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

1. Introduction

Native Australian fruits and other plants could be a rich source of new phytochemicals beneficial for human use. The indigenous people use these plants for medicinal purposes and to develop functional food products [1]. Phytochemicals are widely studied for their biological function, medicinal use, and synergistic therapeutic impact [2,3]. Plant secondary metabolites, mainly phenolic compounds, have attracted significant interest due to their remarkable potential for promoting health and well-being [4]. Their phenolic compounds and other phytochemicals exhibit a wide range of proven biological properties, making them valuable assets for improved health and well-being [5]. Among their bioactive constituents, phenolic compounds such as phenolic acids and flavonoids have emerged as key players, displaying a broad range of proven biological activities. Extensive research in recent decades has shed light on the role of phenolic metabolites in promoting health and preventing diseases. These phytochemicals exhibit vital biological activities, including cellular inhibition, modulation of signal transduction pathways, enzyme regulation, metal chelation, and potent scavenging of free radicals in cells [6]. Their multifaceted actions contribute to their significant impact on human health and well-being. Australian plants are widely employed in pharmaceuticals, nutraceuticals, and functional foods. With a rich history of traditional use, these botanical wonders have been revered for their ability to alleviate ailments such as aches, fractures, inflammation, and wound healing [6,7]. The growing interest in the comprehensive metabolite profiling of Australian native plants aims to explore the full potential of these natural resources. By understanding these plants' unique composition and bioactive properties, researchers can pave the way for developing novel pharmaceuticals, nutraceuticals, and functional foods with enhanced efficacy and safety profiles.

Moreover, their exceptional antioxidant and antimicrobial properties have applications in various industries, including cosmetics, pharmaceuticals, and food production [8]. Embracing diverse health-promoting roles, these medicinal herbs and plants have been recognized for their anti-diabetic, anti-inflammatory, neuroprotective, cardioprotective, and even anti-HIV properties, offering a broad spectrum of benefits [6,9]. Beyond their therapeutic applications, these botanical wonders also find use in the food and beverage industry. Their antioxidant and antimicrobial constituents make them valuable additions to food products, enhancing shelf life and promoting food safety. Myrtles, including aniseed myrtle, lemon myrtle, and cinnamon myrtle, are remarkable Australian native plants that have gained significant attention for their unique properties and versatile applications. Anise myrtle (Syzygium anisatum), also known as ringwood and aniseed myrtle, is valued for its peculiar aroma and licorice-like flavor. It has strong antimicrobial, antifungal, and antioxidant properties [10]. Due to its distinctive flavor, it is widely used in biscuits, cakes, teas, beverages, syrups, and other food products. Lemon myrtle (Backhousia citriodora) is valued for its potent lemon scent and revitalizing flavor. It is abundant with citral (a terpenoid), which has strong antimicrobial, antioxidant, and antifungal properties. Citral is also a flavoring agent, making lemon myrtle a potential additive in foods, beverages, and cosmetics [10,11]. Cinnamon myrtle (Backhousia myrtifolia), also known as grey myrtle, carrol, ironwood and neverbreak belongs to the Myrtaceae family and is valued due to its cinnamon-like aroma [12]. It is reported for its antioxidant, antimicrobial, antifungal, and antiseptic properties [12]. Metabolite profiling of the myrtle species aims to identify the bioactive substances that give them distinctive flavors and medicinal benefits. Researchers can learn more about the potential health benefits of these plants, including their antibacterial and antioxidant characteristics and support for the digestive and respiratory systems, by exploring the complex phytochemical composition of these medicinal plants [14]. These myrtles, being natural products, are increasingly used in the food and beverage sector. Chefs, herbalists, and academics are interested in them because of their unique flavors and health benefits, which have inspired ongoing research into their possible pharmacokinetics, bioavailability, and increased functional food uses.

Though some studies are conducted on Australian myrtles [10,13,14], however, complete profiling of these plants is still lacking due to their complex nature and the unavailability of pure standards of phytochemicals. The emerging interest in the food supply required detailed analytical identification, characterization, and quantification of these pigments to understand their role in food and human health collectively. Therefore, the primary aim of this research was to conduct a thorough analysis of these selected Australian native myrtles, with a specific focus on identifying phenolic and non-phenolic metabolites that hold significant importance for human health. To achieve this objective, state-of-the-art analytical techniques, including LC-ESI-QTOF-MS/MS, were employed to identify and accurately quantify bioactive phenolic compounds extracted from aniseed, lemon, and cinnamon myrtles. Advanced instruments successfully elucidated a detailed and intricate profile of phenolic and non-phenolic metabolites from these commercially utilized herbs and medicinal plants in Australia. LC-ESI-TOF-MS/MS is widely used for untargeted analysis of phytochemicals from complex extracts. This study will provide new opportunities for using these Australian myrtles in functional and nutraceutical products.

2. Results and Discussion

2.1. Quantification of Phenolic Contents from Australian Myrtles

Phenolic compounds in Australian myrtles were measured using TPC, TFC, and TCT assays. Secondary bioactive metabolites like flavonoids and phenolic acids are beneficial to health. They are considered multifunctional metabolites because they act as reducing agents, hydrogen atom donors, free radical scavengers, and metal chelators [15]. In this study, we investigated Australian myrtles including, aniseed myrtle (AM), cinnamon myrtle (CM), and lemon myrtle (LM), for bioactive phenolics. The results of quantified total phenolics are given in Table 1.

The highest TPC (52.49 ± 3.55 mg GAE/g) was measured in aniseed myrtle which is comparable to the previously quantified TPC of Australian myrtles leaves which were dried at 70°C for 105 min (52.47 ± 1.29 mg GAE/g), and 50°C for 315 min (51.63 ± 2.03 mg GAE/g). The second highest TPC range was observed in cinnamon myrtles (41.31 ± 3.23 mg GAE/g). Previously, phenolic compounds in native Australian lemon myrtle and Tasmanian pepper berries were found between 16.9 to 31.4 mg GAE/g. The highest flavonoid content was observed in aniseed myrtles, which have a value of 23.73 ± 2.32 mg QE/g. Cinnamon myrtles followed with a 19.41 ± 1.57 mg QE/g, while lemon myrtles have the lowest flavonoid concentrations, with 15.73 ± 1.34 mg QE/g. Compared to previously reported TFC in the lemon myrtle extract, which ranged from 15.36–33.09 mg GAE/g, were found comparable to Australian myrtles [16]. Previously, Saifullah et al. [14] was quantified the TPC (51.63 ± 2.03 mg/g) in ultrasound-assisted extraction with 50% acetone, which almost two-fold reported in this study. Our results of the TPC are comparable with the results of Konczak et al. [10]. The latter study also used 80% methanol, as we used in this study. The possible variations in results could be attributed towards different plant parts, the geographical location of the samples collected, and extraction conditions (solvent, time, and temperature) [17]. However, compared to the other plants mentioned, cinnamon myrtle has a substantially higher TCT (1.83 ± 0.53 mg CE/g). Aniseed and lemon myrtles have comparable TCT (1.52 ± 0.84 mg CE/g and 1.49 ± 0.14 mg CE/g), respectively. Overall, Australian myrtles are enriched with phenolic compounds. This is a first comprehensive study on quantifying the total polyphenols, flavonoids, and condensed tannins from Australian myrtles along with their antioxidant capacities and individual compounds using HPLC-MS/MS.

2.2. Quantification of Antioxidant Activities from Australian Myrtles

The antioxidant potential of myrtles was quantified using different in-vitro assays. The results are given in Table 2.

The FRAP, ABTS, PMA, FICA, ^•OH-RSA assays have mainly been utilized to determine antioxidant potential of polyphenolic compounds [18]. The ABTS assay is widely acknowledged as a cost-effective and versatile method for assessing the antioxidative potential of diverse samples, including edibles, dietary supplements, and biological specimens [19]. The ABTS assay has widespread recognition in the scientific community. Table 2 shows the ABTS of aniseed myrtles (148.16 ± 3.74 mg AAE/g), followed by cinnamon myrtles (142.66 ± 3.87 mg AAE/g). Lemon myrtles are observed to have the lowest ABTS which is (92.36 ± 0.75 mg AAE/g). The ABTS obtained for Maria Rita myrtle (Myrtus communis) leaves (135.7 ± 10.21 mmol TE/g DW) was observed. However, a previous study investigating ABTS for different cultivars of myrtle (Myrtus communis) leaves (for instance Giovanna (173.91 ± 3.74 mmol TE/g DW), Grazia (188.17 ± 5.83 mmol TE/g DW), Maria Antonetta (242.6 ± 10.93 mmol TE/g DW) and Sofia (245.03 ± 1.21 mmol TE/g DW), respectively have shown significantly higher results compared to the ABTS mentioned in our study [20]. The higher ABTS observed in previous studies compared to our study could be attributed to multiple factors. These include the utilization of myrtle leaves from cultivars with naturally higher levels of antioxidants, variations in environmental conditions during cultivation that favor increased antioxidant production, potential differences in the maturity of the leaves at the time of sampling, discrepancies in the extraction methods employed, and variations in the composition of the samples tested. A higher value of ABTS represents a greater antioxidant capacity of the tested sample [21].

The findings of this research determined that aniseed myrtle leaves had the highest FRAP of 14.30 ± 1.92 mg AAE/g, followed by cinnamon myrtles leaves with 9.21 ± 1.03 mg AAE/g. In contrast, lemon myrtles leaves exhibited the lowest FRAP with measurements of 4.60 ± 0.23 mg AAE/g. The FRAP obtained from (for instance Giovanna (173.91 ± 3.74 mmol TE/g DW), Grazia (188.17 ± 5.83 mmol TE/g DW), Maria Antonetta (242.6 ± 10.93 mmol TE/g DW) and Sofia (245.03 ± 1.21 mmol TE/g DW) were found relatively higher with the FRAP reported in our study [22]. The higher FRAP observed in the previous study compared to our present study could be attributed to variations in sample composition, specifically higher concentrations of antioxidant compounds. These discrepancies may arise from differences in the botanical source or cultivar of the samples, resulting in variations in the levels of polyphenols, flavonoids, and other antioxidant molecules. Additionally, variations in extraction methods, sample preparation, and analytical techniques utilized in the previous study may have contributed to the observed differences in FRAP values [17].

The Phosphomolybdenum antioxidative (PMA) assay is used to measure the molybdenum (VI) to molybdenum (V) reduction capacity induced by antioxidant phenolic compounds. This process gives rise to the development of a striking green molybdenum (V)/phosphate complex. According to the PMA assay findings, cinnamon myrtles leaves (17.09 ± 0.38 mg AAE/g) have substantially greater total antioxidant activity than the other listed myrtle leaves. In comparison, lemon myrtles leaves have considerably lower total antioxidant activity (10.57 ± 0.18 mg AAE/g), respectively. The highest FICA numbers of aniseed myrtle leaves (1.80 ± 0.10 mg AAE/g), followed by cinnamon myrtle leaves (1.63 ± 0.05 mg AAE/g) however, lemon myrtle leaves (0.98 ± 0.03 mg AAE/g) had the lowest FICA. It was discovered that the FICA derived in this study from lemon myrtle leaves was quite similar to the FICA reported for oregano/ lemon myrtle 0.42 to 0.75 g AAE/g) [23]. It is reported that the FICA in herbal compounds is crucial as it reduces the amount of transition metals required for lipid peroxidation, thereby lowering oxidative damage. By inhibiting the formation of a complex between ferrozine and ferrous ions, these selected plant extracts could offer defense against lipid peroxidation by reducing the activity of ferrous ions. The maximum value of ^•OH-RSA was measured in aniseed myrtle (23.62 ± 0.47 mg AAE/g), followed by cinnamon myrtle (21.62 ± 0.21 mg AAE/g), while the lowest was found in lemon myrtle (19.66 ± 0.31 mg AAE/g). The assessment of the scavenging capacity of plants was determined by analyzing ^•OH-RSA. Hydroxyl radicals (^•OH), known for their high reactivity, can cause lipid peroxidation, substantial biological damage, and DNA damage by attacking various molecules in the biological system. Therefore, these selected medicinal plant extracts’ capabilities to scavenge ^•OH radicals present a valuable defense mechanism against the biological harm inflicted by these free radicals.

2.3. Pearson Correlation Analysis between Phenolic Contents and Their Antioxidant Activities

It is widely studied that polyphenols have health benefits due to their potent antioxidant potential. A Pearson’s correlation was conducted between phenolic contents and their antioxidant activities to understand their relationship. The results of Pearson’s correlation are given in Table 3.

It is shown from the results of Table 3 that TPC was positively correlated (p ≤ 0.1) with the TFC (r = 0.99), FRAP (r = 0.92), ABTS (r = 0.92), FICA (r = 0.96) and OH-RSA (r = 0.99) while the TFC positively correlated with FRAP (r = 1.00), FICA (r = 0.93) and OH-RSA (r = 0.99). A biplot (Figure 1) was also conducted to investigate the association between active variables and active observations.

It is depicted that F1 shares 77.62%, while F2 only shares 22.38% variability between these results. It is also clearly indicated that aniseed myrtle has a higher concentration of TPC and TFC which play a significant role in antioxidant activities.

A higher concentration of OH groups in a flavonoid has previously been shown to be advantageous for biological activities. The structural arrangement of each ring, a catechol group in the B ring, its number of hydroxyl groups, and many double bonds in the C ring further affect each ring's ability to act as an antioxidant in extracts. Phenolic compounds and antioxidant activity have been linked in numerous studies of herbs and medicinal plants. In a previous study, we found a link between the antioxidant properties of spices and herbs and their phenolic concentration. Furthermore, two other investigations revealed a positive correlation between the biological activities of native Australian fruits and the phenolic content of other plants. Moreover, the loading variables of biplot indicate that Australian myrtles are unique to each other and have diversity of phenolic contents and their antioxidant activities.

2.4. LC-MS Analysis

The untargeted analysis of Australian aniseed, cinnamon and lemon myrtles was conducted using LC-ESI-QTOF-MS/MS. A total of 145 secondary metabolites were putatively identified using MS/MS spectra (Table 4). Base peak chromatograms and MS/MS spectra of some selected compounds identified in Australian myrtles are given in Figure S1 and Figure S3.

2.4.1. Phenolic Acids

Phenolic acids are widely distributed in fruits, vegetables, herbs, and medicinal plants. Phenolic acids can be further classified into different subclasses based on their chemical structure. Two well-known subclasses are hydroxybenzoic acids and hydroxycinnamic acids [24]. Hydroxybenzoic acids include compounds such as gallic acid and protocatechuic acid, while hydroxycinnamic acids encompass caffeic acid, ferulic acid, and p-coumaric acid, among others. We used LC-ESI-QTOF-MS/MS to identify 25 phenolic acids in our study (Table 4). Phenolic acid species are present in the leaves of all native Australian flora, including myrtles [25]. Hydroxybenzoic acids and derivatives (10) and hydroxycinnamic acids and derivatives (15) are two main classes of phenolic acids.

Hydroxybenzoic and hydroxycinnamic acids, unique subclasses of phenolic acids, offer distinct health benefits. These compounds overall exhibit potent antioxidant, anti-cancerous, and anti-inflammatory properties. Hydroxybenzoic acids have been found to possess potent antimicrobial properties, exhibiting inhibitory effects against various bacteria and fungi [26]. These compounds have shown promise in natural food preservation and the prevention of microbial infections. On the other hand, hydroxycinnamic acids have been studied for their potential neuroprotective effects, with evidence suggesting their ability to support brain health and protect against neurodegenerative diseases [27]. Their unique properties make hydroxybenzoic and hydroxycinnamic acids valuable contributors to antimicrobial strategies and neuroprotection.

Hydroxybenzoic Acids and Derivatives

Ten hydroxybenzoic acids were characterized in the current work. In aniseed and lemon myrtles, compound 1 (gallic acid 4-O-glucoside) and compound 3 (protocatechuic acid 4-O-glucoside) were identified in negative ionization mode at m/z 331.0675 and 315.0737, respectively [66]. Aniseed, cinnamon, and lemon myrtles were frequently found to contain compound 2 (gallic acid, C₇H₆O₅), compound 4 (protocatechuic acid, C₇H₆O₄) and compound 6 (p-hydroxybenzoic acid, C₇H₆O₃) with [M − H]⁻ at m/z 169.0145, 153.0195, and 137.0230, respectively. Gallic acid is a remarkable phytochemical widely found in nature, known for its exceptional properties. It exhibits robust anti-asthmatic, anti-allergic, anti-mutagenic, anti-inflammatory, anti-cancer, and neuroprotective effects, making it a highly valuable compound [28]. With its pervasive presence in various natural sources, gallic acid is a potent agent with multifaceted health benefits [29]. The following two compounds identified in positive ionization mode, compound 5 (m/z 123.0449) and compound 10 (m/z 481.1734) were discovered as benzoic acid, which is observed to be present in aniseed, cinnamon, and lemon myrtles and paeoniflorin found in lemon myrtles. Benzoic acids offer unique health benefits due to their distinct properties. They have been found to possess potent antimicrobial effects (inhibiting the growth of various bacteria and fungi) and antioxidant activity (helping to reduce oxidative stress and protect against cellular damage) [30,31,32]. Another compound 7 was characterized at m/z 167.0353, which was tentatively detected as vanillic acid in the negative mode in lemon and cinnamon myrtle leaves. Vanillic acid exhibits unique health benefits, including anti-cancerous and anti-inflammatory properties, and antioxidant, anti-aging, antimicrobial, anti-ulcerogenic, and anti-depressant qualities [33]. Its diverse range of potent health advantages makes vanillic acid a valuable compound with promising therapeutic potential. While compound 8 (ellagic acid glucoside, C₂₀H₁₆O₁₃) was identified in aniseed myrtle, and compound 9 (syringic acid, C₈H₈O₅) was identified in all these selected myrtles, and both compounds were identified with at ESI⁻ m/z 463.0531 and 197.0521.

Hydroxycinnamic Acids and Derivatives

In this study, Compound 11 to 25 belongs to the class of hydroxybenzoic acids. The compound 11 with M − H]⁻ at m/z 337.0957 was tentatively identified as 3-p-coumaroylquinic acid predominantly found in cinnamon myrtle leaves. While four more compounds 13, 18, 19, and 21 were detected commonly in aniseed, lemon, and cinnamon myrtles and identified to be p-coumaric acid, 3-caffeoylquinic acid, ferulic acid and caffeic acid with the negative mode of ionization at m/z 163.0389, 353.0897, 193.0509, 179.0348, respectively. Extensive studies have explored the potential cardiovascular benefits of ferulic acid, including its ability to lower blood pressure, inhibit blood clot formation, improve lipid levels in the blood, and exhibits anti-cancer, antioxidant, anti-inflammatory, and antimicrobial properties [34]. Caffeic acid, a phenolic compound in various plant-based foods, offers unique health benefits due to its antioxidant and anti-inflammatory properties. Its potent antioxidant activity helps combat oxidative stress and reduces the risk of chronic diseases [35]. Additionally, caffeic acid exhibits anti-inflammatory effects by inhibiting the production of pro-inflammatory enzymes and cytokines, making it potentially beneficial for conditions characterized by inflammation, such as arthritis and inflammatory bowel disease [36]. Compounds 12, 14, 16, 17, 23, 24 and 25 with [M − H]⁻ at m/z 195.0669, 325.0927, 149.0606, 223.0610, 515.1193, 529.1365 and 359.0762 were putatively designated as dihydroferulic acid , p-coumaric acid 4-O-glucoside, cinnamic acid, sinapic acid, 1,5-dicaffeoylquinic acid, 1-caffeoyl-5-feruloylquinic acid and rosmarinic acid and all of these compounds were commonly detected in aniseed myrtles and lemon myrtles. Rosmarinic acid possesses many remarkable health benefits as its antioxidant properties shield against cellular damage induced by oxidative stress. At the same time, its anti-inflammatory effects help diminish inflammation within the body and its antimicrobial capabilities, bolstering the body's defense against harmful microorganisms [37,38]. Notably, its anti-inflammatory property extends to potentially serving as a natural allergy treatment [39]. Cinnamic acid is a truly remarkable compound with tremendous potential and diverse benefits, including antioxidant, antimicrobial, anti-diabetic, anti-cancer, anti-inflammatory properties [40,41,42]. Moreover, studies have suggested that cinnamic acid could serve as a natural sunscreen ingredient, effectively absorbing harmful UV radiation and providing protection for the skin, thereby contributing to its overall health and beauty [43]. Sinapic acid offers a range of unique health benefits, including potent antioxidant effects, anti-inflammatory properties, potential antimicrobial activity, neuroprotective potential, cardiovascular support, liver health promotion, and possible anticancer properties [44]. Two more compounds (20 and 22) were characterized at m/z 209.0811 and 281.0655, which were tentatively detected as 3,4-dimetoxycinnamic acid, p-coumaroyl malic acid in the positive mode in aniseed myrtle leaves. While compound 15 (2-S-glutathionyl caftaric acid, C₂₃H₂₇N₃O₁₅S) was identified in lemon myrtle with M − H]⁻ at m/z 616.1096.

2.4.2. Flavonoids

Flavonoids (C6-C3-C6), belonging to the abundant bioactive compounds found in plants and herbs, offer many health benefits. Renowned for their anti-mutagenic, anti-carcinogenic, anti-microbial, anti-cancer, anti-inflammatory, and antioxidative properties, flavonoids serve as essential components in, nutraceutical, medicinal, cosmetics, functional, and pharmaceutical applications [45]. Our study identified 28 flavonoids, including 23 flavonols, 11 flavanols, 10 flavanones, 29 flavones, and 10 Isoflavonoids.

Flavonols

In this current study, (compounds 26-48) were identified in aniseed, lemon and cinnamon myrtles which are considered to fall under the flavonols (also known as 3-hydroxyflavones) category in negative and positive ionization modes ([M − H]⁻/[M + H]⁺). Four compounds 26, 35, 37, 39 with [M − H]⁻ at m/z 579.1370, 331.0447, 465.1056 and 493.0971 was putatively designated as kaempferol 3-O-xylosyl-glucoside, 3'-O-methylmyricetin (laricitrin), dihydromyricetin 3-O-rhamnoside, and europetin 3-galactoside which was detected in only lemon myrtle. Compound 48 was characterized as 3,7-dimethylquercetin (C₁₇H₁₄O₇) with positive ionization mode at m/z 331.0833 in lemon myrtle leaves. Four more compounds (32, 34, 36, 44) were characterized as myricetin 3-O-arabinoside (C₂₀H₁₈O₁₂), myricetin 3-O-rutinoside (C₂₇H₃₀O₁₇), quercetin 3-O-glucosyl-xyloside (C₂₆H₂₈O₁₆), kaempferol 3,7,4'-O-triglucoside (C₃₃H₄₀O₂₁) at m/z 451.0878, 627.1541, 597.1460, and 773.2140 was identified only in aniseed myrtle. Compounds 43 and 45 were identified in lemon, cinnamon, and aniseed myrtle with a precursor ion at [M − H]⁻ m/z 301.0352 and 315.0530 was designated as quercetin (C₁₅H₁₀O₇) and isorhamnetin (C₁₆H₁₂O₇). Isorhamnetin contains potent anti-inflammatory and anti-cancer capabilities, advantages for the heart, decreased oxidative stress and inflammation and improved high cholesterol and blood pressure levels. In addition to having neuroprotective benefits, isorhamnetin may be used to treat neurological illnesses, including Alzheimer [46]. Quercetin, a potent flavonoid, that offers immune system boosting may have neuroprotective effects, promising anticancer properties, and anti-inflammatory effects [47]. Compound 38 was characterized as rutin (C₂₇H₃₀O₁₆) at m/z 611.1600 in aniseed, lemon, and cinnamon myrtle. Rutin, a bioactive flavonol, offers health benefits including its role as a potent antioxidant that helps combat oxidative stress, anti-inflammatory properties that assist in reducing inflammation within the body, and its potential to support cardiovascular health by promoting healthy blood vessels and circulation [48]. Six compounds (30, 31, 40, 41, 42) were identified at m/z 477.0657, 479.0815, 435.0926, 447.0948, 627.1579 in the negative mode they were tentatively categorized as quercetin 4'-O-glucuronide, isomyricitrin, quercetin 3-O-xyloside, quercetin 3-O-rhamnoside (quercitrin), and taxifolin 4',7-diglucoside in aniseed myrtle only. A metabolite of the flavonoid quercetin, which is present in various plant-based diets, is called quercetin 4'-O-glucuronide. The powerful antioxidant and anti-inflammatory characteristics of quercetin 4'-O-glucuronide, as well as its immune-stimulating and prospective therapeutic effects in treating diabetes, have all been reported [49]. Compounds 27, 29, 33 and 46 were designated as 3-methoxynobiletin (C₂₂H₂₄O₉), myricitrin 3-rhamnoside (myricitrin) (C₂₁H₂₀O₁₂), quercetin 3-O-glucoside (C₂₁H₂₀O₁₂) and 6-hydroxyquercetin (C₁₅H₁₀O₈) at [M − H]⁻ at m/z 431.1326, 463.0888, 463.0808 and 317.0302 in lemon myrtle and aniseed myrtle. Myricitrin, also known as myricitrin 3-rhamnoside possess antioxidant properties, anti-inflammatory effects and potential neuroprotective effects that support brain health [50]. Compounds 28 and 47 were identified at m/z 345.0638 and 403.1396 in the negative mode which were tentatively categorized as limocitrin and 3-methoxysinensetin in lemon myrtle and cinnamon myrtle. Limocitrin which is considered to be not common compound offers health benefits such as potent antioxidant activity and anti-inflammatory properties [51].

Flavanols

Compounds 49-59 belong to the flavanols, a subclass of flavonoids. They are renowned for their antioxidant properties and associated with various health benefits, such as improving heart health and cognitive function. 4'-O-methylepigallocatechin (compound 49) and (-)-epigallocatechin 7-O-glucuronide (compound 57) were putatively characterized in aniseed and lemon myrtles at m/z 319.0816 and 481.0988, respectively in negative ionization mode. Four more compounds (50, 55, 56, 58) are detected in negative modes of ionization ([M − H]⁻) at m/z 897.1892, 451.1239, 303.0863 and 441.0845 and were characterized as prodelphinidin trimer GC-GC-C, Catechin 3'-glucoside, 3'-O-methylcatechin and (-)-epicatechin 3-O-gallate, respectively and all compounds were commonly found in lemon and aniseed myrtles. Compound 59 (theaflavin 3,3'-O-digallate, C₄₃H₃₂O₂₀) was tentatively recognized with [M − H]⁻ at m/z 867.1367 in aniseed myrtle. Theaflavin offers distinct health benefits separate from others. It supports cardiovascular health by reducing LDL cholesterol levels, exhibits antiviral properties that can help fight against viral infections, and aids in weight management by promoting metabolism and fat oxidation [52]. Three more compounds (51, 53, and 54) were putatively characterized as (-)-epigallocatechin, epicatechin and (+)-catechin commonly found in aniseed myrtle, lemon myrtle and cinnamon myrtle with ([M − H]⁻) at m/z 305.0681, 289.0729 and 289.0729. Strong antioxidant activities of the (+)-catechin molecule help protect cells against oxidative stress and free radical damage.

Furthermore, (+)-catechin has been shown to have anti-inflammatory properties, and it may also improve cognitive performance and have a favourable influence on brain health [53]. Epicatechin has been studied for its potential cardiovascular benefits, including promoting healthy blood pressure levels and supporting cardiovascular health. Additionally, epicatechin exhibits antioxidant activity, and may have neuroprotective properties, potentially supporting brain health and cognitive function [54]. Compound 52 was characterized as cinnamtannin A2 with the chemical formula C₆₀H₅₀O₂₄ at m/z 1155.2775 in lemon and cinnamon myrtle.

Flavanones

Compounds 60-69 were also detected with the negative and positive mode of ionization. Three compounds 60 (neoeriocitrin, C₂₇H₃₂O₁₅), 65 (eriodictyol, C₁₅H₁₂O₆), and 68 (kaempferol 7-(6''-galloylglucoside, C₂₈H₂₄O₁₅), were found with negative mode of ionization at m/z 595.1684, 287.0563 and 599.1043 in aniseed and lemon myrtle leaves extract. Because of its potent anti-inflammatory and antioxidant actions, neoeriocitrin has the potential as a natural treatment for ailments involving inflammatory bowel disease and arthritis. Neoeriocitrin is also linked to neuroprotective properties that may lower the likelihood of cognitive decline and neurodegenerative illnesses. By reducing levels of cholesterol and blood pressure, it has a good effect on cardiovascular health as well [55]. Eriodictyol has been found to have potential anti-cancer properties, neuroprotective effects, anti-inflammatory effects, and antioxidant properties [56]. Compound 61 was identified at m/z 459.1301 in negative mode and putatively categorized as 6''-acetylliquiritin in cinnamon and lemon myrtle. A precursor ion of compound 62 (m/z 433.1134 and 433.1135) was identified in aniseed, lemon, and cinnamon myrtle as naringenin 7-O-glucoside in negative mode. Two more compounds (63 and 69) with positive ionization modes at m/z 581.1873 and 465.1398 were characterized as narirutin and hesperetin 5-glucoside in aniseed myrtle. Narirutin, found in citrus fruits, exhibits potent anti-inflammatory properties, making it beneficial for reducing inflammation. It also acts as an antioxidant, protecting against oxidative stress and potential cellular damage [57]. Compounds 64 was putatively recognized as (hesperetin 3'-O-glucuronide, C₂₂H₂₂O₁₂) at m/z 477.1023 in the negative mode in aniseed myrtle. Compound 66 (pinocembrin 7-O-benzoate, m/z 361.1073) was identified in cinnamon myrtle in a positive mode of ionization. 6-Geranylnaringenin (compound 67, C₂₅H₂₈O₅) was only tentatively known at m/z 407.1883 in negative ionization mode in aniseed and cinnamon myrtle.

Flavones

Five flavones (compounds 70-96) were detected in this study. Compounds 70, 72, 89 were characterized as diosmin (diosmetin 7-O-rutinoside), hibiscetin 3-glucoside and kaempferol-3-O-rhamnoside at m/z 383.1498, 497.0926 and 431.1041 in aniseed myrtle. Diosmin has been demonstrated to have positive benefits on vascular and skin health and anti-inflammatory, analgesic, anti-oxidant and anti-inflammatory properties [58]. Compound 71 (artocarpetin B, at ESI⁻ m/z 383.1498) was identified in lemon myrtle, aniseed myrtle and cinnamon myrtle. Compounds (73, 75, 78, 83, 85, 90, 91, 93, 95 and 96) was putatively recognized as kanzonol E, quercetin 3-(2-galloylglucoside), syringetin-3-O-glucoside, quercetin 3-(2''-galloylrhamnoside), 6-hydroxyluteolin 7-O-rhamnoside, kaempferol, apigenin 7-O-glucuronide, diosmetin 7-glucuronide, tricin and 3,5-diacetyltambulin at m/z 387.1600, 615.1026, 509.1261, 599.1037, 447.0945, 285.0414, 445.0771, 475.0873, 329.0684, and 427.1029 in the negative mode in lemon myrtle. Kanzonol E possesses antioxidant, anticancer and anti-inflammatory properties. Tricin exhibits antimicrobial activity, antioxidant properties, and potential anti-cancer properties [59]. Compound 80 was identified in lemon, cinnamon and aniseed myrtle with a precursor ion at [M − H]− m/z 421.1288 and was designated as (multijugin – C₂₄H₂₂O₇). Five more compounds including compound 74 (chrysoeriol 7-O-glucoside, C₂₂H₂₂O₁₁), compound 82 (apigenin 6-C-glucoside, C₂₁H₂₀O₁₀), compound 86 (luteolin 8-C-glucoside (orientin,C₂₁H₂₀O₁₁)), compound 87 (isorhamnetin 3-galactoside, C₂₂H₂₂O₁₂), compound 92 (3,5-dimethylquercetin glucoside, C₂₃H₂₄O₁₂) were identified at m/z 461.1076, 433.1134, 447.0945, 477.1047 and 491.1211 in negative mode and observed to be found in lemon myrtle and aniseed myrtle. Compound 81 (rhoifolin, at m/z 577.1523) was detected in lemon and cinnamon myrtles. Rhoifolin has many health advantages like lowering inflammation, safeguarding cardiovascular health, and improving mental function. Along with having therapeutic effects on diabetes, skin conditions, as well as liver function, It also has anti-allergic qualities and provides skin protection [60]. Hibiscetin 3-glucoside (compound 72, C₂₁H₂₀O₁₄), apigenin 6,8-di-C-glucoside (compound 77, C₂₇H₃₀O₁₅), 6-hydroxyluteolin 7-O-rhamnoside (compound 79, C₂₁H₂₀O₁₁) were tentatively detected at ESI⁺ m/z 497.0926, 595.1673 and 449.1084 in aniseed myrtle. Compounds 76, 78 and 84 showed [M + H]⁺ at m/z 495.0814, 509.1261 and 315.0878 were putatively identified as myricetin 3-glucuronide, syringetin-3-O-glucoside and velutin respectively in cinnamon myrtle and lemon myrtle. Velutin has demonstrated the potential in promoting skin health by protecting against UV-induced damage and supporting collagen synthesis. Velutin exhibits antimicrobial properties, inhibiting the growth of bacteria and fungi. Its versatile nature makes it a promising natural compound for skincare and antimicrobial applications [61]. Compound 94 (7-methoxyflavone, C₁₆H₁₂O₃) with positive ionization mode at m/z 253.0863 were characterized as 7-methoxyflavone in lemon and aniseed myrtles.

Chalcones and Dihydrochalcones

Compound 97 (dihydropedicin, at m/z 333.1341) was identified in positive mode as well as compound 99 (Phloretin, at m/z 273.0780) was discovered in negative ionization mode while both compounds were found in lemon, cinnamon, and aniseed myrtle. Previous research suggests that phloretin may have the potential as anti-inflammatory, antioxidant, skin-lightening and anti-aging properties, making it a promising ingredient in skin care products [62]. Compound 98 (phloretin 2'-O-glucuronide, C₂₁H₂₂O₁₁) and compound 100 (xanthohumol, C₂₁H₂₂O₅) were observed at m/z 449.1084 and 355.1552 in aniseed myrtle in negative and positive mode respectively. Xanthohumol has been found to possess potential chemo-preventive properties. Studies suggest that xanthohumol may help inhibit the initiation and progression of various types of cancer, making it a promising natural compound for cancer prevention and treatment [63]. Compounds 101 and 102 showed [M + H]⁺ and [M − H]⁻ at m/z 285.1130 and 567.1706 were putatively identified as 2'-hydroxy-4',6'-dimethoxychalcone and phloretin 2'-O-xylosyl-glucoside respectively in cinnamon myrtle only.

2.4.3. Isoflavonoids

Eleven isoflavonoid compounds (103-113) were discovered in this study. Compounds 103 ([M + H]⁺), 105 [M − H]⁻, 107 [M − H]⁻ at m/z 487.1245, 273.0752 and 431.0978 were putatively identified as 6''-O-acetylglycitin, 3',4',7-trihydroxyisoflavanone and daidzein 7-O-glucuronide respectively in aniseed and lemon myrtles. Compound 104 (3',4',7-Trihydroxyisoflavan, m/z 259.0974), compound 110 (violanone, m/z 317.0712) and compound 113 (dihydrobiochanin A, m/z 287.0913) were identified in positive mode in cinnamon myrtle. Dihydrobiochanin is potentially beneficial in menopausal symptom relief and hormone balance. Dihydrobiochanin also exhibits antioxidant properties, helping to combat oxidative stress and protect against cellular damage. Previous research also suggests that dihydrobiochanin may have potential neuroprotective effects, supporting brain health and reducing the risk of neurodegenerative diseases [64]. A precursor ion of compound 106 (m/z 273.1129) was identified in aniseed and cinnamon myrtle and aniseed myrtle while compound 108 (m/z 337.0717) was identified in aniseed myrtle, and these compounds were designated as 3'-O-methyequol and dolineone, respectively in positive mode. Compound 111, having chemical formula C₁₆H₁₂O₆ was identified at m/z 301.0712 in the positive mode, which was tentatively categorized as 3'-hydroxymelanettin in aniseed, cinnamon, and lemon myrtle.

2.4.4. Tannins

Twelve compounds from 114 to 125 are identified as tannins. Grandinin (compound 114) and 2-O-galloylpunicalin (compound 116) were putatively characterized in aniseed and cinnamon myrtles at m/z 1065.1090 and 933.0666, respectively in negative ionization mode. Grandinin provides promising potential as a natural vasodilator, helping to relax and widen blood vessels, which may contribute to improved blood flow and cardiovascular health. Additionally, grandinin exhibits antioxidant properties, protecting against oxidative stress and may have anti-inflammatory effects [65]. Four more compounds (115, 117, 119, 121) are detected in negative modes of ionization ([M − H]⁻) at m/z 609.1265, 359.0978, 633.0742 and 577.1373 were characterized as prodelphinidin B3, glucosyringic acid, corilagin and procyanidin B2, respectively and all compounds were commonly found in lemon and aniseed myrtles. Corilagin has shown potential as a natural anti-aging compound. Studies suggest that corilagin may help to reduce the formation of advanced glycation end-products (AGEs), which are associated with aging and age-related diseases. Its distinct ability to target AGEs sets it apart and highlights its potential as a valuable ingredient in anti-aging formulations [66]. Compound 122 (punicafolin, C₄₁H₃₀O₂₆) was tentatively recognized with ([M + H]⁺) at m/z 939.1111 in cinnamon myrtle. Punicafolin has the potential to support gut health. Studies suggest that Punicafolin may help to promote healthy gut microbiota by selectively inhibiting harmful bacteria while promoting the growth of beneficial bacteria. Its distinct ability to positively influence gut health sets it apart and highlights its potential as a valuable natural compound for digestive wellness [67]. Two more compounds (118 and 120) were putatively characterized as kurigalin and potentillin commonly found in aniseed myrtle with ([M − H]⁻) at m/z 635.0905 and 935.0866. Potentillin acts as a natural immune modulator. Studies suggest that potentillin may help to regulate and strengthen the immune system, enhance its response against pathogens, and promote overall immune health [68]. Compound 123 (procyanidin trimer C1) and compound 125 (ellagic acid) were tentatively identified in lemon, aniseed, and cinnamon myrtles at m/z 865.1981 and 300.9985 in negative ionization mode. Ellagic acid stands out as possessing the potential to promote skin health. Studies suggest that ellagic acid may help to reduce skin aging signs, such as wrinkles and fine lines, by protecting against UV-induced damage and promoting collagen synthesis. It also possesses anti-inflammatory and antioxidant properties [69]. Compound 124 (procyanidin B2 3-gallate) was considered at m/z 729.1460 in negative mode from lemon myrtle.

2.4.5. Stilbenes

Based on MS/MS data, three stilbene compounds were discovered in this investigation in the positive mode of ionization. Compound 126 was identified at m/z 303.1236 and putatively categorized as 3'-hydroxy-3,4,5,4'-tetramethoxystilbene found in aniseed and cinnamon myrtle. A precursor ion of compound 127 (m/z 231.1024) was identified in cinnamon myrtle as dihydroresveratrol. One more compound 128 at m/z 391.1393 was characterized as polydatin, observed in lemon myrtle. Polydatin has shown potential as a natural cardioprotective compound, supporting cardiovascular health by reducing oxidative stress and inflammation [70]. Polydatin exhibits neuroprotective properties, potentially supporting brain health and may have anti-aging effects, promoting cellular longevity and potentially delaying the aging [71].

2.4.6. Lignans

The following two compounds identified in negative ionization mode, compound 129 (m/z 415.2121) and compound 132 (m/z 359.1492) were discovered as deoxyschisandrin and lariciresinol found in cinnamon and lemon myrtle. Another compound 133 was characterized at m/z 359.1496, which was tentatively detected as pinoresinol in the positive mode in aniseed and cinnamon myrtle leaves. Pinoresinol has shown potential in supporting liver health. Studies suggest that pinoresinol may help to promote liver function and protect against liver damage. It exhibits inhibitory effects on pro-inflammatory molecules, potentially reducing inflammation and associated conditions [72]. Compound 130 (lariciresinol-sesquilignan, C₃₀H₃₆O₁₀) with [M − H]⁻ at m/z 555.2228, which is observed to be present in lemon myrtles and compound 131 (silibinin, C₂₅H₂₂O₁₀) found in lemon and aniseed myrtles were identified with [M − H]⁻ at m/z 481.1131. Silibinin has been studied for its potential as a natural hepatoprotective compound, supporting liver health and protecting against liver damage. Silibinin exhibits antioxidant properties, helping to neutralize harmful free radicals, and may have anticancer effects, showing promise in inhibiting the growth of cancer cells and potentially contributing to cancer prevention and treatment [73,74].

2.4.7. Other Compounds

Twelve additional other compounds (compounds 134 to 145) were identified from the studied Australian myrtles. Psoralen (compound 134) and scopoletin (compound 143) were found in lemon and aniseed myrtle with [M − H]⁻ mode at m/z 185.0287 and 191.0350. Based on previous research, scopoletin has been demonstrated to have anti-inflammatory properties on the skin. It may also hold promise as a natural remedy for skin disorders including eczema and psoriasis. With its anti-depressant, antioxidant, anti-diabetic, antibacterial, anti-inflammatory, and anxiety-relieving qualities, scopoletin provides a number of potential health advantages [75]. Compounds 136 (caffeine) and 145 (vanillin) were tentatively identified in lemon and aniseed myrtle at m/z 195.0885 and 153.0552 in positive ionization mode. Four more compounds, quinic acid (compound 136 – C₇H₁₂O₆), pyrogallol (compound 137 – C₆H₆O₃), rosmanol (compound 139 – C₂₀H₂₆O₅), carvacrol (compound 144 – C₁₀H₁₄O) were identified commonly in lemon, aniseed, and cinnamon myrtles at m/z 191.0564, 125.0238, 345.1700 and 149.0970, respectively in the negative mode of ionization. Rosmanol has several health benefits including anti-diabetic, antibacterial, antioxidant and anti-inflammatory capabilities. For instance, one study has revealed that rosmanol could potentially have a natural remedy for hair loss since it might aid in encouraging hair follicle cell proliferation, which in turn can assist in stimulating hair growth. Rosmanol may also have the capacity to work as an organic treatment for aged skin [76]. Quinic acid (compound 135) has been shown to possess anti-ulcer activity, potentially assisting in the prevention and treatment of gastric ulcers by promoting the healing of the stomach lining and reducing inflammation. Quinic acid has been found to exhibit potential anti-diabetic properties by helping to regulate blood sugar levels and improve insulin sensitivity [77]. Compounds 140 and 141 were tentatively known as urolithin C and urolithin A by the precursor ion [M−H]⁻ at m/z 243.0293 and 227.0377 in aniseed myrtle only. Compound 138 (catechol) was considered at m/z 111.0448 in negative mode from lemon myrtle. Catechol has been shown to have potential anti-aging effects by promoting collagen synthesis and improving skin elasticity, leading to reduced wrinkles and improved skin appearance. Catechol also possesses anti-cancer and anti-microbial properties [78]. Compound 142 (carnosol C₂₀H₂₆O₄) at ESI⁺ m/z 331.1905 was tentatively characterized in cinnamon myrtles.

2.5. Distribution of Metabolites in Australian Myrtles

The complex distribution of hundreds of phenolic compounds in various samples could be computed using the Venn diagram. In this context, we conducted Venn diagrams of total number of phenolic compounds (A), total number of phenolic acids (B), total number of flavonoids (C), and total number of other compounds (D) in aniseed myrtle (AM), cinnamon myrtle (AM), and lemon myrtles (LM). The results of the Venn diagram are given in Figure 2.

Aniseed and lemon myrtles have the highest number of unique phytochemicals (22% and 18%, respectively). 17% were identified in all three medicinal plants while 27% overlapped in aniseed and lemon myrtles (Figure 2A). Aniseed and lemon myrtles have some percentage of phenolic acids (12%), while 35% overlap between these two plants. 31% overlapped in all these plants (Figure 2B). Aniseed myrtle has the highest percentage of unique flavonoids (27%), while 25% overlapped in these two plants (Figure 2C). Other compounds were identified in the highest percentage which is 14% in lemon myrtle (Figure 2D) while 14% were also overlapped in aniseed myrtle and cinnamon myrtle. 19% of other compounds overlapped in all three selected myrtles. Overall, these selected Australian myrtles contain a diverse range of phytochemicals that could be highly beneficial in the therapeutic and pharmaceutical industries.

2.6. Quantification/Semi-Quantification of Individual Phenolic Compounds from Australian Myrtles

This study quantified the most abundant phenolic compounds from aniseed, lemon, and cinnamon myrtles. The results of quantified phenolic compounds are presented in Table S1.

2.6.1. Phenolic Acids

Phenolic acids are ubiquitous parts of fruits, herbs, vegetables, and medicinal plants where they play an essential role in organoleptic properties, color, sensory attributes, and bioactive and nutraceutical potential. Twelve phenolic acids were quantified in the selected Australian myrtles. Sinapic acid (199.32 ± 11.84 μg/g) and p-coumaric acid (62.08 ± 7.49 μg/g) were only quantified in aniseed myrtle, cinnamon myrtle, and lemon myrtle, respectively. Moreover, all three myrtles were quantified in gallic acid, p-hydroxybenzoic acid, protocatechuic acid, chlorogenic acid, and coumaric acid 4-O-glucoside (Table 4). Previously, Konczak et al. [10] quantified chlorogenic acid (7.8 ± 0.1 mg/g) in anise myrtle. Gallic acid quantified in higher concentrations in lemon myrtle dried leaves ultrasound extracted with 50% acetone, as reported by Saifullah et al. [14]. Compound 5 (cinnamic acid) was quantified in aniseed myrtle (609.08 ± 23.32 μg/g) and lemon myrtle (312.03 ± 21.06 μg/g), respectively. Compound 6 (ferulic acid) was quantified in lemon myrtle (87.13 ± 5.14 μg/g) and aniseed myrtle (21.05 ± 1.32 μg/g), respectively. Syringic acid (compound 8, C₉H₁₀O₅) is the most abundant phenolic acid in aniseed myrtle (1135 ± 44.56 μg/g), lemon myrtle (231.01 ± 26.06 μg/g) and cinnamon myrtle (39.06 ± 6.05 μg/g), respectively. Caffeic acid (compound 9) was quantified in aniseed myrtle (161.12 ± 11.56 μg/g) and cinnamon myrtle (13.46 ± 2.81 μg/g), respectively. Caffeic acid possesses antioxidant, anti-diabetic, anti-inflammatory, anti-cancer, and antibacterial properties, among other potential health advantages [79].

2.6.2. Flavonoids and Non-Flavonoids

Australian myrtles are enriched with flavonoids, the largest class of polyphenols known for their highly beneficial effects, including antioxidant, anti-cancer, anti-inflammatory, anti-diabetic, anti-mutagenic, and neuroprotective effects [80,81]. Epicatechin was the most abundant in aniseed myrtle (4.15 ± 0.12 mg/g) and lemon myrtle (3.4 ± 0.1 mg/g), and cinnamon myrtle (1.2 ± 0.01 mg/g), respectively. Catechin was only quantified in lemon myrtle (4.9 ± 0.3 mg/g). The highest concentration (1174.14 ± 54.43 μg/g) of procyanidin B2 was quantified in lemon myrtle while the lowest concentration (110.32 ± 8.65 μg/g) was quantified in cinnamon myrtle.

The highest concentrations of myricetin 3-arabinoside (943.98 ± 35.19 μg/g) and quercetin (532.21 ± 19.37 μg/g) were quantified in aniseed myrtle. Lemon myrtle also measured with quercetin (301.92 ± 16.22 μg/g). A daily intake of quercetin is 5-40 mg making it the most prevalent flavonoid. Recently, it got much interest due to potent capacity to inhibit the SARS-CoV-2 3CLpro (protease that participates in the viral replication cycle) [82]. Isorhamnetin (C₁₆H₁₂O₇) was quantified in aniseed myrtle (145.32 ± 9.01 μg/g) and lemon myrtle (223.05 ± 11.19 μg/g), respectively. Isorhamnetin is a 3’-O-methyl derivative of quercetin that is measured up to 2.9 to 3.3 mg/g in buckthorn berries and dried parsley. Isorhamnetin reported for antioxidant, anti-cancer, anti-inflammatory, anti-ageing, neuroprotective and skin protective effects [46]. Kaempferol was quantified in aniseed myrtle (41.03 ± 6.13 μg/g), lemon myrtle (25.43 ± 2.42 μg/g) and cinnamon myrtle (24.51 ± 2.09 μg/g), respectively. Isovitexin (compound 23, apigenin 6-C-glucoside) was the only flavone compound quantified in aniseed myrtle (3206.13 ± 132.07 μg/g), lemon myrtle (303.87 ± 9.93 μg/g) and cinnamon myrtle (56.71 ± 7.18 μg/g), respectively. It has been reported for antioxidant, anti-inflammatory, anti-depressant, anti-cancer, anti-hypertensive, antiviral and neuroprotective effects [83]. Five tannins and pyrogallol were also quantified in Australian myrtles. Ellagic acid glucoside (742.02 ± 22.43 μg/g) and grandinin (65.16 ± 6.19 ug/g) were quantified in aniseed myrtle while grandinin (154.12 ± 9.37 μg/g), 2-O-galloylpunicalin (110.21 ± 11.54 μg/g), and potentillin (77.61 ± 6.91 μg/g) were quantified in cinnamon myrtle, respectively.

2.7. Heatmap Clustering, Debiased Sparse Partial Correlation Network and Chemometric Analysis

Heatmap Pearson’s clustering of quantified phenolic compounds in Australian myrtles are given in Figure 3A. It depicts that catechin and epicatechin are the most abundant phenolic compounds in lemon myrtle and cinnamon myrtle, respectively. A more intense red color represents a higher concentration while the light orange represents low concentration and blue color represents with no concentration or very low concentration (Table S1). Debiased sparse partial correlation network (Figure 3B), biplot (Figure 3C), partial least squares-discriminant analysis variable importance in projection (VIP) score (Figure 3D) of abundance phenolic compounds were conducted using the MetaboAnalyst 5.0 to visualize the distribution of these compounds in Australian myrtles.

The compounds with stronger association generated strong cluster with each other while the compounds for example resveratrol, sinapic acid, p-hydroxybenzoic acid, and caffeic acid have less association with other compounds (Figure 3B). Mainly flavonoids have stronger associations with each other. Furthermore, biplot indicates that Australian myrtles contains a diverse range of phenolic acids. For example, caffeic acid, kaempferol, myricetin 3-O-arabinoside, ellagic acid glucoside, syringic acid, isovitexin, cinnamic acid and quercetin are strongly associated with aniseed myrtle. Catechin, sinapic acid, p-hydroxybenzoic acid, ferulic acid, procyanidin B2, gallic acid and isorhamnetin are strongly associated with lemon myrtle. Potentillin, grandinin, chlorogenic acid, p-coumaric acid, 2-O-galloylpunicalin and quercetin-3-O-rhamnoside are strongly associated with cinnamon myrtle. Figure 3D conducted to evaluate the contribution of individual variables to the established discrimination model. The variables with VIP scores and p > 0.05 were selected as significant contributors indicated with red color. Aniseed and lemon myrtles have significant contribution with unique variables. Catechin, myricetin 3-O-arabinoside, isovitexin, (-)-Epicatechin 3-O-gallate, syringic acid, ellagic acid glucoside and procyanidin B2 have significant importance in Australian myrtles.

3. Materials and Methods

3.1. Sample Preparation and Extraction of Phenolic Compounds

Australian myrtles leaves (aniseed, lemon, and cinnamon myrtles) in dried form were purchased from Australian Super Foods (www.australiansuperfoods.com.au). The leaves were ground into fine powder using a laboratory grinder. The extraction procedure of Ali et al. [84] was employed: adding one gram of dried fine powder (1g sample) in 20ml of 80% analytical-grade methanol (acidified with 0.1% analytical-grade formic acid) in triplicate. The samples were put in a benchtop shaking incubator (ZWYR240, LABWIT, Australia) for 16 hours at 150 rpm and 10°C. The centrifugation (Allegra X-12R, CA, USA) was done at 8000×g for 15 minutes. The supernatant was filtered using a 0.45μm non-sterile PTFE syringe filter and stored at −20°C. All analyses were completed within seven days.

3.2. Measurement of Phenolic Contents in Australian Myrtles

3.2.1. Total Phenolics

To begin, 25µl of phenolic extracts were added into 96-well plates along with 200µl of water (Milli-Q) and 25% Folin-Ciocalteu reagent (v/v) in triplicate. The plate was then incubated at room temperature for 5 minutes in the dark. The reaction mixture was then given 25µl of 10% aqueous sodium carbonate (w/w), and it was left in the dark for 60 minutes at room temperature while the absorbance was measured at 765nm. Building a standard curve against gallic acid with concentrations ranging from 0 to 200μg/ml in ethanol allowed for the measurement of the TPC. Milligrams of gallic acid equivalents (mg GAE) per gram dry weight of the samples were used to record the results [17].

3.2.2. Total Flavonoids

In 96-well plates, 80 microliters of the sample extract were combined with 80 microliters of a 2% aluminium chloride aqueous solution and 120 microliters of a sodium acetate aqueous solution (50g/L) in triplicate. After 2.5 hours in dark storage at 25°C, the absorbance at 440nm of the reaction mixture was measured. The results were presented in milligrams of quercetin equivalents (QE, 0-50μg/ml) in methanol for every gram of sample dry weight.

3.2.3. Total Condensed Tannin

A 96-well plate containing 150µl of 4% vanillin solution and 25µl of sample solution was used to perform tannins assay in triplicate. Then, 25μl of 32% H₂SO₄ in methanol was also added and incubated for 15 minutes at 25°C. After 15 minutes of incubation at 25°C, the absorbance was measured at 500nm. All samples were measured in triplicate, and the quantification process involved creating a standard curve using a catechin solution in methanol-containing concentrations ranging from 0-1000μg/ml. The results were represented as mg catechin equivalents (CE) for each gram of dry weight.

3.3. Quantification of Antioxidant Activities

3.3.1. ABTS Radical Scavenging Assay

ABTS assay followed the methods described by Severo et al. [85] with modifications. A 22µl 140mM potassium persulfate solution and 1.25ml 7mM ABTS solution were used to prepare the ABTS dye. The reaction mixture was left in the dark for 16 hours to produce an ABTS⁺ dye. Ethanol was added to the solution to make the ABTS⁺ solution absorbance (0.70 ± 0.02) at 734nm. After that, a 96-well plate containing 10µl of sample phenolic extract and 290µl of ABTS⁺ solution was used to measure the absorbance at 734 nm for 6 minutes of incubation at 25°C in the dark. The radical scavenging capacity was measured by creating a standard curve for ascorbic acid concentrations in water ranging from 0-150µg/ml. The data were presented as mg AAE/g.

3.3.2. FRAP Assay

The FRAP dye was prepared by mixing 20mM ferric chloride, 10mM of TPTZ, and 300mM sodium acetate buffer in the ratio of 10:1:1 (v:v:v). A 96-well plate technique combined 20µl of sample phenolic extract with 280µl of the FRAP dye in triplicate. After 10 minutes of incubation at 37°C, the absorbance at 593nm of the reaction mixture was measured. The standard curve against 0-50µg/ml ascorbic acid in water was constructed to measure each sample's antioxidant capacity. The results are given as milligram ascorbic acid equivalents (mg AAE/g) for every gram of dry weight of the samples following Kiani et al. [86].

3.3.3. OH-RSA

After mixing a 50µl extract with 50µl of 6mM FeSO4.7H₂O and 50µl of 6mM H₂O₂ (30%) in triplicate, the mixture was incubated at 25°C for 10 minutes. 50µl of 6mM 3-hydroxybenzoic acid was added after incubation, and the absorbance was assessed at a wavelength of 510nm. To create a standard curve, ascorbic acid concentrations between 0 and 300µg/ml were employed. Data were then given in mg AAE/g following the method of Kiani et al. [86].

3.3.4. Fe²⁺ Chelating Activity

85µL of water, 50µl of 2mM ferrous chloride (with an additional 1:15 dilution in water), 50µl of 5mM ferrozine (with an additional 1:6), and a total of 15µl of extract were combined. The mixture was then incubated at 25°C for 10 minutes. The absorbance was then assessed at a 562nm wavelength. A standard curve was created using EDTA at values ranging from 0 to 50µg/ml, and the data was given as mg EDTA/g.

3.3.5. PMA Activity

The phosphomolybdate activity (PMA) of Australian myrtles was determined using the technique developed by Ali et al. [17], with some modifications. To conduct the PMA, 40 μL of each phenolic extract was combined with 260 μL of phosphomolybdate reagent, which consisted of 0.6 M H₂SO₄, 0.028 M sodium phosphate, and 0.004 M ammonium molybdate in a ratio of 1:1:1 (v/v/v). The solution was subjected to incubation at a temperature of 95 °C for a duration of 90 minutes. Subsequently, it was allowed to cool down to room temperature, and the absorbance was quantified at a wavelength of 695 nm. A calibration curve was created using concentrations ranging from 0 to 200 μg/mL of ascorbic acid, and the outcomes were reported in terms of milligrams of ascorbic acid equivalents per gram (mg AAE/g).

3.4. HPLC-MS/MS Quantification of Phenolic Compounds

The identification of phenolic compounds from Australian myrtles were performed using the previously described method by Ali et al. (2023). LC-ESI-Q-TOF-MS/MS (Accurate-Mass Q-TOF LC/MS Agilent 6520) equipped with Agilent HPLC 1200 series was used for the analysis of the untargeted phenolic metabolites. The screening of the phenolic extracts was conducted using a Synergi 4 μm Hydro-RP 80 Å LC column (250 × 4.6 mm) protected with C18 ODS (4.0 × 2.0 mm) guard column (Phenomenex, Torrance, California, United States). An aliquot of 10 μL from each phenolic extract was injected while the flow rate of mobile phase A (0.1% formic acid in Milli-Q water) and mobile phase B (0.1% formic acid in acetonitrile) was 600 μL/min with the following gradient; 0 min, 5% B; 10 min, 20% B; 15 min, 30% B; 20 min, 40% B; 25 min, 50% B; 30 min, 60% B; 40 min, 80% B; 45 min, 90% B; 50 min, 100% B; 55 min, 100% B; 58 min, 10% B; 60 min, 5% B. The following LC conditions: scan mode 50–1300 amu, capillary voltage (3500 V), nitrogen gas flow rate (9 L/min) at 325 °C, nebulization 45 psi and collision energies (10, 15 and 30 eV) were used in auto MS/MS mode. Agilent MassHunter Workstation Software Quality Analysis (version B.06.00) was used to identify and characterize phenolic metabolites with the help of Personal Compounds Database and Library (PCDL) for metabolites and GNPS, NIST, FooDB, MassBank libraries and databases using MS-DIAL. A total of 27 phenolic compounds were semi-quantified in this experiment. MS/MS spectra of 40 commercial standards were also acquired in this experiment. A mixture of 26 commercial standards was used to generate equations using LC-MS/MS [6].

3.6. Statistical Analysis

The results of this study were analyzed for analysis of variance and biplot using Minitab and XLSTAT-2019.1.3. Heatmap clustering, debiased sparse partial correlation (DSPC) network, partial least squares-discriminant analysis (PLS-DA) loading plot, partial least squares-discriminant analysis (PLS-DA) variable importance in projection (VIP) score, sparse partial least squares-discriminant analysis (sPLS-DA) loading plot of abundance phenolic compounds were conducted using the MetaboAnalyst 5.0.

4. Conclusions

In this study, we identified a total of 145 phytochemicals and quantified a total of 27 phenolic compounds in leaves of Australian myrtles. Aniseed myrtle was quantified with the highest total phenolic content and a higher antioxidant capacity than other selected myrtles. We identified several unique phytochemicals in these plants. Catechin, epicatechin, isovitexin, myricitrin, isomyricitrin, laricitrin, quercitrin, quercetin, ellagic acid arabinoside, ellagic acid glucoside, and myricetin 3-O-arabinoside are prominent and unique phenolic compounds in these Australian myrtles. These plants could be beneficial in formulating medicinal drugs. Further research should be conducted to investigate the effect of geographical and climatic conditions on phenolic in these Australian myrtles. More studies should be conducted to validate their bio-efficacy and absorption in the human body using cell culture and rodent models.