Metabolite Profiling of Manilkara zapota L. Leaves by High-Resolution Mass Spectrometry Coupled with ESI and APCI and In Vitro Antioxidant Activity, α-Glucosidase, and Elastase Inhibition Assays

High-resolution mass spectrometry equipped with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) sources was used to enhance the characterization of phytochemicals of ethanol extracts of Manilkara zapota L. leaves (ZLE). Sugar compounds, dicarboxylic acids, compounds of phenolic acids and flavonoids groups, and other phytochemicals were detected from the leaves. Antioxidant activity and inhibition potentiality of ZLE against α-glucosidase enzyme, and elastase enzyme activities were evaluated in in vitro analysis. ZLE significantly inhibited activities of α-glucosidase enzyme at a lower concentration (IC50 2.51 ± 0.15 µg/mL). Glucose uptake in C2C12 cells was significantly enhanced by 42.13 ± 0.15% following the treatment with ZLE at 30 µg/mL. It also exhibited potential antioxidant activities and elastase enzyme inhibition activity (IC50 27.51 ± 1.70 µg/mL). Atmospheric pressure chemical ionization mass spectrometry (APCI–MS) detected more m/z peaks than electrospray ionization mass spectrometry (ESI–MS), and both ionization techniques illustrated the biological activities of the detected compounds more thoroughly compared to single-mode analysis. Our findings suggest that APCI along with ESI is a potential ionization technique for metabolite profiling, and ZLE has the potential in managing diabetes by inhibiting α-glucosidase activity and enhancing glucose uptake.


Introduction
Nature is an abundant source of medicinal plants; various natural foodstuffs containing prospective bioactive components which support potential health care are traditionally used as herbal remedies for numerous life-threatening diseases [1]. Medicinal plants for their potential bioactive compounds are used for the ailment of numerous illness like inflammations, oxidative stresses, fatness and hypertensive conditions, diabetic mellitus, skin care, neurological complaints, and cancer, and to regulate expression of various genes [2]. Thus, the demand of discovering and developing new natural therapeutics is of growing interest to the researchers. Screening of plant extracts using different ionization techniques in mass spectrometry is one of the fastest and better ways for the discovery of new bioactive compounds [3].
Manilkara zapota L. is commonly referred to as sapodilla and also locally known as sofeda in Bangladesh, belonging to the Sapotaceae family. It is a long-living, evergreen plant commonly found in India, the south part of Mexico, and Central America [4]. Sapodilla is edible and has a sweet taste with rich flavor. There have been some studies on the fruits, seeds, barks, and leaves of M. zapota for phytochemical characterization and assessment of biological activities such as antioxidant, antimicrobial, antihyperglycemic, hypocholesterolemic, antityrosinase, and antidiabetic properties [4][5][6][7][8][9]. However, there have been few studies on thorough phytoconstituent identification based on advanced mass spectrometry and assessment of α-glucosidase enzyme inhibition activity of M. zapota leaves. Hence, characterization of bioactive secondary metabolites of M. zapota L. leaves and evaluation of their potential biological activities are of greater interest for further investigating the prospective use of sapodilla because of climacteric change, and as this becomes possible, the popularity of this fruit in many countries is rapidly increasing.
Mass spectrometry (MS)-based analytical methods are highly sensitive for the identification, quantification, and structural interpretation of metabolites. Sample ionization is one of the most important steps for mass spectrometry-based metabolomics. Electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and electron impact ionization (EI) are typically used in the field of metabolomics [10]. ESI ionizes competently the strong and moderate polar metabolites, whereas APCI is typically considered for nonpolar and less-polar metabolites [11]. Mass spectrometers using different ionizations provide a mass-to-charge ratio (m/z peaks) of ions by generating mass spectrum, and tandem mass spectrometry (MS/MS) split precursor ions into smaller fragmented ions for the structural elucidation of unknown compounds [12,13].
In this study, direct injection negative-mode ESI-MS and APCI-MS were undertaken to explore a broad range of metabolites from M. zapota L. leaves. Furthermore, various in vitro tests were performed to assess its α-glucosidase inhibition activity, uptake of glucose in C2C12 myotubes, and its antioxidant and elastase inhibition activities. In this study, the experimental findings were also compared with those of other previous studies. Here, a systematic approach for metabolite profiling and bioactivity studies of ZLE was employed.

Identification of Secondary Metabolites
Obtained mass spectra of the extracts from negative-mode ESI-MS and APCI-MS are displayed in Figure 1. Using negative-mode ESI-MS and APCI-MS, 174 and 225 peaks were detected, respectively; 65 peaks were commonly found with both ionization techniques ( Figure S1). Peak lists detected from mass spectra in APCI-MS and ESI-MS are provided in Supplementary Data (Table S1). Proposed compounds were first assumed by comparing their experimental masses in high-resolution mass spectrometry with theoretical calculated masses.
Identification of compounds was carried out using tandem mass spectrometry (MS/MS), which is an eminent technique to elucidate the structure of compounds and thereby very effective to identify unknown compounds comprising complex mixtures. MS/MS fragmentations of all targeted m/z peaks were carried out using the higher-energy collisional dissociation (HCD) activation mode at collision energy (CE) values of 10, 20, and 30 eV.

Antioxidant Effects of ZLE
In this study, antioxidant activity of ZLE was evaluated following DPPH • and ABTS •+ scavenging assays, and the results of the assays are shown in Figure 3A,B. It was found that the percentage inhibition of DPPH • was 25.31%, 36.07%, 56.06%, 80.21%, and 86.28% at 1, 3, 10, 30, and 100 µg/mL, accordingly. There was a progressive radical scavenging trend with a higher concentration of ZLE. IC 50 of ZLE was determined at 7.93 ± 1.43 µg/mL, where ascorbic acid had IC 50 : 5.34 ± 0.27 µg/mL, which indicated that the leaf extracts possessed a powerful DPPH • scavenging activity. Experimental results of the ABTS •+ scavenging assay are displayed in Figure 3B. At 100 µg/mL, the inhibition of ABTS •+ by the leaf extracts was 56.15% and IC 50 was 72.85 µg/mL, which suggested that ZLE had a capacity to donate both electrons and hydrogen, resulting in having antioxidant potential. With the purpose of determining the reducing potentiality of ZLE, ferric-reducing antioxidant power (FRAP) and cupricreducing antioxidant capacity (CUPRAC) assays were conducted. ZLE (100 µg/mL) demonstrated strong reducing power with 53.30 ± 2.85 µM ascorbic acid equivalent FRAP and 40.09 ± 3.61 µM ascorbic acid equivalent CUPRAC value, respectively. These results suggested that ZLE also had a strong electron donating capacity, resulting in having powerful antioxidant capacity ( Figure 3C,D).

α-Glucosidase Inhibitory Activity of ZLE
α-Glucosidase enzymes aid in hydrolysis of carbohydrates including starches during digestion into glucose in the intestine which, in turn, contribute to the increase of blood glucose levels. Inhibiting the α-glucosidase enzyme activity with type 2 diabetes patients may suppress postprandial hyperglycemia [35]. ZLE's ability to inhibit α-glucosidase is presented in Figure 4A and the results showed that ZLE inhibited the activity of α-glucosidase in concentration dependently. ZLE significantly inhibited α-glucosidase activity even at 1 µg/mL and the calculated IC 50 was found very low (2.51 ± 0.15 µg/mL), whereas acarbose had a higher value (IC 50 216.26 ± 1.63 µg/mL). ('ns' indicated nonsignificant; '-' indicated sample was not added and '+' indicated sample was added). Results were as mean ± standard deviation of three analyses (** p < 0.05 versus control using one-way ANOVA).

Effects of ZLE on Cell Viability and Glucose Uptake in C2C12 Myotubes
The effects of ZLE on cell viability in C2C12 cells are presented in Figure 4B. ZLE exhibited moderate to high cytotoxicity at 100 and 200 µg/mL, respectively, whereas at up to 50 µg/mL, there were no cytotoxic effects observed. Glucose uptake potential of ZLE in C2C12 myotubes was measured following the treatment with ZLE at the designated time-points ( Figure 4C). The effect of ZLE (at 30 µg/mL) on basal-or insulin-induced glucose uptake initiated at 0.5 h, which was utmost at 2 h then gradually reduced. Hence, we considered 2 h time period for ZLE administration in the sequential assays. We observed that basal-or insulin-administered glucose uptake augmented in a concentration proportional fashion ( Figure 4D). Basal-and insulin-administered glucose uptake improved remarkably by 42.13 ± 0.27% and 57.74 ± 0.17%, respectively, at the highest concentration of ZLE (30 µg/mL).
Rosiglitazone, a commonly recognized thiazolidinedione group antidiabetic agent, was applied here in the form of positive control. It also substantially augmented basal-and insulin-applied glucose uptake levels by 28.49 ± 0.49% and 50.38 ± 0.32%, respectively.

Elastase Inhibition Activity of ZLE
Experimental results showed that elastase inhibition activity was increased with the increased concentration of ZLE ( Figure S9). IC 50 for elastase inhibition activity was measured at a concentration of 27.51 ± 1.70 µg/mL of ZLE, whereas less inhibition activity was observed for EGCG at a higher concentration.

Discussion
Previous studies found that ethanol extracts of plant materials exhibit good biological activities and contain more bioactive components [36][37][38]. In this study, we performed ethanol extraction of M. zapota leaves (ZLE) and used ZLE for the subsequent experiments.
Mass spectrometry analysis employing direct sample solution injection is a rapid and cost-effective technique compared to other separation procedures [39]. Determination of precise mass and its fragmentation peak using tandem mass spectrometry (MS/MS) aids in the elemental composition information for unknown compound identification. However, for accurate and isomeric identification, we needed to follow a sequential analysis using liquid chromatography mass spectrometry (LC MS), infrared (IR) spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy [40,41].
Today, the ESI technique is widely used for the analysis of singly-charged small molecules to multiply charged large molecules in complex substances [42,43]. APCI is also applicable for the analysis of less polar compounds with a molecular weight of <1500 Da [44]. In this study, direct injection APCI was used along with the ESI technique equipped with high-resolution MS for the phytochemical screening of ZLE. We used negative-mode electrospray ionization (ESI) in mass spectrometry for the metabolite profiling of M. zapota leaves because it yielded enhanced sensitivity and more observable peaks in the mass spectra [45]. Negative-mode ESI-MS presented extensive insights of polyketide classes (flavones, flavanols, flavanones, isoflavones, flavonols, phloroglucinols, anthraquinones, bisanthraquinones, and stilbenes) compounds particularly through the loss of H 2 O, CO, CH 2 CO, and CO in tandem mass spectrometry (MS/MS) [46].
In this study, more m/z peaks were observed in the APCI technique (225 peaks) than ESI technique (174 peaks), and 65 m/z peaks were found common. Using both ESI and APCI, 334 m/z peaks of ZLE were detected, which specified the advantage of using both techniques for enhanced metabolite profiling. As a result, a higher number of compounds could be identified in this study compared to previous ones. One study reported the identification of six compounds, including apigenin-7-O-α-L-rhamnoside, oleanolic acid, caffeic acid, lupeol acetate, myricetin-3-O-α-L-rhamnoside, some hydrocarbons, and fatty acids [5], and another study reported the presence of myricetin-3-O-α-L-rhamnoside and myricetin [47] in the M. zapota leaves. However, in this study, 39 compounds including sugar, dicarboxylic acids, phenolic acids, flavonoids, and other phytochemicals were identified in ZLE (Table 1). DPPH • and ABTS •+ scavenging assays are widely practiced procedures for the determination of antioxidant prospective of natural supplements (e.g., foods, plants). DPPH • and ABTS •+ scavenging methods measure the transfer of hydrogen or electrons from potential antioxidants to free radicals. In this study, ZLE exhibited more significant DPPH • scavenging activity than the ABTS •+ scavenging activity ( Figure 3A,B). FRAP and CUPRAC assays were performed to measure the reducing potentiality of antioxidants in which the reduction is performed with the assistance of compounds which break free-radical chain through donating hydrogen atom [48]. Experimental findings suggest that ZLE exhibited a noticeable antioxidant potential (ascorbic acid equivalent CUPRAC and FRAP values), increased with increasing concentration of the samples ( Figure 3C,D). Taken together, the results demonstrated that ZLE has a strong potential antioxidant activity. The DPPH • and ABTS •+ scavenging abilities of Manilkara zapota fruit with IC 50 37.63 ± 1.18 µg/mL and 73.14 ± 2.84 µg/mL, respectively, have been reported [49]. However, in our present study, we found the DPPH • and ABTS •+ scavenging activities of ZLE with IC 50 7.93 ± 1.43 and 72.85 µg/mL, accordingly, which indicated that M. zapota leaves might be a promising bioactive agent.
It has been reported that caffeic acid and chlorogenic acid (compounds found in ZLE) have remarkable α-glucosidase inhibition ability with IC 50 values of 4.98 and 9.24 µg/mL, respectively [63]. Other phenolic compounds like ferulic acid (IC 50 10.80 ± 0.90 mg/mL) [64] and epicatechin (IC 50 5.86 µg/mL) [65] also possess α-glucosidase inhibition activity. In this study, we identified norathyriol and myricetin from ZLE using negative-mode APCI-MS which possesses a potential α-glucosidase inhibition activity with IC 50 values of 0.81 and 2.09 µg/mL, accordingly [66,67]. Therefore, it was assumed that ZLE showed a significant α-glucosidase inhibition potential due to the combined effect of these compounds.
As we found that ZLE possesses significant α-glucosidase inhibition activity, we carried out the uptake of glucose in C2C12 myotubes for determining the prospects of ZLE as an anti-diabetic agent. Skeletal muscle is an important site for the insulin-mediated glucose uptake, transport, and repository. Furthermore, an increase in glucose uptake in this cell is considered to play a key function in the control of glucose homeostasis and regulation of T2D [68]. ZLE significantly improved the uptake of glucose in C2C12 cells in concentration dependently. We observed synergistic effect of ZLE on glucose uptake in C2C12 cells on exposure to insulin, which suggested the role of ZLE to imitate the insulin-like actions ( Figure 4D).
Collagen and elastin being the prominent proteins in the body modulate important biological and mechanical functions. Collagen is the principal part of the extracellular matrix (ECM), and it provides structural support and strength. On the other hand, elastin is the most prevalent constituent of ECM, which contributes to elasticity in living tissues [69][70][71]. Elastin is degraded by the activation of elastase upon UV irradiation [72]. We found in this study that ZLE strongly inhibited elastase enzyme activity (Supplementary Figure S9). In our previous study, it was found that different combinations of epicatechin, syringic acid, and vanillic acid had a substantial interactive effect on inhibitory ability of elastase [73]. Sirinya et al. found the elastase inhibition activity of Manilkara zapota fruit with IC 50 35.73 ± 0.61 µg/mL [49], whereas in our study ZLE exhibited elastase inhibitory action with IC 50 27.51 ± 1.70 µg/mL. Therefore, M. zapota leaves possessed more significant elastase inhibition activity than the fruits.

Plant Materials and Extraction
Leaves of M. zapota L. were collected from the Khulna area of Bangladesh in June 2018. Taxonomic identification (accession number DACB-23801) was carried out at the Bangladesh National Herbarium. Shade-dried fresh leaves were ground into fine homogenized powder using a mortar and pestle. Of the fine power, 100 g was processed in three cycles with a reflux system of the extraction process of 6 h using ethanol. A Whatman filter paper (Schleicher and Schuell, Keene, NH, USA) was used to filter the extracts. Evaporation of the solvent was done using a rotary evaporator (SB-1000, Tokyo Rikakikai Co. Ltd., Tokyo, Japan). At last, we obtained powdered extracts of the sample after using a freeze-dryer (MCFD 8518, Ilshin Biobase Co. Ltd., Goyang, Korea).

Sample Preparation
Stock solution of ZLE was prepared by dissolving the extract in 100% ethanol (10 mg/mL), and subsequently, the stock solution was diluted using ethanol and water for MS analysis. Homogenization of the prepared samples solution was made following 1 min vortex and 5 min sonication in a sonication bath (Powersonic 410, Hwashin Technology Co., Gyeonggi, South Korea). For in vitro analysis, the extracts at a concentration of 30 mg/mL were dissolved in DMSO and deionized water (50:50, v/v), and subsequent diluted solutions were made using deionized water.

Mass Spectrometry Analysis
Negative-mode ESI-MS and APCI-MS experiments were executed with Q-Exactive™ Orbitrap Mass Spectrometer (Thermo Fisher Scientific Inc., San Jose, CA, USA). A 500-µL syringe (Hamilton Company Inc., Reno, NV, USA) and a syringe pump (model 11, Harvard, Holliston, MA, USA) were used to infuse the sample solution into the ESI source at 20 µL/min. Typical negative-mode ESI-MS conditions were at mass resolution 140,000 (full width at half maximum, FWHM), sheath gas flow rate 6, auxiliary gas flow rate 2, sweep gas flow rate 0 (arbitrary units), spray voltage 4.00 kV, capillary temperature 400 • C, S-lens RF level 50, and automatic gain control 5 E 6 . Highly pure (99.99 %) nitrogen obtained from the evaporation of liquid nitrogen was applied as the sheath, auxiliary, and sweep gas. Negative-mode external calibration was performed using Pierce Velos solution (Thermo Fisher Scientific, Rockford, IL, USA) into the ESI interface.
Mass spectra were acquisitioned from m/z 50 to m/z 750 for both ionizations and 10-30 eV collision energy was applied in the tandem mass spectrometry (ESI-MS/MS and APCI-MS/MS).
Experimental conditions for ESI and APCI mass spectrometry were optimized considering the most abundant peak's intensity to obtain a good quality spectrum. Operating parameters for ESI-MS and APCI-MS were optimized and briefed in the Supplementary Data ( Figures S10-12, Tables S2 and S3).

Data Processing
Obtained mass spectrometry data were handled using Xcalibur 3.1 with Foundation 3.1 (Thermo Fisher Scientific, USA). A signal-to-noise ratio (S/N) value of 100 was considered to detect m/z peaks, and exported peak lists were further processed using Microsoft Excel. Elemental formulas for the detected m/z peaks were assigned within typical conditions of C c H h N n O o , counting c unlimited, h unlimited, and 0 ≤ n ≤ 10, and 0 ≤ o ≤ 50 using MIDAS Formula Calculator. ChemDraw Professional 15.0 (PerkinElmer, Waltham, MA, USA) was used to draw the structures of the identified compounds. A Venn diagram for the overlapped peaks was generated by FunRich 3.1.3 [74].

Antioxidant Activity Assays
The DPPH • scavenging capability of ZLE was determined following a previous methodology [75]. Into 2-µL sample solutions of different concentrations (1, 3, 10, 30, and 100 µg/mL), 198 µL of 0.2 mM solution of DPPH in 50% ethanol was added. The mixture was allowed to stand for 10 min at 25 • C, and the absorbance was recorded by a microplate reader (Multiskan GO 51119200; Thermo Fisher Scientific Oy, Ratastie, 01620 Vantaa, Finland) at 517 nm. Ascorbic acid was used as a reference antioxidant compound.
The ABTS •+ scavenging activity was determined adapting the method described by Re et al. [76] with slight modifications, and 2 µL of different concentrations of ZLE (1, 3, 10, 30, and 100 µg/mL) was mixed with 198 µL ABTS solution, and the absorbance was recorded at 734 nm. Ascorbic acid was used as a reference antioxidant compound, and both DPPH • and ABTS •+ scavenging activities were calculated using Equation (1). (1) where A control is the control's absorbance and A sample is the sample's absorbance. Data were obtained for three samples.

Radical-scavenging activity (% inhibition) = [(A control −
The ferric-reducing antioxidant power (FRAP) assay and cupric-reducing antioxidant capacity (CUPRAC) assay were performed following the reported methods [77,78]. Then, 2 µL of the sample solution of ZLE at different concentrations (1, 3, 10, 30, and 100 µg/mL) was mixed with 198 µL of the FRAP reagent, and the absorbance was measured at 595 nm. Ascorbic acid was also used as a reference compound. In the case of the CUPRAC assay, the samples at different concentrations (1,3,10,30, and 100 µg/mL) were added with the mixture of 10 mM CuCl 2 , 7.5 mM neocuproine, and 1 M ammonium acetate buffer (pH 7.0). The mixture was incubated for 1 h at 25 • C temperature, and the absorbance was determined at 450 nm. Ascorbic acid was used as a reference compound, and the reducing capability was marked as ascorbic acid equivalent (in micromolar).

Inhibitory Assay of α-Glucosidase
The capability of ZLE to restrain α-glucosidase activity was determined in accordance with the reported method [79]. With 0.2 U/mL of α-glucosidase in 0.1 M sodium phosphate buffer (pH 7.0), the sample solutions of 2 µL of different concentrations (1, 3, 10, 30, and 100 µg/mL) were mixed, and incubated for 10 min at 37 • C. Then, pNPG (substrate) was mixed into the prepared solution to terminate enzyme-substrate activity at 37 • C for 1 h. The enzyme-substrate reaction was carried out at 37 • C for 30 min. Absorbance of the mixture was figured out utilizing a 96-well plate microplate reader (Multiskan GO 51119200; Thermo Fisher Scientific Oy, Ratastie, 01620 Vantaa, Finland) at 405 nm. Acarbose as a positive control was employed and inhibitory percentage was calculated following the Equation (2).

Inhibition of enzyme activity (%) = [(A control −
where A control is the control's absorbance and A sample is the sample's absorbance. Three sets of the sample's data were obtained.

Cell Culture and Cell Viability Assay
C2C12 myotubes (American Type Culture Collection, Manassas, VA, USA) were grown in Dulbecco's modified eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and streptomycin-penicillin (100 µg/mL and 100 U/mL, accordingly; Hyclone, Mordialloc, VIC, Australia) at 37 • C under a 5% CO 2 atmosphere. The viability of C2C12 cells were detected following the tetrazolium dye colorimetric test (MTT) assay. At first C2C12 cells were grown at a density of 1 × 10 5 cells/well in 96-well plates for 24 h. They were treated with different concentrations of ZLE after reaching 90% confluence. After 24 h of incubation, an MTT reagent was added to each well and the plate was incubated for 1 h at 37 • C. The media was removed and then the wells were washed two times with phosphate-buffered saline (PBS) (pH 7.4). The intracellular formazan was solubilized in 100% DMSO. The absorbance was recorded by microplate reader (Perkin Elmer, Wallac Victor 3, MA, USA) at 570 nm and the viability percentage was determined.

Muscle Differentiation and Glucose Uptake Assay
C2C12 cells were grown at a density of 1 × 10 5 cells/well in 96-well plates with DMEM supplemented with 10% fetal bovine serum (FBS) and 1% streptomycin-penicillin at 37 • C under 5% CO 2 atmosphere. The cells were differentiated in DMEM containing 2% horse serum for 5 days after reaching confluence. The cells were allowed to starve in low glucose serum-free DMEM for 24 h. Thereafter, 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4yl) amino]-D-glucose (2-NBDG) assay was carried out to determine glucose uptake [80]. Briefly, cells were treated with different concentrations of ZLE and insulin (100 nM) for 30 min, followed by 20 µM of 2-NBDG for 6 h. The cells were rinsed three times using cold PBS after incubation and uptake of 2-NBDG uptake recorded with a microplate reader (Perkin Elmer, Wallac Victor 3, MA, USA) at an excitation wavelength of 490 nm and emission wavelengths of 535 nm, accordingly.

Elastase Inhibition Assay
ZLE's potentiality to hinder elastase activity was employed based on a previous study [73]. The reaction was performed in a mixture of 0.04 U elastase and 0.1 M Tris-HCl buffer (pH 8.0) with 0.78 mM N-succinyl-(Ala)-3-p-nitroanilide (substrate). With 100 µL of the substrate solution, different concentrations of the sample solution (1, 3, 10, 30, and 100 µg/mL) were mixed and then added to 100-µL enzyme solution. Then, mixer absorbance was figured out using a microplate reader (Multiskan GO 51119200; Thermo Fisher Scientific Oy, Ratastie, 01620 Vantaa, Finland) at 405 nm. Epigallocatechin gallate (EGCG) as a positive control was used, and the elastase inhibition percentage was calculated following above mentioned Equation (2).

Statistical Analysis
All results were as mean ± standard deviation. Linear regression analyses were carried out to calculate half maximal inhibitory concentration values (IC 50 ). Statistical analyses were accomplished through one-way analysis of variance (ANOVA), following Dennett's test by SigmaPlot 12.5 (Systat Software Inc., San Jose, CA, USA). Differences were counted significant if p < 0.05.

Conclusions
In this study, we found that combining ESI and APCI techniques with mass spectrometry maximized the coverage of m/z peak detection rather than using a single ionization technique. The combination of ESI and APCI ionization techniques in mass spectrometry provided us with more detailed characteristic features of metabolites and biological activities of natural compounds. We rapidly determined the secondary metabolites of M. zapota leaves using direct injection mode mass spectrometry. We identified 39 different phytochemicals of M. zapota leaves using negative-mode ESI-MS and APCI-MS. It was found that the leaf extracts of M. zapota had a significant α-glucosidase enzyme inhibition activity and also dose-dependently augmented glucose uptake alone or in combination with insulin in C2C12 myotubes. M. zapota leaves extracts also showed strong antioxidant and elastase inhibition potential. Compounds identified using negative-mode APCI-MS together with negative-mode ESI-MS strongly demonstrated the α-glucosidase activity of ZLE. It can be concluded that combining ESI and APCI with high-resolution mass spectrometry is an enhanced approach for metabolite profiling and biological activity studies. The findings of this work suggest that M. zapota leaves can be a potential resource for antidiabetic agents, antioxidants, and elastase enzyme inhibitors. Furthermore, we need to carry out liquid chromatography mass spectrometry (LC-MS) to determine the concentration of bioactive compounds. In vivo biological assays are also needed to be performed for the better characterization of antidiabetic ability of M. zapota leaves. Finally, we are interested to continue research on selective bioactive compounds for enhanced antidiabetic activity.  Figure S1: Venn diagram showing the overlapping peaks detected in (-) mode electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) mass spectrometry (MS). Figure S2: (-) mode electrospray ionization tandem mass spectrometry (ESI MS/MS) fragmentation pattern of succinic acid. Figure S3: (-) mode electrospray ionization tandem mass spectrometry (ESI MS/MS) fragmentation pattern of malic acid. Figure S4: (-) mode electrospray ionization tandem mass spectrometry (ESI MS/MS) fragments of gallic acid. Figure S5: (-) mode electrospray ionization tandem mass spectrometry (ESI MS/MS) fragments of epicatechin. Figure S6: (-) mode atmospheric pressure chemical ionization tandem mass spectrometry (APCI MS/MS) fragments of myricetin. Figure S7: (-) mode electrospray ionization tandem mass spectrometry (ESI MS/MS) fragments of quinic acid. Figure S8: (-) mode atmospheric pressure chemical ionization tandem mass spectrometry (APCI MS/MS) fragments of norathyriol. Figure S9: Elastase-inhibition activities of Manilkara zapota leaves ethanol extracts (ZLE). Figure S10: Optimization of negative-mode electrospray ionization mass spectrometry (ESI-MS) operating parameters. Figure S11: Optimization of (-) mode atmospheric pressure chemical ionization mass spectrometry (APCI-MS) operating parameters. Figure S12: (-) mode atmospheric pressure chemical ionization (APCI) mass spectra at different vaporizer temperatures. Table S1. Detected peak lists in APCI and ESI MS. Table S2. Optimized operational conditions for negative-mode electrospray ionization mass spectrometry (ESI-MS). Table S3. Optimized operational conditions for (-) mode atmospheric pressure chemical ionization mass spectrometry (APCI-MS).