars increased from 24.23% to 30.05% and from 3.57% to 7.98%, respectively.

Anti-inflammatory and anti-oxidative activities of lemon myrtle (Backhousia citriodora) leaf extract.

In this present study, we examined the anti-inflammatory and anti-oxidative properties of alcoholic lemon myrtle extract (LME). The total polyphenol and flavonoid content of LME were determined as 118.77 and 14.53 mg/g extract, respectively. LME showed anti-oxidative properties, such as 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) and 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) radical scavenging activity. The anti-inflammatory activities of LME were investigated using the lipopolysaccharide-stimulated murine macrophage RAW 264.7 cells. Pretreatment with LME was performed at non-cytotoxic concentrations of 10-100 µg/mL. LME inhibited the production of inflammatory mediators such as nitric oxide (NO). Enzyme-linked immunosorbent assay and reverse-transcriptase polymerase chain reaction (RT-PCR) revealed that pretreatment with LME suppressed the protein expression and mRNA levels of pro-inflammatory cytokines such as interleukin IL-6, and tumor necrosis factor (TNF)-α in a concentration-dependent manner, respectively. These results suggest that LME could be used as a potential therapeutic agent having potent anti-inflammatory effects that could be used to treat inflammatory bowel disease.

Effects of different drying methods on extractable phenolic compounds and antioxidant properties from lemon myrtle dried leaves.

Lemon myrtle (Backhousia citriodora) is one of the most commercially grown native herbs in Australia. This study aimed to evaluate the effects of different drying methods on phenolic compounds and antioxidant properties of lemon myrtle leaves to identify the most suitable drying conditions. The drying methods include hot air drying, vacuum drying, microwave drying, sun drying, shade drying and freeze drying. The results showed that drying conditions significantly (p < 0.05) affected the retention of total phenolic content (TPC), total flavonoids (TFC), proanthocyanidins, gallic acid, hesperetin, and antioxidant properties of lemon myrtle leaves. The optimal conditions for hot air drying and vacuum drying are 90 °C for 75 min and 90 °C for 120 min, respectively; whereas optimal drying conditions for microwave drying are 960 W for 7 min, and the time required for sun drying and shade drying are 2 days and 12 days, respectively. The freeze dried leaves contained the highest level of TPC, TFC, proanthocyanidins, gallic acid and hesperetin (74.11 ± 2.87 mg GAE/g dw, 87.15 ± 2.70 mg CE/g dw, 123.49 ± 6.12 mg CE/g dw, 53.77 ± 0.22 mg/g dw and 38.99 ± 0.26 mg/g dw, respectively). The freeze dried leaves also contained higher antioxidant capacity as compared to other samples. No significant difference in phenolic compounds and antioxidant capacity was observed between tested other drying methods. Therefore, any of these methods can be selected for dehydration of lemon myrtle leaves for industrial purposes. However, microwave drying can be selected for drying of lemon myrtle leaves for an industrial scale as it was the most time and/or energy efficient technique.

Formulation, characterisation and antibacterial activity of lemon myrtle and anise myrtle essential oil in water nanoemulsion.

This study focussed on the formulation, characterisation of lemon myrtle (LM) and anise myrtle (AM) essential oil (EO) in water nanoemulsion and their antibacterial activity. The required hydrophilic lipophilic balance (rHLB) value of LM EO and AM EO was 14 and 12, respectively. The Central Composite Rotatable Design (CCRD) model produces the smallest droplet size and polydispersity index (PDI) for LMEO (d ≈ 16.07 nm; PDI ≈ 0.209) and AMEO (d ≈ 30.23 nm; PDI ≈ 0.216) at 1% EO and 10% surfactant mixture (Smix) ratio using ultrasonication for 5 min. Whereas, increased in EO, decrease in Smix concentrations and ultrasonication time produces higher droplet size of nanoemulsions. LMEO (LM-15, LM-17) nanoemulsions was clear and transparent compared to AMEO (AM-15, AM-17). All the selected nanoemulsions showed good stability at 4, 25 and 40 °C during storage, except LM-15 at 40 °C. LMEO nanoemulsion showed enhanced antibacterial activity compared to LMEO alone (P < 0.05).

A Rapid Screening Analysis of Antioxidant Compounds in Native Australian Food Plants Using Multiplexed Detection with Active Flow Technology Columns.

Conventional techniques for identifying antioxidant and phenolic compounds in native Australian food plants are laborious and time-consuming. Here, we present a multiplexed detection technique that reduces analysis time without compromising separation performance. This technique is achieved using Active Flow Technology-Parallel Segmented Flow (AFT-PSF) columns. Extracts from cinnamon myrtle (Backhousia myrtifolia) and lemon myrtle (Backhousia citriodora) leaves were analysed via multiplexed detection using an AFT-PSF column with underivatised UV-VIS, mass spectroscopy (MS), and the 2,2-diphenyl-1-picrylhydrazyl (DPPH(•)) derivatisation for antioxidants as detection methods. A number of antioxidant compounds were detected in the extracts of each leaf extract.

Anti-inflammatory potential of native Australian herbs polyphenols.

The anti-inflammatory potential of hydrophilic polyphenolic-rich extracts obtained from native Australian herbs: anise myrtle, lemon myrtle and Tasmannia pepper leaf, and a reference sample bay leaf, was evaluated using the lipopolysaccharide (LPS)-activated murine macrophage RAW 264.7 model. Pretreatment with all herbal extracts at non-cytotoxic concentrations reduced the LPS-induced protein levels of pro-inflammatory enzymes, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Concomitant decrease in accumulation of their products, prostaglandin E2 (PGE2) and nitric oxide (NO), respectively, was observed. A suppression of LPS-induced expression of COX-2 and iNOS and decrease of NO and PGE2 levels suggests potential anti-inflammatory properties of the extracts. Anise myrtle, lemon myrtle and bay leaf selectively inhibited COX-2 and iNOS enzymes, while Tasmannia pepper leaf extract exhibited a pronounced inhibitory activity toward COX-1 and was the least effective inhibitor of iNOS. Anise myrtle and lemon myrtle are potentially more efficient anti-inflammatory agents than Tasmannia pepper leaf.