LC-MS/MS Characterization of Phenolic Metabolites and Their Antioxidant Activities from Australian Native Plants

Polyphenols are considered vital bioactive compounds beneficial for human health. The Australian flora is enriched with polyphenols which are not fully characterized yet. Thus, the main objective of this study was to identify and characterize the Australian native sandalwood nuts, wattle seeds, lemongrass, and old man saltbush for phenolic compounds and their antioxidant activities. In this study, we tentatively identified a total of 155 phenolic compounds including 25 phenolic acids, 55 flavonoids, 22 isoflavonoids, 22 tannins, 22 lignans, 33 stilbenes, 33 coumarins and derivatives, 12 tyrosols and derivatives, and 6 phenolic terpenes. The highest total phenolic content (TPC) (15.09 ± 0.88 mg GAE/g) was quantified in lemongrass, while the lowest TPC (4.17 ± 0.33 mg GAE/g) was measured in wattle seeds. The highest total flavonoid content (TFC) and total condensed tannins (TCT) were measured in lemongrass and wattle seeds, respectively. A total of 18 phenolic metabolites were quantified/semi-quantified in this experiment. Lemongrass contains a vast number of phenolic metabolites.


Introduction
Australian native plants offer a substantial potential source of new antioxidant chemicals for use in medicines or functional products [1,2]. Because of their long history, the indigenous people in the area have contributed to ongoing improvements in our understanding of the characteristics and potency of numerous plants and food provenance [3]. The predominant plants in southeastern Australia are wattle trees (Acacia sp.), and the indigenous people use their seedlings as a staple diet. Wattle (Acacia victoriae) seeds are one of the commercially accessible local spices, and many people consider them to be a standard item in the culinary business. The toasted and pulverized seeds are used in baked products, mustards, flour mixes, sweet sauces, dressings, and drinks because of their 'nutty' taste [3]. They were suggested for incorporation in diabetic and other specialty diets because they are abundant in proteins and possess a lower glycemic index [4]. Triterpenes saponins extracted from A. victoriae seeds have been shown to reduce the growth of cancerous cells and prevent nuclear factor-kappa B (NFkB) activity [5]. The edible Acacia species' seeds have proven to be both very nutrient-dense and safe to consume in both human clinical trials and laboratory investigations [4]. Wattle seeds are also identified as prickly wattle, gundabluie, elegant wattle, and bramble wattle. The flour of wattle seeds is pea-like flour and is also used in bread making. Due to its high protein profile, it has been consumed as a source of food in dairy products, seasonings, and bakery items for many years [4]. Multiple health advantages, including anti-cancer and anti-tumor properties, have been noted. There has not been much research into the content of phenolic and non-phenolic compounds in wattle seeds. Sandalwood (Santalum spicatum) nuts are precious gifts along with fragrant heartwood. These nuts are highly versatile with a unique flavor and amazing texture. Indigenous Australians used these nuts for the treatment of colds, stiffness, and traacetic acid (EDTA), ferrozine, ammonium molybdate, 3-hydroxybenzoic acid, catechin, iron (II) sulfate heptahydrate, potassium ferrocyanide(III), DPPH, 2,4,6 tripyridyl-s-triazine (TPTZ), and ABTS were purchased from Sigma Aldrich (Castle Hill, NSW, Australia). From Chem-Supply Pty Ltd. (Adelaide, SA, Australia), we purchased sodium carbonate anhydrous and hydrogen peroxide (30%), and we also purchased 98% H 2 SO 4 from RCI Labscan (Rongmuang, Thailand). Thermo Fisher Scientific Inc. supplied methanol, ethanol, acetonitrile, formic acid, glacial acetic acid, iron (III) chloride anhydrous, and HPLC and LC-MS-grade chemicals (Scoresby, VIC, Australia). Thermo Fisher Scientific provided 96-well plates for various in vitro bioactivities and antioxidant tests (Scoresby, VIC, Australia). HPLC vials (1 mL) were also purchased from Agilent equipment (Melbourne, VIC, Australia).

Extraction and Preparation of Samples
Australian native wattle seeds were purchased from Natif (www.natif.coma.au, accessed on 21 September 2021), native lemongrass from Tucker Bush (www.tuckerbush.com. au, accessed on 21 September 2021), while old man saltbush and sandalwood nuts were purchased from Australian super Foods (www.australiansuperfoods.com.au, accessed on 21 September 2021). Wattle seeds and sandalwood nuts were crushed and dried at 50 • C in the oven for 4 days and again ground and defatted with n-hexane before the phenolic extraction. Phenolic compounds were extracted by following the method of Ali et al. [11] in triplicate.

Measurement of TPC, TFC, and TCT
The TPC, TFC, and TCT of Australian native plants were measured by following the methods of Ali et al. [11], Zahid et al. [12] and Ali et al. [13] while all experiments were conducted in triplicate.

Measurement of Antioxidant Activities
The DPPH of selected plant extracts was measured by following the method of Zahid et al. [14] while the ABTS value of selected plants was quantified using the method of Bashmil et al. [15] in triplicate. The FRAP was measured by following the method of Ali et al. [13]. The • OH-RSA of plant extracts was measured by following the method of Chou et al. [16] with modifications. To do this, 50 µL of plant extract, 50 µL 6 mM aqueous solution of FeSO 4 . H 2 O, and 50 µL 6 mM H 2 O 2 solution in water were mixed and incubated at 25 • C for 20 min. After that, 50 µL of 3-hydroxybenzoic acid 6 mM solution in water was added and again incubated for 20 min before the absorbance reading at 510 nm. Ascorbic acid (0-300 µg/mL) in Milli-Q water was used as a reference standard to generate the equation. The PMA of plant extracts was measured by following the method of [13] with minor modifications. Briefly, 40 µL of plant extracts was mixed in 260 µL phosphomolybdate dye (0.6 M H 2 SO 4 in H 2 O, 28 mM trisodium phosphate solution in H 2 O and 4 mM ammonium molybdate solution in water were mixed in a ratio of 1:1:1 (v/v) to make phosphomolybdate dye) and incubated at 90 • C for 90 min in a water bath after properly wrapping the 96-well plates in aluminum foil. Then, the plates were cooled, and absorbance was recorded at 695 nm, while ascorbic acid (0-200 µg/mL) was used to generate a standard curve. The FICA of plant extracts was measured by following the method of [11].

Statistical Analysis
XLSTST-2019.1.3 was used for biplot analysis while the Minitab Program for Windows version was used for a one-way analysis of variance (ANOVA) followed by Tukey's honestly significant test.

Measurement of Total Polyphenols (TPC, TFC, and TCT)
Phenolic metabolites are vital for human health and are widely present in fruits and medicinal plants [9]. The screening and characterization of polyphenols has attracted much attention due to their wide use in the food, feed, pharmacological, and medicinal industries. The results for the measurement of total phenolic content (TPC), total flavonoid content, and total condensed tannins (TCT) are given in Table 1. The TPC represents phenolic acids, flavonoids, isoflavonoids, lignans, stilbenes, and other polyphenols. In this experiment, the highest TPC (15.09 ± 0.88 mg GAE/g) was measured in native lemongrass while the lowest TPC (4.17 ± 0.33 mg GAE/g) was measured in wattle seeds. The TPC of lemongrass was comparable to basil, thyme, bay, and nutmeg while the TPC of wattle seeds, sandalwood nuts, and old man saltbush was comparable to black cumin, black cardamom, cumin, fennel, black pepper, dill, parsley, and fenugreek [9,13]. Previously, Ee et al. [18] studied roasted and raw wattle seeds, and total polyphenols were measured from 3.53 ± 0.05 to 12.19 ± 0.37 mg GAE/g. Meanwhile, Hannachi et al. [19] reported an average of 6.32 mg GAE/g in wattle seeds from Tunisia. Furthermore, Konczak et al. [4] measured 0.8 ± 0.12 mg GAE/g total phenolics in Australian native wattle seeds, while Sommano et al. [20] reported 2.65 mg GAE/g total phenolics in Australian native wattle sees. Previously, Irfan et al. [21] measured total phenolic contents in lemongrass from Pakistan in the range of 32.9 to 61.2 mg GAE/g in acetone and ethanol extracts, while Godwin et al. [22] measured total phenolic content in lemongrass in the range of 1.3 to 7.3 mg GAE/g in cold and hot water. Moreover, Juntachote et al. [23] measured the total phenolic content in lemongrass in the range of 0.53 to 1.0 mg GAE/g in ethanolic fractions. The TPC of old man saltbush and wattle seeds is comparable to mountain pepper (5.91 ± 0.32 mg GAE/g) and tamarind (3.72 ± 0.12 mg GAE/g), respectively, as reported by Cáceres-Vélez et al. [24]. The variation in total phenolics reflects the diversity of phenolic compounds and their ability to reduce the F-C reagent. Moreover, the variations in total phenolics can be attributed to different extraction conditions in the current or later studies, type of solvent, solvent concentrations, solvent-to-sample ratio, time and temperature combinations, and geographical locations where these Australian native plants were grown [9,13].
Flavonoids are the vital and the most abundant plant secondary metabolites found in fruits, herbs, and medicinal plants. More than 10,000 flavonoids have been discovered in nature [8]. The highest TFC (3.07 ± 0.08 mg QE/g) was quantified in lemongrass while the lowest TFC (0.67 ± 0.05 mg QE/g) was in wattle seeds. The TFC of sandalwood nuts and old man saltbush was measured at 2.81 ± 0.21 mg QE/g and 2.32 ± 0.12 mg QE/g, respectively. Interestingly, the highest TCT (2.88 ± 0.10 mg CE/g) was measured in wattle seeds while the lowest TCT (1.12 ± 0.06 mg CE/g) was measured in sandalwood nuts. Previously, Ee et al., [18] measured the total flavonoid content of wattle seeds in the range of 0.23 to 1.93 mg CE/g under different roasting conditions. The flavonoids in wattle seeds are comparable to lentils, soy, common beans, and kidney beans which contained total flavonoids in the range of 0.85 to 1.14 mg QE/g [18,25]. The TFC of lemongrass was found comparable to flavonoids in cumin, basil, and dill while the value of sandalwood nuts and old man saltbush was found comparable to flavonoids in bay leaf, fenugreek, and black cumin [9,13]. Previously, a limited number of studies have been conducted to measure the total flavonoid content in these selected plants. The variations in total flavonoid content in the current and later studies may be attributed to the different factors mentioned above. Moreover, proper screening, identification, and characterization with LC-MS/MS can provide more reliable information regarding the presence of individual phenolic metabolites in these selected Australian native plants.

Antioxidant Potential of Australian Native Plants
Antioxidants are the chemical constituents in the human diet which are capable of deactivating the free radicals in the human body and these antioxidants are obtained from herbs, spices, fruits, and vegetables. In this study, the antioxidant potential of Australian native plants was measured through DPPH, ABTS, FICA, FRAP, PMA, and • OH-RSA. The results of quantified antioxidant activities are given in Figure 1 and Table S1.
DPPH is a low-cost assay and is used to estimate the ability of samples to scavenge the free radicals in biological systems as it works based on the ability to donate electrons or hydrogen ions. DPPH is a free radical which contains stable nitrogen in its center and reduces its bluish-purple color when mixed with the extract of selected plants. These are known as radical scavengers as any substance that causes this reaction can be categorized as an antioxidant. Table S1 indicates that the DPPH for lemongrass (18.73 ± 2.8 mg AAE/g) and sandalwood nuts (10.30 ± 0.9 mg AAE/g) were higher (p < 0.05) than other selected plants. As the flavonoid content of lemongrass was higher, this could be the reason for its higher DPPH. Many studies have reported that the radical scavenging activity of lemongrass is higher than that of many other medicinal plants. The ABTS is also a widely used assay for estimating the free radical scavenging capacity of plant extracts, including hydrophilic and lipophilic constituents, based on the polyphenols' hydrogen ion donating ability. ABTS + radical cation inhibition is based on the characteristic wavelength which is 734 nm [26]. The ABTS values of lemongrass (98.81 ± 6.19 mg AAE/g) and old man saltbush (74.76 ± 1.61 mg AAE/g) were estimated to be higher than those of other selected plants, while the lowest ABTS value was found in sandalwood nuts (37.72 ± 1.40 mg AAE/g).  DPPH is a low-cost assay and is used to estimate the ability of samples to scavenge t free radicals in biological systems as it works based on the ability to donate electrons hydrogen ions. DPPH is a free radical which contains stable nitrogen in its center and duces its bluish-purple color when mixed with the extract of selected plants. These a known as radical scavengers as any substance that causes this reaction can be categoriz as an antioxidant. Table S1 indicates that the DPPH for lemongrass (18.73 ± 2.8 mg AAE and sandalwood nuts (10.30 ± 0.9 mg AAE/g) were higher (p < 0.05) than other select plants. As the flavonoid content of lemongrass was higher, this could be the reason for higher DPPH. Many studies have reported that the radical scavenging activity lemongrass is higher than that of many other medicinal plants. The ABTS is also a wide used assay for estimating the free radical scavenging capacity of plant extracts, includi hydrophilic and lipophilic constituents, based on the polyphenols' hydrogen ion donati ability. ABTS + radical cation inhibition is based on the characteristic wavelength which 734 nm [26]. The ABTS values of lemongrass (98.81 ± 6.19 mg AAE/g) and old man saltbu (74.76 ± 1.61 mg AAE/g) were estimated to be higher than those of other selected plan while the lowest ABTS value was found in sandalwood nuts (37.72 ± 1.40 mg AAE/g).
The functional group of iron used in the biological system is responsible for the ab ity of iron chelation in Australian native plants. The highest value of FICA was found lemongrass (2.48 ± 0.24 mg EDTA/g) compared to other selected plants on the list. Lip peroxidation is responsible for catalyzing and FICA reduces the concentration of tran tion metals, which makes FICA a vital component. Chelating agents stabilize the me ions' oxidized form by forming s-bonds with metal, and in this way, the redox potent is reduced. Ferrous ions can increase lipid peroxidation by Fenton's reaction and this p cess is carried out by dismantling the lipid peroxides and hydrogen to free radicals. Wh ferrous ion decomposes lipid hydroperoxides into alkoxyl and peroxyl radicals, lipid p roxidation is increased. In this reaction, a complex bond is formed between ferrozine a ferrous ion, and the herbal extracts resist this complex formation. In this way, the herb extracts minimize ferrous ions and protect against oxidative damage. The functional group of iron used in the biological system is responsible for the ability of iron chelation in Australian native plants. The highest value of FICA was found in lemongrass (2.48 ± 0.24 mg EDTA/g) compared to other selected plants on the list. Lipid peroxidation is responsible for catalyzing and FICA reduces the concentration of transition metals, which makes FICA a vital component. Chelating agents stabilize the metal ions' oxidized form by forming s-bonds with metal, and in this way, the redox potential is reduced. Ferrous ions can increase lipid peroxidation by Fenton's reaction and this process is carried out by dismantling the lipid peroxides and hydrogen to free radicals. When ferrous ion decomposes lipid hydroperoxides into alkoxyl and peroxyl radicals, lipid peroxidation is increased. In this reaction, a complex bond is formed between ferrozine and ferrous ion, and the herbal extracts resist this complex formation. In this way, the herbal extracts minimize ferrous ions and protect against oxidative damage.
The Fe +3 -TPTZ complex reducing the ability of antioxidant compounds to Fe +2 -TPTZ complex in the biological system was evaluated through the FRAP assay [9,13]. The results showed that sandalwood nuts and lemongrass have significantly higher FRAP than the other selected Australian native plants (p < 0.05). The highest FRAP was found in sandalwood nuts (19.48 ± 3.04 mg AAE/g) and lemongrass (14.55 ± 1.32 mg AAE/g) while wattle seeds were found with the lowest FRAP (2.52 ± 1.97 mg AAE/g). Previously, A positive correlation of flavonoids with antioxidant activities indicated that flavonoids are the main antioxidant constituents [26]. Australian native plants can contain different reducing agents which can bind with free radicals to terminate or stabilize the chain reactions in the biological systems [27]. Thus, the higher reduction power of selected Australian plant extracts indicates their higher antioxidant capacity. The reduction capacity of molybdenum (VI) to molybdenum (V) is measured by using the Phosphomolybdenum antioxidative power assay (PMA assay). This process is performed with an antioxidant phenolic compound followed by the formation of a green molybdenum (V)/phosphate complex. It is indicated from the results that sandalwood nuts have higher PMA (14.43 ± 1.86 mg AAE/g) than other selected Australian native plants.
The anti-radical capacity of selected Australian native plants was also measured by using • OH-RSA. The highest value of • OH-RSA was found in lemongrass (104.34 ± 6.92 mg AAE/g) while the minimum value was found in wattle seeds (19.25 ± 0.92 mg AAE/g). Hydroxyl radicals ( • OH) are one of the most reactive species that are involved in DNA damage, lipid peroxidation, and biological damage by attacking each molecule found in the biological system. Protection from biological damage against free radicals could be prevented by the scavenging of • OH radicals.
It is reported that antioxidant activities vary in selected Australian native plants due to their complex mixture of bioactive compounds, and mainly depend on the method used for extraction. To determine the antioxidant potential of plants, there is a list of methods with their benefits and limitations [15,28]. Due to the complex nature of phenolic compounds and multiple mechanisms of reactions in the biological system, no defined method truly reflects the same antioxidant potential of these bioactive compounds [29]. Various studies have been conducted to estimate the antioxidant activities of different plants from different geographical locations [30][31][32][33][34][35] but studies on Australian native plants are limited. Total polyphenols in Australian native plants and their antioxidant capacities demonstrate that further research is needed to identify and verify the actual contribution of polyphenols towards antioxidant potential while eliminating or minimizing the contribution of nonphenolic metabolites.

Pearson Correlation and Biplot Analysi of Phenolic Contents and Antioxidant Activities
A Pearson correlation analysis was conducted between phenolic contents and antioxidant activities of Australian native plants given in Table 2. It indicates that a highly significant correlation of TPC was observed with DPPH (r = 0.98), RPA (r = 0.99), FICA (r = 0.97), and • OH-RSA (r = 0.98) while TCT negatively correlated with other antioxidant activities ( Table 2). These results indicate that mainly total phenolic content and total flavonoid content in selected Australian native plants are responsible for these antioxidant activities. The variation in antioxidant activities indicates the diversity of phenolic and non-phenolic compounds in these selected Australian native plants. It has been established that the antioxidant potential of flavonoids depends on the availability of an OH-group on the ring B and whether it can donate electrons or hydrogen atoms to a free radical in a biological system [11]. Furthermore, the mechanism of antioxidant reactions, experimental conditions, and the synergistic/antagonistic reactions of different compounds in the extract can affect the antioxidant activity and relationship with total phenolic and flavonoid contents [8,9]. In addition, a biplot analysis further elaborates the correlation between selected Australian native plants, phenolic contents, and their antioxidant activities ( Figure 2). It depicts that F1 has a higher contribution (72.96%) than F2, which has a lower contribution (23.51%) to the antioxidant activities of Australian native plants. Both components (F1 and F2) explained the total variability (96.47%) in these selected antioxidant activities of selected plants. Additionally, it indicates that a higher amount of total condensed tannins in wattle seeds and old man saltbush negatively correlated with the PMA, FRAP, and TFC. Overall, the TCT value did not observe any correlation with the other antioxidant activities. Moreover, the higher concentration of total phenolic content in lemongrass is observed a strong positive correlation with the RPA, • OH-RSA, DPPH, FICA, and ABTS activities while the higher concentrations of flavonoid contents in sandalwood nuts are positively correlated with the PMA, FRAP and RPA activities. The structure of flavonoids significantly affects antioxidant reactions. The presence of more OH-groups in flavonoids is favorable for antioxidant reactions, while antioxidant activity will also increase if the C3-C4 position in the ring B is replaced with OH-groups [11].
flavonoid contents [8,9]. In addition, a biplot analysis further elaborates the correlation between selected Australian native plants, phenolic contents, and their antioxidant activities ( Figure 2). It depicts that F1 has a higher contribution (72.96%) than F2, which has a lower contribution (23.51%) to the antioxidant activities of Australian native plants. Both components (F1 and F2) explained the total variability (96.47%) in these selected antioxidant activities of selected plants. Additionally, it indicates that a higher amount of total condensed tannins in wattle seeds and old man saltbush negatively correlated with the PMA, FRAP, and TFC. Overall, the TCT value did not observe any correlation with the other antioxidant activities. Moreover, the higher concentration of total phenolic content in lemongrass is observed a strong positive correlation with the RPA, • OH-RSA, DPPH, FICA, and ABTS activities while the higher concentrations of flavonoid contents in sandalwood nuts are positively correlated with the PMA, FRAP and RPA activities. The structure of flavonoids significantly affects antioxidant reactions. The presence of more OH-groups in flavonoids is favorable for antioxidant reactions, while antioxidant activity will also increase if the C3-C4 position in the ring B is replaced with OH-groups [11].

LC-MS/MS Identification of Bioactive Phenolic Metabolites from Australian Native Plants
The untargeted screening and characterization of phenolic metabolites in Sandalwood nuts, native lemongrass, old man saltbush, and wattle seeds were identified and characterized by using LC-ESI-QTOF-MS/MS, and MS/MS spectra were compared with libraries and published literature to confirm the phenolic metabolites ( Figures S1 and S2). A total of 155 phenolic metabolites were tentatively identified in these selected Australian native plants (Table 3).     LG * = compounds were identified through pure standards; ** = compounds were identified in both modes (positive and negative); lemongrass (LG), sandalwood nuts (SWN), wattle seeds (WS) and old man saltbush (OSB).

Phenolic Acids
Phenolic acids (hydroxybenzoic acids and hydroxycinnamic acids) are widely present in plants [36]. Their main applications are in cosmetics, medicinal industries, health, and pharmacology due to their antioxidant, anti-aging, and anti-microbial properties [37]. These are aromatic secondary metabolites that have health benefits. In this study, a total of 33 phenolic acids (7 hydroxybenzoic acids, 24 hydroxycinnamic acids, and 2 hydroxyphenyl acetic acids) were characterized by MS/MS which was used for the confirmation of their fragmentation patterns ( Table 3). The fragmentation pattern of phenolic acids generally is shown by the removal of carbon dioxide and hexosyl moiety from their parent ions.

Benzoic Acids and Their Derivatives
Benzoic acids and derivatives are also called benzenoids and are widely present in plants. A total of seven hydroxybenzoic acids were tentatively identified in these Australian native plants. Compounds 1 (protocatechuic acid), 2 (gallic acid), and 4 (p-hydroxybenzoic acid) produced fragment ions at m/z 109, m/z 125, and m/z 93 after the loss of CO 2 (44 Da) from the precursor ions, respectively [9,13] , respectively from the parent ion. Compound 18 was tentatively identified as ferulic acid. Ferulic acid is well known due to its antioxidant, anti-diabetic, anti-cancer, anti-aging activity, radioprotective effect, pulmonary protective, neuro-protective effect, and hypotensive effect [39]. Compound 10 at ESI − m/z 869.2495 generated product ions at m/z 693 (C 32 H 38 O 17 ) and m/z 517 (C 22 H 30 O 14 ) after the removal of one feruloyl unit and two feruloyl units, respectively from the precursor ion. Compound 10 was tentatively identified as 1,2,2 -triferuloylgentiobiose in wattle seeds. Previously, Passo Tsamo et al. [40] reported 1,2,2 -triferuloylgentiobiose in banana cultivars with the same MS/MS spectra.

Flavonoids
Flavonoids are the most abundant class of phenolic compounds. More than 10,000 flavonoids have been reported in nature [8]. We putatively identified a total of 62 flavonoids including 11 flavanols, 9 flavanones, 16 flavones 25 flavonols, and 4 chalcones and dihydrochalcones in selected Australian native plants (Table 3).  [41]. They are well-known for their antioxidant, anti-inflammatory, anti-cancer, and cardio-protective properties [42]. Catechins are the building blocks of condensed tannins commonly known as proanthocyanidins which have a wide range of pharmacological properties [43].

Flavones and Flavanones
In this context, a total of 24 flavonoids were putatively identified in these selected Australian native plants as flavones and flavanones (Table 3) [44]. Previously, neoeriocitrin was identified in the exocarpium citri grandis extract [44], while compound 51 (naringin) generated product ions at m/z 459, 313, and 271 through the neutral loss of C 8 H 8 O (120 Da), C 8 H 8 O plus rhamnoside (266 Da) and rhamnoside plus glucoside (308 Da) from the parent ions, respectively. Naringin was tentatively identified in lemongrass, old man saltbush, and sandalwood nuts. Compounds 58 (swertisin) and 71 (diosmin) were identified through MS/MS spectra of pure standards.

Isoflavonoids
In isoflavonoids, ring A (phenyl ring) is fused with C-ring (six-membered heterocyclic ring) and another phenyl B-ring at the C3 position, while the B-ring is substituted to the C2 position in flavonoids [45]. These plants' secondary metabolites contain a 3-phenylchroman skeleton which is biogenetically derived from the 2-phenylchroman (a basic skeleton of flavonoids) and more than 2400 isoflavonoids have been identified in plants [46]. It is the first time that we tentatively identified a total of 18 isoflavonoids in these selected Australian native plants.

Lignans and Stilbenes
Stilbenes are natural phytochemicals that contain a 1,2-diphenylethylene (a basic skeleton of stilbenoids) and have various pharmacological properties including antioxidant, antimicrobial, anti-cancer, anti-inflammatory, anti-diabetic, anti-aging, cardio-protective, and neuro-protective properties [48]. In this study, a total of five stilbenes were tentatively identified in selected Australian native plants (Table 3) Compound 121 was tentatively identified as a piceatannol and was only identified in wattle seeds. Previously, it was identified in dill leaves and fenugreek [9].
Lignans are a subgroup of non-flavonoid phenolic compounds which comprised two phenylpropane units (C6-C3). In this study, a total of 11 lignans were tentatively identified in selected Australian native plants (Table 3). Compounds 124 and 134 were only identified in wattle seeds and sandalwood nuts, respectively. Todolactol A (compound 124) at ESI − m/z 375.1443 was only identified in wattle seeds while compound 134 (7hydroxysecoisolariciresinol) at ESI − m/z 373.2017 was only identified in sandalwood nuts. Compounds 126 (7-oxomatairesinol), 127 (conidendrin), and 131 (schisandrin) were only identified in the old man saltbush, while compounds 128 (sesaminol 2-O-triglucoside) and 130 (1-acetoxypinoresinol) were only detected in lemongrass. Stilbenes and lignans are widely distributed in plants and have beneficial health properties.

Other Polyphenols
In this context, a total of 21 other polyphenols including 6 coumarins and derivatives, phenolic terpenes (3), tyrosols (4), hydroxybenzoketones (1), hydroxyphenylpropenes (1), cyclitol (1), and other polyphenols (5) were putatively identified in selected Australian native plants (Table 3). Umbelliferone (compound 137) was tentatively identified in lemongrass only at ESI − m/z 161.0246, which generated two product ions at m/z 133 and m/z 117 after the loss of CO (28 Da) and CO 2 (44 Da) from the precursor ion, respectively. Compounds 144, 145, and 146 generated product ions at m/z 287, 105, and 301 after the loss of CO 2 [M−H−44] − from their precursor ions, respectively. Compounds 144, 145, and 146 were tentatively identified as carnosic acid, carvacrol, and rosmanol, respectively. These compounds are phenolic terpenes which have been reported for their antioxidant activity [49]. Pyrogallol (compound 151) was identified in lemongrass, wattle seeds, and old man saltbush which produced fragment ions at m/z 107 and 97 after the loss of a water molecule (18 Da) and CO (28 Da), respectively from the precursor ion. Previously, pyrogallol was confirmed through LC-QTOF-MS/MS and NMR by Zhao et al. [50].

Distribution of Phenolic Metabolites in Australian Native Plants
The distribution of phenolic metabolites in Australian native lemongrass, old man saltbush, wattle seeds, and sandalwood nuts was achieved statistically by using a Venn diagram in R studio, given in Figure 3.

Other Polyphenols
In this context, a total of 21 other polyphenols including 6 coumarins and derivatives, phenolic terpenes (3), tyrosols (4), hydroxybenzoketones (1), hydroxyphenylpropenes (1), cyclitol (1), and other polyphenols (5) were putatively identified in selected Australian native plants (Table 3). Umbelliferone (compound 137) was tentatively identified in lemongrass only at ESI − m/z 161.0246, which generated two product ions at m/z 133 and m/z 117 after the loss of CO (28 Da) and CO2 (44 Da) from the precursor ion, respectively. Compounds 144, 145, and 146 generated product ions at m/z 287, 105, and 301 after the loss of CO2 [M−H−44] − from their precursor ions, respectively. Compounds 144, 145, and 146 were tentatively identified as carnosic acid, carvacrol, and rosmanol, respectively. These compounds are phenolic terpenes which have been reported for their antioxidant activity [49]. Pyrogallol (compound 151) was identified in lemongrass, wattle seeds, and old man saltbush which produced fragment ions at m/z 107 and 97 after the loss of a water molecule (18 Da) and CO (28 Da), respectively from the precursor ion. Previously, pyrogallol was confirmed through LC-QTOF-MS/MS and NMR by Zhao et al. [50].

Distribution of Phenolic Metabolites in Australian Native Plants
The distribution of phenolic metabolites in Australian native lemongrass, old man saltbush, wattle seeds, and sandalwood nuts was achieved statistically by using a Venn diagram in R studio, given in Figure 3.  The Venn diagram ( Figure 3A) indicates that a total of 25 (16%) unique phenolic compounds were identified in native lemongrass, while a total of 11 (7%), 12 (8%), and 12 (8%) unique phenolic metabolites were identified in wattle seeds, sandalwood nuts, and old man saltbush, respectively. This indicates that lemongrass has a more diverse range of phenolic metabolites that may contribute to its higher TPC, TFC, and antioxidant potential compared to other plant extracts (Table 1, Figure 1). A Venn diagram ( Figure 3B) depicts the total phenolic acids in selected Australian native plants. It was observed that lemongrass and sandalwood nuts have a greater variety (15.2%) of unique phenolic acids (5) as compared to old man saltbush and wattle seeds, in which only one (3.0%) and two (6.0%) unique phenolic acids were observed. This diagram further depicts that four (12.1%) of phenolic acids in both lemongrass and sandalwood nuts were similar while only two (6.0%) phenolic acids overlapped in all four plants. Figure 3C represents the total number of flavonoids in Australian native plants. It shows that the highest number of unique flavonoids, 13 (15.4%), was observed in lemongrass while the lowest number of unique flavonoids, 5 (6.0%), was in wattle seeds and sandalwood nuts. Moreover, sandalwood nuts contain a total of 8 (9.5%) unique flavonoids. A total of 10 (11.9%) flavonoids were overlapped in lemongrass and old man saltbush while a total of 5 (6.0%) were overlapped in lemongrass and wattle seeds, and wattle seeds and old man saltbush. A total of 3 (3.6%) flavonoids overlapped in all four selected plants. Figure 3D shows the total number of other phenolic metabolites in Australian native lemongrass, wattle seeds, sandalwood nuts, and old man saltbush. The highest numbers of unique other phenolic metabolites 7 (18.9%) were observed in lemongrass while the lowest numbers of unique phenolic metabolites 2 (5.4%) were observed in sandalwood nuts. Interestingly, a total of three (8.1%) other phenolic metabolites overlapped in lemongrass and wattle seeds, lemongrass and old man saltbush, wattle seeds and old man saltbush, and lemongrass, old man saltbush, and wattle seeds. It was observed that none of the other phenolic metabolites overlapped in all four plants. The Venn diagram is a useful, powerful, and versatile tool that can quickly analyze a large set of data and converts it into simple and digestible information.

Conclusions
These data indicate that the selected Australian native plants contained a diverse range of phenolic metabolites. A total of 155 phenolic metabolites (100 in lemongrass, 56 in sandalwood nuts, 64 in wattle seeds, and 70 in old man saltbush) were tentatively identified. Phenolic metabolites have significant health potential; therefore, these plants could be utilized in the pharmaceutical, medicinal, and food industries. Chlorogenic acid, pcoumaric acid, caffeic acid, protocatechuic acid, quinic acid, sinapic acid, gallic acid, quercetin 3-glucoside, pyrogallol, and cinnamic acid are abundant phenolic metabolites in selected Australian native plants. The significant antioxidant potential and in-depth phytochemical composition of these selected Australian native plants will further explore the use of these plants in medicinal, cosmetic, food, and feed industries.