Antioxidant Activity, α-Glucosidase Inhibition and UHPLC–ESI–MS/MS Profile of Shmar (Arbutus pavarii Pamp)

The genus Arbutus (Ericaceae) has been traditionally used in folk medicine due to its phytomedicinal properties, especially Arbutus pavarii Pamp. However, this plant has not been evaluated for its efficacy, quality, and consistency to support the traditional uses, potentially in treating diabetes. Despite previous studies that revealed the biological activities of A. pavarii as antioxidant and α-glucosidase inhibitory agents, scientific reports on the bioactive compounds that contribute to its health benefits are still scarce. Therefore, this research focused on the evaluation of antioxidant and α-glucosidase inhibitory activities of the methanol crude extracts and various fractions of the leaf and stem bark, as well as on metabolite profiling of the methanol crude extracts. The extracts and fractions were evaluated for total phenolic (TPC) and total flavonoid (TFC) contents, as well as the DPPH free radical scavenging, ferric reducing antioxidant power (FRAP), and α-glucosidase inhibitory activities. Methanol crude extracts of the leaf and stem bark were then subjected to UHPLC–ESI–MS/MS. To the best of our knowledge, the comparative evaluation of the antioxidant and α-glucosidase inhibitory activities of the leaf and stem bark of A. pavarii, as well as of the respective solvent fractions, is reported herein for the first time. Out of these extracts, the methanolic crude extracts and polar fractions (ethyl acetate and butanol fractions) showed significant bioactivities. The DPPH free radical and α-glucosidase inhibitions was highest in the leaf ethyl acetate fraction, with IC50 of 6.39 and 4.93 µg/mL, respectively, while the leaf methanol crude extract and butanol fraction exhibited the highest FRAP with 82.95 and 82.17 mmol Fe (II)/g extract. The UHPLC–ESI–MS/MS analysis resulted in the putative identification of a total of 76 compounds from the leaf and stem bark, comprising a large proportion of plant phenolics (flavonoids and phenolic acids), terpenoids, and fatty acid derivatives. Results from the present study showed that the different parts of A. pavarii had potent antioxidant and α-glucosidase inhibitory activities, which could potentially prevent oxidative damage or diabetes-related problems. These findings may strengthen the traditional claim on the medicinal value of A. pavarii.


Introduction
Medicinal plants have always been known as healthy and natural sources of combating drugs. Historically, for thousands of years, many of such plants have been used for treating various diseases [1]. The World Health Organization (WHO) reported that nearly 80% of the populations of developing countries rely on traditional medicine and consider medicinal plants as their primary sources of medication [2]. Plant extracts are mixtures, rich in natural product compounds, such as flavonoids, alkaloids, terpenoids, and tannins, many of which ing antioxidant power (FRAP). Although the use of colorimetric-based total phenolic and antioxidant assays for describing the bioactivity of chemical constituents in the absence of cell-based or in vivo test has been controversial, studies have revealed the positive correlation between results obtained from these colorimetric methods and cell-based assays [18][19][20]. Hence, these colorimetric methods are still essential screening tools for the assessment of antioxidant potential. The contents of phenolic compounds in the methanolic crude extracts and solvent fractions of A. pavarii leaf and stem bark were determined by using the Folin-Ciocalteu reagent [21] and the results are presented in Table 1. The leaf methanol crude extract contained 886.57 mg GAE/g extract of TPC while the TPC of its respective solvent fractions ranged from 62.54 to 790.76 mg GAE/g fraction. Among the four solvent fractions, the ethyl acetate fraction possessed the highest TPC (790.76 mg GAE/g fraction) followed by butanol (390.47 mg GAE/g fraction), chloroform (186.94 mg GAE/g fraction), and hexane fractions (62.54 mg GAE/g fraction). The results revealed that most of the phenolic compounds were distributed in the ethyl acetate fraction of the leaf methanol crude extract and suggested that the phenolic compounds are of moderate polarity. Meanwhile, the stem bark of A. pavarii also contained high TPC with 795.55 mg GAE/g extract. The TPC of its fraction ranged from 199.14 to 707.61 mg GAE/g fraction. However, unlike the leaf extract, the highest TPC of the stem bark methanol crude extract was in the butanol fraction (707.61 mg GAE/g fraction), followed by the ethyl acetate (480.21 mg GAE/g fraction), chloroform (322.68 mg GAE/g fraction) and hexane fractions (199.14 mg GAE/g fraction). This indicated that the phenolic constituents of the stem bark were mainly distributed in the butanol fraction and suggested that the compounds were of high polarity. The different trends in the results of the leaf and stem bark phenolic contents could be attributed to a different composition of the phenolic constituents of the different parts of the plant [22].

Total Flavonoid Content (TFC)
To determine the total flavonoid content (TFC) in the leaf and stem bark extract and solvent fractions of A. pavarii, a colorimetric approach based on flavonoid-aluminum chloride complexation was employed [23], and the results are presented in Table 1. The results revealed the presence of high flavonoid content in the leaf and stem bark extracts of A. pavarii. The methanol crude extract of A. pavarii leaf contained 442.06 mg QE/g extract while the TFC of its respective solvent fractions ranged from 58.21 to 369.52 mg QE/g fraction. The highest TFC was found in the ethyl acetate fraction, which contained 369.52 mg QE/g fraction, followed by butanol, chloroform and hexane fractions, which contained 277.72, 109.09, and 58.21 mg QE/g fraction, respectively. These results were of the same trend as the TPC results, suggesting that flavonoids could very well be the major class of phenolic constituents in the leaves of A. pavarii.
On the other hand, the methanol crude extract of A. pavarii stem bark contained 638.93 mg QE/g of total flavonoid, which was significantly higher than in the leaf. The difference in the TFC of the leaf and stem bark could be due to the production and accumulation of different secondary metabolites in the leaf and stem bark [24]. Besides, similar to the fractions of the leaf extract, the ethyl acetate fraction of the stem bark extract contained the highest TFC with 707.61 mg QE/g fraction, followed by butanol, chloroform, and hexane fractions, with 213.32, 204.83, and 38.41 mg QE/g fraction, respectively. These results were different than that of the TPC results. Although it contained the highest TPC, the butanol fraction had relatively lower TFC, suggesting that it contains phenolic compounds other than flavonoids, which also revealed the diversity of phenolic compounds present in A. pavarii stem bark [25].

2,2-diphenyl-1-picrylhydrazyl (DPPH) Radical Scavenging Activity
The free radical scavenging activity of the plant extracts and their respective fractions was determined using the DPPH free radical scavenging assay [21]. The presence of antioxidants leads to the reduction of the DPPH free radicals, and hence the dark violetcolored solution is transformed to yellow. Table 1 show the DPPH free radical scavenging activity of the leaf and stem bark of A. pavarii, expressed as IC 50 values. The leaf methanol crude extract inhibited the DPPH free radicals with IC 50 value of 17.57 µg/mL. For the leaf solvent fractions, the ethyl acetate fraction exhibited the most potent DPPH scavenging activity, with IC 50 value of 6.39 µg/mL. This was followed by butanol, chloroform, and hexane fractions, with IC 50 values of 27.69, 39.50, and 95.82 µg/mL, respectively. It is noteworthy that the IC 50 value of the ethyl acetate fraction was even lower than that of quercetin (IC 50 = 8.60 µg/mL), which was used as positive control in the assay. The more potent DPPH scavenging activity of the ethyl acetate fraction as compared to the methanolic crude extract could be explained by the high concentration of free radical scavenging compounds in the fraction after the fractionation process. In addition, the potent DPPH scavenging activity of the ethyl acetate fraction could be contributed by the presence of high amount of phenolic compounds. A positive relationship between phenolic content and DPPH scavenging activity has been reported in previous studies [19,20,26].
Meanwhile, the stem bark methanol crude extract exhibited high DPPH scavenging activity with an IC 50 value as low as 8.67 µg/mL. Among its solvent fractions, the ethyl acetate and butanol fractions showed the more potent activities, with IC 50 values (8.35 and 8.71 µg/mL, respectively) close to that of the methanol crude extract, and comparable with that of quercetin (IC 50 = 8.60 µg/mL). The potent activity exhibited by both the ethyl acetate and butanol fractions indicated that the constituents distributed in ethyl acetate and butanol fractions are structurally effective for scavenging DPPH free radicals. While the phenolic compounds could be the DPPH free radical scavengers in these fractions, the activity of the stem bark butanol fraction could be contributed by phenolic compounds with relatively higher polarity, perhaps those with more polar functional groups or sugar attachment. In addition, the DPPH scavenging activity of stem bark is also more significant as compared to the leaf of A. pavarii, as revealed by the lower IC 50 value of the stem bark. The better activity of the stem bark could be possibly due to its more diverse phenolic compositions.

Ferric Reducing Antioxidant Power (FRAP)
The ferric reducing antioxidant power (FRAP) assay allows the examination of the reducing power of samples. The reducing ability of a sample may reflect its electron transferring capability, which is an important mechanism of antioxidants [27][28][29][30] The FRAP values (mM Fe (II) equivalent) of A. pavarii leaf and stem bark extracts, as well as their respective fractions have been calculated by constructing a standard curve between the absorbance and the concentration of FeSO 4 standard [31]. The results are shown in Table 1. The A. pavarii leaf methanolic extract exhibited reducing power with FRAP value of 82.95 mM Fe (II)/g extract, while the FRAP values of its fractions ranged from 46.76 to 86.33 mM Fe (II)/g extract. Unlike the results of the aforementioned assays, both the ethyl acetate and butanol fractions of A. pavarii leaf exhibited almost similar reducing power and are close to the FRAP value of the methanolic extract. The results of the stem bark showed the same trend as the leaf, although overall it showed slightly lower reducing power than the leaf. These results indicated the presence of strong electron donating antioxidants in the extracts as well as the ethyl acetate and butanol fractions which reduced ferric ions into ferrous ions under the reaction conditions [29]. The phenolic compounds, both flavonoids and non-flavonoid compounds, could be responsible for the reducing ability of A. pavarii leaf and stem bark. This is in agreement with previous studies that reported significant correlations between both TPC and TFC, and FRAP of grape by-products [19], and Clinacanthus nutans [26].

α-Glucosidase Inhibitory Activity
According to the results of α-glucosidase inhibitory activity of A. pavarii leaf and stem bark extracts and fractions, which are presented in Table 1, the leaf methanolic extract inhibited the α-glucosidase enzyme with IC 50 value of 8.75 µg/mL. However, for the leaf fractions, the ethyl acetate fraction exhibited the most potent inhibitory activity with IC 50 value of 4.93 µg/mL, followed by butanol (IC 50 value 10.44 µg/mL) and chloroform (IC 50 value 62.64 µg/mL) fractions. The IC 50 of hexane fraction was not able to be determined as the inhibition against the α-glucosidase enzyme was less than 50% at all the concentrations used. On the other hand, the stem bark methanolic extract inhibited the α-glucosidase enzyme with IC 50 value of 6.78 µg/mL, which was significantly more potent as compared to the leaf methanol extract. For stem bark fractions, the trend was found to be similar to the fractions of the leaf methanolic extract, with the exception at the insignificant different of the activity of ethyl acetate and butanol fractions. Moreover, based on the IC 50 values, the ethyl acetate fractions of both leaf and stem bark, and the butanol fraction of stem bark showed higher α-glucosidase inhibitory activity compared to quercetin, which was used as positive control in the assay. These results revealed an increasing activity with an increasing polarity of the fraction, which could be explained by the existence of highly polar compounds. In other words, it can be due to the amount of phenolic compounds and the type of phenolics present in the sample that may be responsible for the strong inhibition activity against α-glucosidase enzyme. The results obtained in the present study are in good agreement with previous work, which reported the potency of phenolic rich samples in inhibiting α-glucosidase enzyme, in addition to strong antioxidant activity [26]. Besides, previous research has also reported the increased inhibitory effect against α-glucosidase enzyme with increasing polarity of the plant extracts or fractions, with more polar and lower molecular weight phenolic constituents, such as phenolic acids as inhibitors of the enzyme [32]. Furthermore, various studies have outlined that the Arbutus genus could be a great natural source of phenolic and flavonoid compounds which are well known to have a strong hypoglycemic potential [15]. To the best of our knowledge, this is the first report on α-glucosidase inhibitory effect of leaf, stem bark extracts, and fractions of A. pavarii. Its potential therapeutic use for treating or managing diabetes could be worthy of further pharmacological investigations.

Putative LCMS Profiles of A. pavarii Crude Leaf and Stem Bark Methanol Extracts
Analyses of medicinal plants have benefited from the application of liquid chromatography coupled with mass spectrometry (LC-MS) due to the increasingly improved separation and detection abilities of the instruments [33]. The leaf and stem bark methanol extracts of A. pavarii showed good activities in all assays (antioxidant and α-glucosidase inhibitory activities). Hence, these extracts were further characterized using LC-MS/MS to gain better insight into the components that may be contributing to the studied activities. The base peak chromatograms of A. pavarii leaf and stem bark methanol extracts are displayed in Figure 1; Figure 2, respectively; while Table 2 summarizes the retention time (Rt), ionization mode (−ve/+ve), experimental and theoretical parent ion (m/z), error (ppm), MS/MS data, and presence of the identified compounds. A total of 76 compounds were putatively identified based on the MS/MS data in comparison with literature. The base peak chromatograms showed that most of the prominent peaks were attributed to the presence of phenolic compounds. This could support the high TPC and TFC values, and hence the potent antioxidant and α-glucosidase inhibitory activities of the A. pavarii leaf and stem bark.
in the negative mode. Based on the elution order in previous report, compounds 24 and 32 were identified as catechin and epicatechin, receptively [44]. They exhibited the similar MS/MS fragmentation. Fragment ions at m/z 165.02 were resulted from HRF fragmentation, while m/z 151.04 and 137.02 from RDA fragmentation. Besides, the fragments at m/z 271.06 and 245.08 resulted from the loss of water and carbon dioxide, respectively [45,46].      In this work, compounds that are putatively identified as phenolic acids and phenolic acid glycoside derivatives were classified as either gallic acid and its derivatives, or other phenolic acids and glycoside derivatives. Compounds 3, 4, 5, and 8 were identified as isomers of gallic acid monoglucoside. They had pseudomolecular ion at m/z 331.0671, m/z 331.0675, m/z 331.0672, and m/z 331.0667, respectively. Their fragment ion at m/z 169.01 [M-H-162] is due to the neutral loss of a glucosyl moiety (162 Da) ( Table 2). This agrees with previous reports [34,35].  [35,36]. Compound 47 showed a pseudomolecular ion at m/z 300.9984 in negative mode at Rt = 1.96 min (C 14 H 6 O 8 ). It was assigned as ellagic acid based on comparison with the previous report [36].

Identification of Flavonoids and Derivatives
Flavonoids (C6-C3-C6) are phytoconstituents, which contain 15 carbons with two aromatic rings associated by a three-carbon bridge. Based on hydroxylation and different functionalities in chromane (ring C), these polyphenols are further divided into flavones, flavonols, flavan-3-ols, isoflavones, flavanones, and anthocyanidins [39]. In plants, these substances work as regulatory compounds, colorants, and protecting the newly developed plants cells against UV light, wound, pathogens, and herbivores [40,41]. Furthermore, flavonoids are famous for antibacterial, antioxidants, antidiabetics, and various other bioactive activities that have been interested due to its benefits for human health, curing, and preventing of many serious diseases [8,42]. The current study is the first report for the identification of flavonoids in the stem bark of A. pavarii.

Identification of Flavan-3-ols and Derivatives
Analysis of the LCMS profiles of A. pavarii leaf and stem bark methanol extracts indicate the presence of monomers, dimers, trimers, and tetramers of proanthocyanidin (PAs). Three compounds (13, 24, and 32) were putatively identified as PA monomers.  18] was produced via RDA mechanism and successive loss of water molecule. These fragments are characteristic for (epi)gallocatechin. Furthermore, the RDA reaction on the top unit of dimer has been reported to be more energetically favorable, due to the formation of larger π-π hyperconjugated system [53]. Hence, (epi)gallocatechin was suggested as the top unit of this dimer. The (epi)catechin as the base unit was further confirmed by the presence of the fragment ion at m/z 289.07, which was the result of QM cleavage. The ion m/z 287 would presence if the (epi)catechin was the top unit [53]. Besides, the fragments at m/z 125.02 and m/z 467.10 were attributed to the HRF on the top unit. The HRF on the top unit was also more favorable due to the same reason as the RDA [54]. With the same principle applied, compounds 38, 43 and 55 were identified as isomers of (epi)catechin gallate + (epi)cat, while compound 56 was assigned as (epi)afzelechin + (epi)catechin. The fragmentation pathways of these compounds are also illustrated in Scheme 1 [55].
Three isomers were putatively assigned as B-type tetrameric PA in the methanol extract of the stem bark. Compounds 27, 37, and 41 showed pseudomolecular ions at m/z 1153.2627, m/z 1153.2617, and m/z 1153.2635, respectively, in the negative mode with molecular formal C 60 H 50 O 24 . These compounds produced similar fragmentation patterns with similar intensities of ions. Based on the presence of fragment ions at m/z 865.20, 577.14, 407.08, and 289.07, they were identified as ((epi)catechin + (epi)catechin + (epi)catechin + (epi)catechin isomers. Fragmentation pathway is shown in Scheme 1 [62,63].

Identification of Anthocyanins and Derivatives
Anthocyanins are categorized as a class of flavonoids, and they are known for their beneficial effects on both humans and animals. They are usually pigments that give colors to many plants, including A. pavarii. They are present in nature as glycosides [64]. One of the most notable and distinguishing features of anthocyanins group is the ability of its structure to change under distinct pH conditions, leading to a change of color [65]. In this study, the anthocyanins were identified in the methanol extracts of leaf and stem bark of A. pavarii in the positive ionization mode. The fragmentation of the [M+H] ion allows the identification of the anthocyanin aglycone and the glycone. Seven anthocyanins derivatives were putatively assigned in leaf and stem bark of the A. pavarii (compounds  49, 52, 53, 61, 65, 67, and 68). They showed the characteristic fragment ions of delphinidin, cyanidin, petunidin, peonidin, and malvidin aglycones at m/z 303.05, 287.05, 317.06, 301.07, and 331.07, respectively. Five of these anthocyanins had glucose attached to the aglycon [M+H-162] + and one had coumaroyl glucose [M+H-308] + attachment. The assignments of these compounds were in good agreement with those previously reported [64,[66][67][68].

Identification of Flavonol
In both leaf and stem bark methanol extracts, a total of seven compounds (45, 48, 51,  58, 62, 64, and 69 [57]. In this current study, three derivatives of kaempferol were putatively identified in the methanol extracts of the leaf and stem bark. , and 133.10 (-C 10 H 13 ), which were due to RDA fragmentation. A typical RDA fragmentation can be used to identify the presence of 12-13 double bonds in triterpenes (∆ 12 -ursine) [78]. Identification of these two compounds was carried out by comparing their mass spectra and elution order with the literature [79][80][81]. The first eluted compound 74 was identified a betulinic acid while compound 75, which was eluted later was identified as ursolic acid.  [76]. Moreover, compound 2 (Rt = 0.93, C 12 H 16 O 7 ), with a pseudomolecular ion at m/z 271.0801 in the negative mode, was identified as arbutin, which is a glycosylated hydroquinone. Its fragment ion at m/z 108.02 was due to a glucosyl moiety loss [84].

Plant Materials
The plant materials were obtained from Al Jabal, Al Akhdar (Green Mountain) region, North Eastern part of Libya and all samples were harvested during spring 2016. Plant authentication was carried out by Dr. Abdulamid Alzerbi of the Herbarium Unit, Department of Biology, Benghazi University, Libya. The leaf and stem barks of the plant were dried under shade for 15 and 28 days, respectively prior to mechanical grinding into a fine powder. The resulting particles were sieved using a stainless-steel sieve (80 mesh, Retsch, Haan, Germany) to obtain a fine and homogeneous powder. The finely powdered leaf and stem bark were weighed separately and all samples were stored at −20 • C (chiller) until needed.

Preparation of Leaf and Stem Bark Extracts and Their Respective Fractions
For extraction, the dried leaf (1500 g) and stem barks (500 g) were separately mixed with absolute methanol (CH 3 OH) with the solid to liquid ratio of 1:10 (w/v). The mixtures were subjected to sonication (at a controlled temperature) in an ultrasonic bath (Branson, 141 8510E-MTH models, Danbury, CT, USA) for an hour under a frequency of 53 KHz and power of 100 W. The extraction was repeated three times, each time with fresh solvent. The respective extracts were filtered using Whatman filter paper (GE Healthcare, Buckinghamshire, UK) and the collected filtrates were concentrated under reduced pressure using a rotary evaporator (Buchi, New Castle, DE, USA) at 40 • C. The leaf and stem bark methanolic extracts were then liquid-liquid partitioned, performed in a separating funnel, with solvents of increasing polarities, starting with hexane, chloroform, ethyl acetate and n-butanol, to yield the different fractions. In each case, the respective crude extract was resuspended in an adequate amount of methanol, and sonicated to aid the dissolution, before adding distilled water to bring the solution up to a workable volume for solvent fractionation. The extract solution was fractionated first with hexane at an extract solution:organic solvent ratio of 1:3. After vigorous shaking, the mixture was set aside until two layers were formed. The upper layer was collected as hexane fraction. To obtain the chloroform fraction, chloroform was added into the remaining fraction in the separating funnel, followed by vigorous shaking. The chloroform fraction was then collected. The same procedure was repeated using ethyl acetate and subsequently n-butanol to yield the ethyl acetate and n-butanol fractions. After partitioning with butanol, material left in the separating funnel was considered as the residual aqueous fraction. Workflow of the liquid-liquid partition of each of the crude extracts, together with information on yields and physical appearances of the extracts and their respective fraction, are shown in the Supplementary Information (Supplementary Figures S1 and S2, Supplementary Table S1). The solvent fractions were concentrated using rotary evaporator and finally lyophilized and kept in a chiller at −20°C prior to further analysis. The aqueous fraction was not used for the analysis of the antioxidant and α-glucosidase inhibition due to the reason that it could not dissolve in the solvent used for sample preparation in the assays.

Total Phenolic Content (TPC) Assay
The TPC in the methanolic extracts of A. pavarii leaf and stem bark as well as their respective fractions was determined using the Folin-Ciocalteu reagent, as described by Lee et al. [21], with slight modifications. An aliquot of 20 µL of extract or fraction at a concentration of (1000 µg/mL) was transferred into 96-well microplates, followed by 100 µL of the Folin-Ciocalteu reagent. After incubation at room temperature for 5 min, 80 µL of 7.5% sodium carbonate was added into the mixture and incubated for another 30 min. Finally, the absorbance was measured through a microplate reader (SPECTRAmax PLUS) at 765 nm. The analysis was performed in three replications for each sample. The standard curve of gallic acid was constructed to determine the TPC and the results were expressed as mg GAE/g extract for extracts and mg GAE/g fraction for fractions.

Total Flavonoid Content (TFC) Assay
The TFC was measured by using the colorimetric method as described by Kim et al. with slight modifications [23]. A volume of 25 µL of the extracts or fractions at a concentration of 1000 µL/mL were mixed with 100 µL distilled water and 7.5 µL 5% NaNO 2 in a 96-well microplate. Then, 7.5 µL 10% AlCl 3 .6H 2 O was added and the resultant mixtures were incubated at room temperature. After 5 min, 50 µL 1 M NaOH was added into the mixture and the plate was incubated for another 15 min at room temperature. The absorbance was determined by using microplate reader at the wavelength of 415 nm. A standard curve of quercetin was used to calculate the TFC. The experiment was performed in triplicates and the TFC results were expressed as mg QE/g extract for extracts and mg QE/g fraction for fractions.

Free Radical Scavenging (DPPH) Assay
The DPPH assay was conducted in accordance with Lee et al. method [21]. Initially, 100 µL of DPPH (5.9 mg in 100 mL ethanol) was mixed with 50 µL of the extracts or fractions. The mixtures were then kept in dark for 30 min at room temperature. The absorbance was measured using microplate reader at 517 nm. Quercetin was used as a positive control in the assay. The scavenging activity was calculated using the equation Scavenging Activity (%) = [(Ac -As)/Ac] × 100%, where (Ac) is the absorbance of the blank reagent while (As) is the absorbance of the tested samples. The analysis was performed in triplicates and the results were described as IC 50 value.

Ferric Reducing Antioxidant Power (FRAP) Assay
The FRAP of extracts and fractions were examined according to Kadum et al. method with slight modifications [31]. The FRAP reagent was prepared by mixing the solutions of 2,4,6-tripyridyl-s-triazine (TPTZ) and FeCl 3 in the acetic acid buffer (pH 3.6) in the ratio of 1:1:10 (v/v/v). A volume of 10 µL of methanol extract or fractions was transferred into 96-well microplates, followed by addition of 200 µL FRAP reagent and incubation at 37 • C for 30 min. The absorbance was checked through microplate reader (SPECTRAmax PLUS) at 593 nm. The absorbances of ferrous sulfate solution (FeSO 4 ·7H 2 O), with a range of concentrations between 0.1 and 1 mM were used for plotting a calibration curve. The ascorbic acid was used as positive control and FRAP was presented as mM Fe (II)/g extract for extracts and mM Fe (II)/g fraction for fractions.

α-Glucosidase Inhibitory Assay
The inhibitory activity of the extracts and fractions on α-glucosidase was assayed using method of Lee et al. with some slight modifications [21]. The α-glucosidase enzyme (0.02 U/well), and p-nitrophenyl-α-D-glucopyranose (PNPG) substrate (1 mM) were prepared in 50 mM phosphate buffer (pH 6.5). A total of 10 µL of extract or fraction was mixed with 130 µL of phosphate buffer (30 mM) and 10 µL of enzyme in a 96-well microplate. After incubation of the plate for 5 min, 50 µL of PNPG was added to each sample-containing well, blank substrate, negative control, and positive control, followed by further incubation for 15 min at room temperature to start the reaction. Subsequently, 50 µL of 2M glycine was added into each well to stop the reaction. The enzymatic activity was determined by calculating the p-nitrophenol that has been released from PNPG at the wavelength of 405 nm by using a spectrophotometer (SPECTRAmax PLUS, Sunnyvale, CA, USA). The inhibition percentage (%) was calculated using the following formula: % Inhibition = [(∆Ac − ∆Ae)/∆Ac], where ∆Ac is the absorbance difference between the negative control and blank control whereas ∆Ae is the absorbance difference between sample and the blank sample. Eventually, the results were expressed as IC 50 value in µg/mL.

UHPLC-MS/MS Analysis
The UHPLC-MS/MS analysis was conducted using a Dionex Ultimate 3000 UHPLC system attached to a Q Exactive TM Focus Hybrid Quadrupole Orbitrap mass spectrometer (Thermo Scientific, San Jose, CA, USA). Analyte separation was carried out using a Hypersil Gold C18 column (2.1 × 100 mM, 1.9 µm, Thermo Scientific, San Jose, CA, USA) with a mobile phase consisting of LCMS grade water (solvent A) and acetonitrile (solvent B), each containing 0.1% formic acid and flowing at 0.4 mL/min. The programmed gradient consisted of 0 min (95% A), 5 min (95% A), 25 min (60% A), 55 min (0% A), 65 min (0% A), 67 min (95% A), and 70 min (95% A). Samples were prepared at the concentration of 1 mg/mL (w/v) by dissolving 1 mg of a dried sample of the active extract with the 1mL of LC-MS grade methanol. The resultant mixture was then filtered using 0.22 um nylon membranes and 10 µL of the filtrate was auto injected for the analysis. The MS analysis was done with the parameters set as follow: negative and positive ion mode (done separately), collision energy of 30 eV, spray voltage 4.2 kV (positive mode) and 3.5 kV (negative mode), capillary temperature 350 • C, auxiliary gas heater temperature 0 • C, sheath gas flow rate of 45 (arbitrary units) for the positive mode and 40 (arbitrary units) for the negative mode, and auxiliary nitrogen (99% pure) at a flow rate of 10 and 8 units for positive and negative mode, respectively. Then, the mass resolution was set to 70,000 full width at half maximum (FWHM) and a full scan from 150 to 2000 amu. The identification analysis was carried out by comparing between the available data of MS/MS from the literature. The results were expressed as the mean ± standard deviation of three replicates. One-way ANOVA with Tukey comparison test was employed to determine the significant difference among the samples. The p < 0.05 value was considered to be statistically significant, and vice versa.

Conclusions
In this study, TPC and TFC, as well as the antioxidant and α-glucosidase inhibitory activities of methanol extracts and fractions of A. pavarii leaf and stem bark were examined. The phytochemical profiles of the methanol extracts of A. pavarii leaf and stem bark were also characterized using UHPLC-ESI-MS/MS. The results revealed that methanol extracts and fractions of A. pavarii exhibited different antioxidant and α-glucosidase inhibitory activities. Overall, the methanol extracts and polar fractions (ethyl acetate and butanol) exhibited remarkable TPC and TFC, as well as antioxidant and α-glucosidase inhibitory activities. In addition, plant phenolics, both flavonoids and non-flavonoid constituents, could be responsible for the antioxidant and α-glucosidase inhibitory activities of A. pavarii leaf and stem bark. Via the UHPLC-ESI-MS/MS analysis, a total of 76 compounds were putatively identified, in which a large proportion of them were phenolic compounds, which could be the constituents contributing to the antioxidant and α-glucosidase inhibitory activities of A. pavarii leaf and stem bark. However, the exact identity of the bioactive candidates for the two activities will require further rigorous investigations via bioassay-guided isolation and purification approach or other contemporary methods, such as multi-platform metabolomics. Nevertheless, the findings of this present study indicate the potential of A. pavarii as an antidiabetic agent. Further investigations of this therapeutical potential through in depth pharmacological studies, followed by research into its development as phytomedicinal preparations or health supplements for diabetes, will help pave the way towards its valorization. To the best of our knowledge, this study is the first report on the TPC, TFC, antioxidant activity, α-glucosidase inhibitory, and LC-MS/MS profiling for the stem bark of A. pavarii.