Antioxidant, Alpha-Glucosidase Inhibition Activities, In Silico Molecular Docking and Pharmacokinetics Study of Phenolic Compounds from Native Australian Fruits and Spices

Native Australian fruits and spices are enriched with beneficial phytochemicals, especially phenolic compounds, which are not fully elucidated. Therefore, this study aimed to analyze native Australian mountain-pepper berries (Tasmannia lanceolata), rosella (Hibiscus sabdariffa), lemon aspen (Acronychia acidula), and strawberry gum (Eucalyptus olida) for phenolic and non-phenolic metabolites and their antioxidant and alpha-glucosidase inhibition activities. Liquid chromatography–mass spectrometry–electrospray ionization coupled with quadrupole time of flight (LC-ESI-QTOF-MS/MS) was applied to elucidate the composition, identities, and quantities of bioactive phenolic metabolites in Australian native commercial fruits and spices. This study identified 143 phenolic compounds, including 31 phenolic acids, 70 flavonoids, 10 isoflavonoids, 7 tannins, 3 stilbenes, 7 lignans, 10 other compounds, and 5 limonoids. Strawberry gum was found to have the highest total phenolic content (TPC—36.57 ± 1.34 milligram gallic acid equivalent per gram (mg GAE/g), whereas lemon aspen contained the least TPC (4.40 ± 0.38 mg GAE/g). Moreover, strawberry gum and mountain pepper berries were found to have the highest antioxidant and anti-diabetic potential. In silico molecular docking and pharmacokinetics screening were also conducted to predict the potential of the most abundant phenolic compounds in these selected plants. A positive correlation was observed between phenolic contents and biological activities. This study will encourage further research to identify the nutraceutical and phytopharmaceutical potential of these native Australian fruits.


Introduction
Diabetes mellitus is one of the leading causes of death around the globe [1] and is characterized by high blood glucose levels. Alpha-glucosidase (α-glucosidase) is the main enzyme with a significant role in hydroxylation, digestion, and absorption of sugars in the human body. Therefore, inhibiting α-glucosidase is an effective strategy for treating and minimizing type 2 diabetes. There is increasing interest in using natural sources to treat diabetes. Various nutraceuticals and bioactive compounds have been investigated to control/inhibit the complications of diabetes. Using phenolic metabolites is a therapeutic approach to suppressing the prevalence of pre-or post-diabetic conditions [1]. Therefore, detailed characterization and identification of phenolic metabolites is required to understand the potent role of polyphenols in food and human health.
Fruits, vegetables, herbs, spices, and medicinal plants contain large amounts of phytochemicals, including polyphenols. When they encounter living tissues, they exhibit a beneficial effect on human health [1]. Flavonoids are the largest subclass of polyphenols, with more than ten thousand compounds being reported [2]. According to nutritionists, foods high in polyphenols may reduce or remove the risk for certain malignancies, degenerative diseases, cardiovascular ailments, and chronic inflammation in humans [3].

Molecular Docking and Pharmacokinetic Properties of Abundant Phenolic Compounds
The pharmacokinetic properties of the most abundant phenolic compounds tentatively identified in the plants were predicted by following the methods of Ali et al. [16] and Daina et al. [17]. Oral bioavailability, absorption, distribution, metabolism, excretion, and toxicity of the abundant phenolic compounds were predicted. Moreover, in silico molecular docking was also conducted to predict the α-glucosidase potential of the selected phenolic compounds from native Australian fruits and spices, as described by Ali et al. [1]. Grid box dimensions were x = −12.95, y = −36.99, and y = 87.77 while docking ligands with a length lower than 20 Å. Phytochemicals, especially plants' secondary metabolites, are vital for human health [18]. Phenolic acids and flavonoids are critical secondary bioactive metabolites with various health benefits. They are considered multi-functional metabolites, as metal chelators, hydrogen atom donators, free radical scavengers, and reducing agents [18].
In this study, we investigated Australian mountain pepper berries, rosella, lemon aspen, and strawberry gum for phenolic and non-phenolic compounds. TPC, TFC, TCT, and TMAC quantified in these native Australian plants are given in Table 1.
Total phenols represent phenolic acids, flavonoids, stilbenes, lignans, coumarins and derivatives, tyrosols, and other small molecules. In this context, strawberry gum was found to have the highest TPC (36.57 ± 1.34 mg GAE/g) of the selected Australian native plants. The TPC of strawberry gum was comparable to the previously quantified TPC of Australiangrown thyme (43.16 ± 1.54 mg GAE/g), basil (39.91 ± 1.39 mg GAE/g), and allspice (40.49 ± 1.92 mg GAE/g) [10,18]. Previously, the levels of phenolic compounds in Australian native lemon myrtle and Tasmanian pepper berry were found to be in the range of 16.9 to 31.4 mg GAE/g [19]. Moreover, the TPC of mountain pepper berries was comparable to the TPC reported by Cáceres-Vélez et al. [20] and Vélez et al. [21]. The concentrations of phenolic contents in mountain pepper berry, rosella, and lemon aspen are 2 to 3-fold higher than in Australian-grown cherries [22]. Previously, Lukmanto et al. [23] also measured the TPC of 8.63 mg GAE/g, which is comparatively higher than our results. The TPC of strawberry gum is also comparable to that of villous amomum fruit (46.02 ± 1.12 mg GAE/g) and that of citron fruit (46.22 ± 1.01 mg GAE/g) reported by Liu et al. [24].  The highest TFC (15.69 ± 2.69 mg QE/g) was quantified in strawberry gum, and the lowest TFC (0.79 ± 0.04 mg QE/g) was quantified in lemon aspen. The highest TCT (8.05 ± 0.52 mg CE/g) was also measured in strawberry gum, and the lowest TCT (1.26 ± 1.13 mg CE/g) was measured in rosella. The TMAC was only measured in mountain pepper berry (0.17 ± 0.03 mg/g) and rosella (0.08 ± 0.02 mg/g). Previously, we measured higher amounts of total anthocyanins in the Davidson plum and quandong peach than berries [1]. Flavonoids are the most abundant class of phenolic compounds in fruits, herbs, and medicinal plants, and they have gained much interest due to their health properties. Previously, a limited number of studies have been conducted to investigate the total flavonoid contents in these plants. There are significant differences in total phenolics, and flavonoids found in each study conducted on these plants due to the aforementioned factors.

Biological Activities of Native Australian Fruits and Spices
Phenolic compounds include diverse antioxidant constituents present in plants that have various health effects. According to several studies, certain plants' antioxidant properties vary due to their diverse bioactive components and mostly depend on the extraction technique and method used to quantify them. Numerous studies have been carried out to estimate the antioxidant activities of plants from different geographical locations [25][26][27][28][29][30], but the information on native Australian plants is limited. Therefore, we conducted various antioxidant activity tests to understand the targeted antioxidant potential of native Australian fruits. Various antioxidant activity tests should help in understanding the potential of these native Australian herbs and medicinal plants.
In this study, seven in vitro antioxidant assays (DPPH, ABTS, FRAP, RPA, • OH-RSA, FICA, and PMA) were conducted, and α-glucosidase inhibition activity was tested, to measure the antioxidant and anti-diabetic potential of native Australian native mountain pepper berries, rosella, strawberry gum, and lemon aspen (Table S1, Figure 1). DPPH and ABTS are the widely used in vitro antioxidant assays for total antioxidant potential measurement of plant extracts based on scavenging the free radicals in the biological system. The highest DPPH (49.70 ± 3.21 mg AAE/g) was measured in strawberry gum, and the lowest DPPH (24.94 ± 0.70 mg AAE/g) was quantified in sea lemon aspen. ABTS + radical cation inhibition is based on the characteristic wavelength of 734 nm [31]. The ABTS values of strawberry gum (87.65 ± 3.17 mg AAE/g) and mountain pepper berries (85.60 ± 2.32 mg AAE/g) were higher than those of rosella (59.27 ± 1.50 mg AAE/g) and lemon aspen (46.18 ± 0.38 mg AAE/g). Some other studies also reported higher ABTS values for rosemary, oregano, and mint [18]. This indicates that strawberry gum has a higher antioxidant potential than mountain pepper berries, rosella, and lemon aspen. The Fe +3 -TPTZ complex, which reduces the antioxidant compounds' ability to form an Fe +2 -TPTZ complex in the biological system, was evaluated through the FRAP assay [10]. The FRAP values of strawberry gum (26.57 ± 3.10 mg AAE/g) and rosella (14.30 ± 1.92 mg AAE/g) were higher than those of the other selected fruits and spices (p < 0.05). Previously, the highest FRAP values were found in rosemary (17.21 ± 0.54 mg AAE/g) and oregano (10.72 ± 1.44 mg AAE/g).
Fenugreek was found to have the lowest value of FRAP (1.48 ± 1.21 mg AAE/g). Furthermore, Wojdyło et al. [32] also reported higher FRAP for rosemary than the other plants selected in our study.
DPPH and ABTS are the widely used in vitro antioxidant assays for total antioxidant potential measurement of plant extracts based on scavenging the free radicals in the biological system. The highest DPPH (49.70 ± 3.21 mg AAE/g) was measured in strawberry gum, and the lowest DPPH (24.94 ± 0.70 mg AAE/g) was quantified in sea lemon aspen. ABTS + radical cation inhibition is based on the characteristic wavelength of 734 nm [31].
Excessive amounts of different reactive oxygen species (ROS), such as hydrogen peroxide (H 2 O 2 ), hydroxy radicals ( • OH), and super-oxide radicals (O 2 • ), cause various pathologies. The • OH radicals cause lipid peroxidation and DNA damage due to high oxidative stress, and the daily consumption of antioxidant-rich fruits is crucial in order to protect the human body from these pathologies [18]. The highest • OH-RSA value (93.29 ± 2.20 mg AAE/g) was achieved by strawberry gum. This is vital because • OH scavenging inhibits lipid peroxidation by inhibiting the transition of oxidized metal ions [33,34]. The metal chelating ability of native Australian fruits and spices was estimated by using the ferrous ion chelating assay (FICA), and the highest FICA result (9.05 ± 0.27 mg EDTA/g) was achieved by strawberry gum. The principal antioxidant ingredients are flavonoids, according to a significant association between the antioxidant properties and flavonoids. These fruits and spices can contain different reducing agents which can bind with free radicals to terminate or stabilize the chain reactions in the biological systems [35]. Thus, high reduction power for a fruit extract indicates high antioxidant capacity. Free radicals can be produced by metabolic processes within bodily tissues and brought from outside sources such as food, medications, and pollution. Natural antioxidants are increasingly being used as food additives to neutralize free radicals. This is due to their scavenging abilities and the fact that they are all-natural, non-synthetic items that are well-liked by consumers. Furthermore, α-glucosidase inhibition activity of strawberry gum (81.01 ± 4.6 %) and mountain pepper berries (65.78 ± 5.01 %) was quantified higher than acarbose (standard), rosella, and lemon aspen. Previously, Syabana et al. [36] quantified the IC 50 of Cosmos caudatus (61.33 ± 1.21 µg/mL), Etlingera elaitor (53.13 ± 2.87 µg/mL), Pluchea indica (12.17 ± 0.18 µg/mL), and Syzygium polyanthum (11.76 ± 0.32 µg/mL). The inhibitory activity of strawberry gum (12.01 ± 1.2 µg/mL) was higher than the activity of Cosmos caudatus and Etlingera elaitor and comparable to the activity of Pluchea indica and Syzygium polyanthum ( Table S1). The inhibitory activity levels of rosella (79.09 ± 7.52 µg/mL) and lemon aspen (83.07 ± 9.03 µg/mL) were lower than those of these plants (Table S1). Moreover, the inhibitory activity of strawberry gum was comparable to that of the methanolic extract of Satureja cuneifolia (10.66 µg/mL) reported by Taslimi et al. [37].
Fruits, herbs, spices, and medicinal plants are used as antioxidant sources in the human diet because they inhibit or deactivate the free radicals in the body [38]. Generally, phenolic molecules are regarded as the active antioxidant components in fruits, herbs, and medicinal plants, thereby having potent health benefits. They act as metal chelators, anti-radicals, hydrogen-ion donators, and reducing agents in the biological system [10]. It has been reported that there are many methods to measure a plant extract's total antioxidant potential due to the diverse nature of antioxidant compounds, mainly phenolic constituents [1,18]. The plant's bioactive compounds, mainly polyphenols, depend on the type of cultivar, area, and climatic conditions. There are several techniques to assess the antioxidant capacities of bioactive phenolic metabolites, each with its advantages and disadvantages [11,14,19]. Generally, no approach measures the exact antioxidant capacity of bioactive phenolic compounds because of the complexity of phenolic compounds and the variety of processes of reactions in the human body [39]. These results demonstrate that further research is required to identify and quantify the individual phenolic compounds in these selected native Australian plants. Thus, LC-MS/MS was used to elucidate plant extracts' structure, composition, and bioactive metabolites. The proper quantification and identification of individual phenolic compounds via the process of LC-MS/MS in these plants might help make the essential role of these bioactive metabolites in antioxidant activities understandable.

Correlation Analysis
Correlation analysis was executed between the phenolic contents (TPC and TFC) of the Australian native herbs and their antioxidant activities generated by the eight different assays ( Table 2). It is observed that a positive correlation (p ≤ 0.1) of TPC was observed with the TFC (r = 1.00), TCT (r = 0.99), FRAP (r = 0.92), • OH-RSA (r = 0.97), and RPA (r = 0.99); the TFC had a significant positive correlation with TCT (r = 0.99), DPPH (r = 0.98), FRAP (r = 0.92), • OH-RSA (r = 0.97), and RPA (r = 0.99). This appears to show a direct association between the phenolic compounds in the strawberry gum and the antioxidant processes of peroxyl inhibition, ferric chelation, and free radical scavenging. There was a strong correlation of flavonoids with hydroxyl inhibition, but there were lesser ones with the free radical scavenging, the phosphomolybdate assay outcome, and ferric ion chelation activity. This also indicates the diversity of phenolic and non-phenolic metabolites present in the extracts of native Australian plants. This may be connected to the fact that the flavonoid's ability to operate as an antioxidant often depends on where the hydroxyl group is located on the B-ring and whether it can provide a free radical, either a hydrogen or an electron [1]. Additionally, the experimental conditions, the mechanisms of the antioxidant reactions, and the synergistic or antagonistic effects of various compounds present in the reaction mixture can all impact the associations between antioxidant activity and phenolic compounds [15,18].
A biplot ( Figure 2) exhibits that the higher TPC, TFC, and TCT in strawberry gum significantly contributed to all antioxidant activities except that shown by the phosphomolybdate assay. Furthermore, it is depicted that mountain pepper berries have higher concentrations of total monomeric anthocyanin than other plants. Interestingly, rosella and lemon aspen are negatively correlated with all biological activities, which indicates that these have low concentrations of phenolic compounds and flavonoids. Previously, it has been demonstrated that a greater number of OH groups in a flavonoid is favorable for biological activities. Furthermore, each ring's structural arrangement its number of hydroxyl groups, a catechol group in the B ring, and several double bonds in the C ring determine the antioxidant capacity of phenolic metabolites in extracts [40]. Numerous investigations of herbs and medicinal plants have shown a significant, positive association between phenolic content and antioxidant activity [41]. Previously, we reported a positive correlation between phenolic contents of herbs and spices and their antioxidant activities [10,18]. Additionally, two other studies showed that phenolic contents in native Australian fruits and other plants also had positive relationships with their biological activities [1,15].

LC-MS Analysis
Nutritionists and food scientists have concentrated on exploring the thorough characterization of fruits, spices, and medicinal plants in response to the growing interest in and understanding the antioxidant potential and associated health benefits of phenolic chemicals. The untargeted characterization and screening of phenolic compounds from Australian native fruits and medicinal plants (mountain pepper berries, strawberry gum, rosella, and lemon aspen) were achieved using LC-ESI-QTOF-MS/MS. In this context, a total of 143 phenolic and non-phenolic metabolites, including 31 phenolic acids, 70 flavonoids, 10 isoflavonoids, 7 tannins, 3 stilbenes, 7 lignans, 10 other compounds, and 5 limonoids, were tentatively characterized through the analysis of their MS/MS spectra (Table 3, Figures S1 and S2).

Phenolic Acids
Phenolic acids are diverse plant metabolites from the secondary class produced via the phenylpropanoid pathway by shikimic acid [42]. They are broadly utilized in beauty, health, pharmacology, and medicinal industries due to their anti-aging, antioxidant, antimicrobial, anti-cancer, cardio-protective, antitumor, and anti-inflammatory properties [43]. Australian fruits and other plants also had positive relationships with their biological activities [1,15].

LC-MS Analysis
Nutritionists and food scientists have concentrated on exploring the thorough characterization of fruits, spices, and medicinal plants in response to the growing interest in and understanding the antioxidant potential and associated health benefits of phenolic chemicals. The untargeted characterization and screening of phenolic compounds from Australian native fruits and medicinal plants (mountain pepper berries, strawberry gum, rosella, and lemon aspen) were achieved using LC-ESI-QTOF-MS/MS. In this context, a total of 143 phenolic and non-phenolic metabolites, including 31 phenolic acids, 70 flavonoids, 10 isoflavonoids, 7 tannins, 3 stilbenes, 7 lignans, 10 other compounds, and 5 limonoids, were tentatively characterized through the analysis of their MS/MS spectra (Table  3, Figure S1, Figure S2).

Benzoic acid and Its Derivatives
Benzoic acid is the simplest aromatic carboxylic acid and has a range of derivatives. Eight hydroxybenzoic acids in total were tentatively identified in these native Australian plants. Compounds 1, 3, and 8 produced fragment ions at m/z 169, 153, and 137 after the loss of the glycosyl moiety [M−H−162] − from the precursor ions, respectively. Compounds 1, 3, and 8 were tentatively identified as gallic acid 4-O-glucoside, protocatechuic acid 4-O-glucoside, and 4-hydroxybenzoic acid 4-O-glucoside, respectively. Compounds 2, 4, and 5 generated product ions at m/z 125, 109, and 93 after the loss of CO 2 (44 Da) from the parent ions, respectively [10]. Compounds 2, 4, and 5 were identified through pure standards as gallic acid, protocatechuic acid, and p-hydroxybenzoic acid. Compound 2 (gallic acid-C 7 H 6 O 5 ) and compound 4 (protocatechuic acid-C 7 H 6 O 4 ) were identified in mountain pepper berries and strawberry gum. Gallic acid 4-O-glucoside was only identified in mountain pepper berries, whereas benzoic acid (compound 4) was identified in all four fruits. The presence of the unique phenolic acid protocatechuic acid has been reported in many therapeutic plants [44]. It has said to possess several health benefits, including anti-inflammatory, antioxidant, anti-cancer, anti-ulcer, anti-diabetic, hepato-protective, and neuro-protective activities [44].

Cinnamic Acids and Derivatives
The most prevalent phenolic acid class is hydroxycinnamic in fruits, herbs, and medicinal plants. Sixteen hydroxycinnamic acids were identified, and their fragmentation patterns were verified using MS/MS. The removal of CO 2 and the hexosyl moiety from the parent ions is the primary way that phenolic acids exhibit the fragmentation pattern [10]. Rosmarinic acid, caffeic acid, sinapic acid, p-coumaric acid, 3-caffeoylquinic acid (chlorogenic acid), and cinnamic acid were confirmed through pure standards. The quinic acid , and both from the precursor ion, respectively. In contrast, a product ion at m/z 119 is a specific fragment ion of p-coumaric acid. As a result, compound 31 was tentatively identified as p-coumaroyl glycolic acid in rosella and mountain pepper berries. Previously, Kadam et al. [47] also reported p-coumaroyl glycolic in Lepidium sativum seedcake.

Flavonoids
Flavonoids are widely used in nutraceutical, pharmaceutical, and cosmetic industries due to their anti-carcinogenic, antimicrobial, anti-inflammatory, anti-mutagenic, and antioxidant properties. In this study, we tentatively identified seventy flavonoids ( Table 2).

Anthocyanins
Anthocyanins are water-soluble, colored plant pigments. The main positions of their hydroxyls are 3, 5, and 7 in ring A and 3 and 5 in ring B [48]. The screening, identification, and characterization of anthocyanins in native Australian rosella and mountain pepper berries were conducted. This work identified nine anthocyanins using their MS/MS spectra ( Table 2). The native Australian quandong peach and Davidson plum were used as reference plants to understand anthocyanins' structural and spectral characteristics further; these fruits are abundant in anthocyanins [1]. The removal of sugar units from anthocyanins (162 Da for hexoses, 150 Da for xyloses, 132 Da for pentoses, and 308 Da for the rutinoside moiety from the basic aglycone of corresponding anthocyanins) results in the formation of MS/MS product ions (303 Da for delphinidin, 331 Da for malvidin, 301 Da for peonidin, 317 Da for petunidin, and 287 Da for cyanidin) [1]. Compounds 33, 35, and 36 at ESI + m/z 581.1526, 595.1660, and 449.0994 generated a characteristic fragment ion at m/z 287 (cyanidin). Thus, compounds 33, 35, and 36 were tentatively identified as cyanidin 3-sambubioside, cyanidin 3-rutinoside, and cyanidin-3-O-glucoside, respectively. Compound 36 (cyanidin 3-O-glucoside) was identified in mountain pepper berries and rosella. Cyanidin-3-O-glucoside was quantified in grapes from 2.7 to 51.7 µg/mL by Oh et al. [49]. Compounds 32, 38, 39, 40, and 41 produced a distinctive fragment of delphinidin at m/z 303 in positive-ion mode ( Table 2). Compounds 33, 38, and 39 were only identified in rosella. Due to their positively charged oxygen atom, anthocyanins have higher antioxidant activity than other flavonoids [50].

Flavanols
We identified monomeric flavanols in our samples, including epicatechin, epigallocatechin, and derivatives [48]. In this study, seven flavanols, including polymerized and derivative substances, were tentatively identified in mountain pepper berries, rosella, strawberry gum, and lemon aspen. Flavanols are also called catechins, having no double bond between C2 and C3, and there is no carbonyl group in ring C (C4) [51]. Compound 44 at ESI − m/z 305.0650 generated product ions at m/z 289, 169, and 125 from the ion precursor.

Flavones and Isoflavones
Flavones are characterized by a non-saturated C3 chain and have a double bond between C2 and C3 [48]. Sixteen compounds were characterized as flavones and flavanones in mountain pepper berries, rosella, strawberry gum, and lemon aspen.  [53]. The resulting ions at m/z 300 and 271, which correspond to the loss of CH 3 and CO 2 from the precursor [1,18], were used to identify isorhamnetin (compound 93 at ESI − m/z 315.0504), which was identified in mountain pepper berries, lemon aspen, and strawberry gum. In addition to repairing iron-induced DNA oxidation, myricetin 3-O-rhamnoside (compound 85) also inhibits the activity of digestive, lipid, fecal, and colonic bacterial enzymes and functions as an anti-allergenic, anti-obesity, and anti-cancer compound [54]. Flavonols are also frequently found in Australian native fruits and medicinal plants. According to a comparison of the flavonoid literature, the aglycone derivatives of kaempferol, myricetin, and quercetin are the most often found flavonols in these plants. These aglycone derivatives are renowned for having highly effective anti-diabetic properties. These aglycone compounds are eight times more potent than the diabetic medication acarbose, according to some research [55]. In many earlier investigations, quercetin and kaempferol were connected to rutinoside, galactosides, and glucosides; previously, these flavonoid-3-O-glycosides were not described in selected native Australian plants. Three phenolic compounds, 99, 100, and 101, were only identified in strawberry gum.

Lignans and Stilbenes
Stilbenes are natural phytochemicals that contain a 1,2-diphenylethylene (a basic skeleton of stilbenoids), and lignans are a group of diphenol derivatives with dibenzylbutane skeleton structures [15]. Due to their diverse structural makeup and established advantages for human health, lignans and stilbenes are among the most studied secondary plant metabolites [15]. Ten metabolites that fit into these classes were putatively discovered in this investigation. A total of three stilbenes (piceatannol, polydatin, and piceatannol 3-O-glucoside) and seven lignans were tentatively identified in these selected Australian native fruits and medicinal plants. , respectively, from the precursor ion. Compound 127 was characterized as enterolactone. Enterolactone has been acknowledged for its antioxidant [58] and anti-cancer activities [59]. Compounds 122 (sesamin), 124 (silibinin), and 128 (2-hydroxyenterodiol) were only identified in mountain pepper berries.
The screening and profiling of the phenolic compounds give an overall idea of antioxidation compounds in selected Australian native plants. Strawberry gum is an excellent source of phenolic compounds, especially flavonoids used in the food, feed, cosmetics, and pharmaceutical industries because several of them have already been shown to possess high antioxidation capabilities.

Molecular Docking
In silico molecular docking was conducted to predict the roles of abundant phenolic compounds in α-glucosidase inhibition activity. The estimated binding geometry 2D and 3D structures of myricitrin and chlorogenic acid in α-glucosidase protein (5NN8) are given in Figure 4A,B; and the calculated binding energy, glide energy, and binding geometry 2D of selected phenolic compounds are given in Table S3 and Figure S3.

Molecular Docking
In silico molecular docking was conducted to predict the roles of abundant phenolic compounds in α-glucosidase inhibition activity. The estimated binding geometry 2D and 3D structures of myricitrin and chlorogenic acid in α-glucosidase protein (5NN8) are given in Figure 4A,B; and the calculated binding energy, glide energy, and binding geometry 2D of selected phenolic compounds are given in Table S3 and Figure S3.
interactions between target proteins (5NN8) and potential inhibitors. Therefore, it is critical to assess the inhibitory activities of individual purified phenolic compounds to establish the precise roles of individual bioactive compounds in the inhibition of α-glucosidase. Moreover, the insights into inhibitory mechanisms of bioactive polyphenolic compounds against α-glucosidase and other proteins involved in diabetic conditions can be revealed through advanced molecular dynamics techniques and free-energy calculations, and through inverse molecular docking [61].

Pharmacokinetics Study of Selected Phenolic Compounds
Using computational methods to test the potential drug metabolites helps reduce the number of experimental studies and improve the success rate in pharmacokinetics studies. Absorption, distribution, metabolism, excretion, and toxicological (ADMET) screening were also conducted to validate this study for drug discovery. The interaction of inhibitors with a target receptor cannot guarantee the suitability of phenolic metabolites as drugs for the target pathology. Therefore, ADMET screening of compounds is critical in drug discovery. Unfavorable characteristics of ADMET in the biological system are the main reasons for the failure of drug molecules during clinical experiments [7]. This study evaluated the most abundant phenolic compounds identified in selected plants for AD-MET properties.

Pharmacokinetics Study of Selected Phenolic Compounds
Using computational methods to test the potential drug metabolites helps reduce the number of experimental studies and improve the success rate in pharmacokinetics studies. Absorption, distribution, metabolism, excretion, and toxicological (ADMET) screening were also conducted to validate this study for drug discovery. The interaction of inhibitors with a target receptor cannot guarantee the suitability of phenolic metabolites as drugs for the target pathology. Therefore, ADMET screening of compounds is critical in drug discovery. Unfavorable characteristics of ADMET in the biological system are the main reasons for the failure of drug molecules during clinical experiments [7]. This study evaluated the most abundant phenolic compounds identified in selected plants for ADMET properties.

Absorption and Distribution
The absorption of the phenolic compounds was predicted through the BIOLED-Egg method and using the pkCSM platform. The results of absorption are given in Figure 5 and Table S4 and Table S5. Evaluation of abundant phenolic compounds through the BOILED-Egg method. The blue dots indicate molecules predicted to be expelled from the CNS by P-glycoprotein, and the red dots indicate molecules predicted not to be expelled from the CNS by P-glycoprotein. The egg-yolk area predicts the phenolic metabolites that will passively penetrate the blood-brain barrier. In contrast, the egg-white area predicts which phenolic compounds will be absorbed through the gastrointestinal tract. Figure 5 predicts that cinnamic acid, coumarin, p-coumaric acid, p-hydroxybenzoic acid, chrysin, [6]-gingerol, and 3',4',5'-trimethoxyflavone pass through the blood-brain barrier; and gallic acid, protocatechuic acid, caffeic acid, pyrogallol, cyanidin, taxifolin, epicatechin, delphinidin, naringenin, genistein, phloretin, quercetin, diosmetin, isorhamnetin, limocitrin, and eriodictyol should be absorbed through the gastrointestinal tract. Moreover, the results predict that the cinnamic acid found in mountain pepper berries and strawberry gum will more readily cross the blood-brain barrier than other phenolic compounds (Table S4) (Table S4). Therefore, we can predict that anthocyanins with sugar moieties may play a role in gut modulation after the breakdown through colonic fermentation into their basic aglycones, or they will play a role as Figure 5. Evaluation of abundant phenolic compounds through the BOILED-Egg method. The blue dots indicate molecules predicted to be expelled from the CNS by P-glycoprotein, and the red dots indicate molecules predicted not to be expelled from the CNS by P-glycoprotein. The egg-yolk area predicts the phenolic metabolites that will passively penetrate the blood-brain barrier. In contrast, the egg-white area predicts which phenolic compounds will be absorbed through the gastrointestinal tract. Figure 5 predicts that cinnamic acid, coumarin, p-coumaric acid, p-hydroxybenzoic acid, chrysin, [6]-gingerol, and 3 ,4 ,5 -trimethoxyflavone pass through the blood-brain barrier; and gallic acid, protocatechuic acid, caffeic acid, pyrogallol, cyanidin, taxifolin, epicatechin, delphinidin, naringenin, genistein, phloretin, quercetin, diosmetin, isorhamnetin, limocitrin, and eriodictyol should be absorbed through the gastrointestinal tract. Moreover, the results predict that the cinnamic acid found in mountain pepper berries and strawberry gum will more readily cross the blood-brain barrier than other phenolic compounds (Table S4) (Table S4). Therefore, we can predict that anthocyanins with sugar moieties may play a role in gut modulation after the breakdown through colonic fermentation into their basic aglycones, or they will play a role as prebiotic polyphenols. Additionally, cinnamic (1.72), coumarin (1.65), 3 ,4 ,5 -trimethoxyflavone (1.39), p-coumaric acid (1.21), p-hydroxybenzoic acid (1.15), pyrogallol (1.12), naringenin (1.03), chrysin (0.95), [6]-gingerol (0.94), and taxifolin (0.92), are predicted to have the highest Caco-2 cell permeability. If the Caco2 permeability value is higher than 0.90, a compound is considered to have high Caco-2 permeability. Furthermore, the compounds which have Caco-2 permeability, gastrointestinal absorption, a good bioavailability score, and obey Lipinski's rule of five while not being able to pass through the BBB, not acting as P-gp substrates, and having poor skin permeability should be successful drugs [62].
Most of the flavonoids that are not absorbed in the gastrointestinal tract can be metabolized by gut microbiota into small phenolic metabolites, where they tend to be absorbed in the colon [48]. Flavonoids are bound to albumin and transported to the liver through the portal vein after absorption. However, the bioavailability of flavonoids is low due to the limited absorption, extensive metabolism, and rapid excretion [63].

Drug-Likeness
The bioavailability radars of selected compounds were obtained by following the method of Daina et al. [17] to predict the drug-likeness to assess the oral bioavailability of compounds ( Figure 6). , are predicted to have the highest Caco-2 cell permeability. If the Caco2 permeability value is higher than 0.90, a compound is considered to have high Caco-2 permeability. Furthermore, the compounds which have Caco-2 permeability, gastrointestinal absorption, a good bioavailability score, and obey Lipinski's rule of five while not being able to pass through the BBB, not acting as P-gp substrates, and having poor skin permeability should be successful drugs [62].
Most of the flavonoids that are not absorbed in the gastrointestinal tract can be metabolized by gut microbiota into small phenolic metabolites, where they tend to be absorbed in the colon [48]. Flavonoids are bound to albumin and transported to the liver through the portal vein after absorption. However, the bioavailability of flavonoids is low due to the limited absorption, extensive metabolism, and rapid excretion [63].

Drug-Likeness
The bioavailability radars of selected compounds were obtained by following the method of Daina et al. [17] to predict the drug-likeness to assess the oral bioavailability of compounds ( Figure 6).   Table S6 depict that no compound predicted oral bioavailability except quinic acid. To predict the oral bioavailability of selected compounds, six physiochemical properties (size, polarity, lipophilicity, flexibility, saturation, and solubility) were considered and analyzed through the bioavailability radar.   Table S6 depict that no compound predicted oral bioavailability except quinic acid. To predict the oral bioavailability of selected compounds, six physiochemical properties (size, polarity, lipophilicity, flexibility, saturation, and solubility) were considered and analyzed through the bioavailability radar.

Metabolism, Excretion, and Toxicity
Cytochrome P450 (CYP) plays a vital role in the metabolism of bioactive compounds (drugs) [63]. The predicted metabolism and excretion of the phenolic compounds are given in Table S7. Metabolism was predicted through the CYP model for substrate or inhibitor (CYP1A2, CYP2D6, CYP3A4, CYP2C9, and CYP2C19). Bioactive compounds that inhibit the CYP pathway may cause elevated concentrations of other bioactive compounds, resulting in higher toxicity of that compound and vice versa. Bioactive compounds with higher total clearance are predicted to have higher bioavailability and metabolism in the liver (Table S5). Virtual toxicological screening of the bioactive compounds is provided in Table  S8. The predicted results indicate that all bioactive compounds do not inhibit the hERG 1 channel, and no compound predicted AIME toxicity, hepatotoxicity, skin sensitization, Tetrahymena pyriformis toxicity, or Minnow toxicity except 3 ,4 ,5 -trimethoxyflavone, which predicted toxicity in Minnow.

Conclusions
In this study, native Australian fruits and spices were comprehensively analyzed for polyphenols, and a total of 143 metabolites were identified. Twenty-six of these compounds were quantified. Strawberry gum had higher total phenolic content, antioxidant capacity, and α-glucosidase inhibition activity than rosella, lemon aspen, and mountain pepper berries. Furthermore, in silico molecular docking predicted that flavonoids have a significant role in the inhibition of α-glucosidase. Additionally, simulated pharmacokinetics predicted that all screening phenolic compounds from native Australian fruits and spices are safe and do not have any toxicity, and small phenolic metabolites such as phenolic acids have higher absorption in Caco-2 cells and the gastrointestinal tract than other phenolic compounds. This study demonstrates that strawberry gum has a significant medicinal and pharmaceutical potential that could be utilized in food, feed, cosmetic, and pharmaceutical industries with the further proved in vivo data.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/antiox12020254/s1. Figure S1: Base peak chromatograms (BPC) of mountain pepper berries, rosella, lemon aspen, and strawberry gum in positive (black) and negative (blue) modes of ionization. Figure S2: MS/MS spectra of some selected compounds. Figure S3: Two-dimensional binding geometry of some selected compounds. Table S1: Antioxidant activities of native Australian fruits and spices. Table S2: Quantification/semi-quantification of phenolic metabolites in Australian native fruits and spices (µg/g). Table S3: The calculated binding energies of selected compounds. Table S4: Predicted absorption and distribution of selected compounds. Table S5: Pharmacokinetic properties of selected compounds. Table S6: Radar bioavailability properties of selected compounds. Table S7: Metabolism and excretion of selected compounds. Table S8: Predicted toxicity of abundant phenolic compounds.
Author Contributions: Conceptualization, methodology, formal analysis, investigation, software, validation, data curation, visualization, writing-original draft preparation, A.A.; writing-review and editing, supervision, resources, project administration, funding acquisition, F.R.D. and J.J.C. All authors have read and agreed to the published version of the manuscript.