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. 2022 Nov 1;11(11):2171. doi: 10.3390/antiox11112171

A Study on Chemical Characterization and Biological Abilities of Alstonia boonei Extracts Obtained by Different Techniques

Adriano Mollica 1, Gokhan Zengin 2, Kouadio Ibrahime Sinan 2, Marcella Marletta 3, Stefano Pieretti 4, Azzurra Stefanucci 1,*, Ouattara Katinan Etienne 5, József Jekő 6, Zoltán Cziáky 6, Mir Babak Bahadori 7, Carene Picot-Allain 8, Mohamad Fawzi Mahomoodally 8,9,10
Editor: Stanley Omaye
PMCID: PMC9686497  PMID: 36358543

Abstract

In the quest for novel therapeutic agents from plants, the choice of extraction solvent and technique plays a key role. In this study, the possible differences in the phytochemical profile and bioactivity (antioxidant and enzyme inhibitory activity) of the Alstonia boonei leaves and stem bark extracted using water, ethyl acetate and methanol, and different techniques, namely infusion, maceration and Soxhlet extraction, were investigated. Data collected showed that methanol extracts of both A. boonei leaves (48.34–53.08 mg gallic acid equivalent [GAE]/g dry extract) and stem bark (37.08–45.72 mg GAE/g dry extract) possessed higher phenolic content compared to the ethyl acetate extracts (leaves: 30.64–40.19 mg GAE/g; stem bark: 34.25–35.64 mg GAE/g). The methanol extracts of A. boonei leaves showed higher radical scavenging and reducing capacity, and these findings were in accordance with phenolic content results. In general, water extracts of A. boonei leaves and stem bark obtained by infusion were poor inhibitors of acetylcholinesterase, α-amylase, α-glucosidase, and tyrosinase, except for butyrylcholinesterase. The chemical profiles of the extracts were determined by UHPLC–MS and the presence of several compounds, such as phenolic acids (caffeic, chlorogenic and ferulic acids, etc.), flavonoids (rutin and isoquercetin) and flavonolignans (Cinchonain isomers). Cell viability was tested using the human peripheral blood monocytic cell line (THP-1), and the extracts were safe up to 25 μg/mL. In addition, anti-inflammatory effects were investigated with the releasing of IL-6 TNF-α and IL-1β. In particular, stem bark extracts exhibited significant anti-inflammatory effects. Data presented in this study highlight the key role of solvent choice in the extraction of bioactive secondary metabolites from plants. In addition, this study appraises the antioxidant and enzyme inhibitory action of A. boonei leaves and stem bark, which are extensively used in traditional medicine.

Keywords: Alstonia, phenolics, extraction methods, anti-inflammatory, natural agents

1. Introduction

Alstonia boonei, belonging to the Apocynaceae family, has been extensively used in traditional medicine. This ethnomedicinal plant, commonly found in tropical and subtropical Africa, Australia, Southeast Asia, and Central America, was found to exhibit several biological and pharmacological actions [1]. A. boonei is a large deciduous tree, measuring up to 45 m, with a deeply fluted trunk that can reach 1.2 m in diameter and a greyish-green or grey bark, from which, a copious milky latex is exuded [2]. In ethnomedicine, A. boonei is used to treat malaria, sore throat, cough, toothache, snake bites, ulcer, jaundice, skin conditions, arthritis, rheumatism and hypertension, and is also used as antihelminthic [1,2,3]. Pharmacological studies conducted on A. boonei stem bark methanol extract established its anti-inflammatory, analgesic and antipyretic activities [4]. A. boonei combined with Khaya ivorensis exhibited antiplasmodial activity in the murine malaria model, thereby validating its traditional use in the treatment of malaria [5]. Traditional use of A. boonei as an anti-inflammatory agent was validated by a study conducted by Enechi, Odo and Onyekwelu [2], who reported that the ethanol extract of the stem bark of A. boonei exhibited a remarkable inhibitory effect on leucocyte migration. An aqueous fraction of 70% methanol extract of A. boonei leaves demonstrated significant anti-inflammatory and antioxidant activities in carrageenan and formaldehyde-induced arthritic rats [1]. A. boonei stem bark ethanol extract showed inhibitory action against Escherichia coli [6]. The ethyl acetate extract of A. boonei leaves showed potent inhibitory activity against key enzymes targeted in the management of diabetes type II, namely, α-amylase (IC50: 3.17 mg/mL) and α-glucosidase (IC50: 0.70 mg/mL). Besides, administration of ethyl acetate extract to starch-loaded Wistar rats showed a significant reduction in the blood glucose level of the rats within 2 h [7].

The plant secondary metabolites present possess versatile therapeutic actions and have been shown to exhibit inhibitory action on several enzymes. In this sense, plant secondary metabolites capable of mitigating the activity of enzymes targeted in the management of diabetes type II, Alzheimer’s disease and epidermal hyperpigmentation represent interesting possibilities for new drug development. Diabetes type II, the most common type of diabetes, is a chronic metabolic condition, which is characterised by hyperglycemia as a result of defective insulin secretion or functioning [8]. Alzheimer’s disease is the most common neurodegenerative geriatric condition, characterized by progressive memory impairment and cognitive deficits [9]. The incidence and prevalence of diabetes type II and Alzheimer’s disease are rapidly growing, and are affecting millions of individuals globally. It has been claimed that anti-diabetic agents possessing low or no inhibitory action against α-amylase were favorable, since high α-amylase inhibition has been associated with poor digestion of ingested carbohydrates, causing abdominal discomfort [10]. In this sense, one of the therapeutic strategies used to manage diabetes type II focuses on the inhibition of α-glucosidase, which is situated in the epithelial mucosa of the small intestine [11]. Tyrosinase, an enzyme containing copper, is essential for the biosynthesis of melanin, a brown pigment that shields human skin from ultraviolet radiation [12]. However, epidermal hyperpigmentation problems and dermatological conditions are more likely to develop when melanin production and accumulation are excessive. Cosmetic and dermatological tyrosinase inhibitors are used to treat hyperpigmented skin conditions such as acne scars, age spots and melasma [13].

In the quest for novel therapeutic agents from natural sources, namely plants, extraction is a fundamental step that will determine the phytochemical profile of the extracts, and subsequently, their bioactivity. In fact, the choice of the extraction solvent and technique has always been a challenge for researchers. Several studies have shown the significant difference in bioactivity of plant extracts prepared from different solvents. Indeed, the choice of the extraction solvent depends on the nature of phytochemicals being targeted, in case the compound is known. However, in the quest for novel bioactive compounds with unknown structures, extracting using solvents of different polarities might provide better insight into the phytochemical profile of a plant, and eventually help identify interesting bioactive compounds.

In this regard, the present study set out to investigate the possible differences in the phytochemical profile and bioactivity (antioxidant, enzyme inhibitory and anti-inflammatory activity) of the A. boonei leaves and stem bark extracted using different solvents, namely water, ethyl acetate and methanol, and using different extraction techniques, namely, infusion, maceration and Soxhlet extraction. The chemical compounds of the extracts were characterized by the UHPLC–MS technique. It is expected that data gathered from the present investigation will provide an insight into the possible effects of extraction solvents and methods on the observed bioactivity of A. boonei leaves and stem bark. The obtained results could open a new horizon in the production of functional applications with A. boonei leaves or stem bark.

2. Materials and Methods

2.1. Plant Material and Preparation of Extracts

In the summer of 2019, the leaves and stem bark of A. boonei were harvested in the village of Prikro (Brobo City, Côte d’Ivoire). The National Floristic Centre (The Université Félix Houphout-Boigny, Abidjan, Côte d’Ivoire) identified the plant. Deposits of voucher specimens were made at the herbarium of the aforementioned institution. Leaves and stem barks were carefully separated, and they were dried under dark conditions for one week at room temperature.

Ethyl acetate, methanol and water were used as solvents in the present study. Methanol allows for the extraction of both hydrophilic and hydrophobic compounds from plant materials, and thus, we could gain more insights for plant extracts. It has been shown in the literature that methanol is commonly used and is more effective as an extraction solvent for Alstonia boonei [14,15,16,17,18,19]. With this in mind, methanol was selected as one solvent for the current study.

In the preparation of plant extracts, we used three techniques: maceration, Soxhlet and infusion. The maceration technique was performed either stirred or not stirred. Using the technique, the plant materials (10 g) were stirred with 200 mL of the solvents (ethyl acetate or methanol) at 250 rpm for 24 h at room temperature. Without stirring, the plant materials (10 g) were kept in the solvents (ethyl acetate or methanol) for 24 h in the dark at room temperature. With the Soxhlet method, the plant materials (10 g) were extracted with the solvents (ethyl acetate or methanol) in a Soxhlet apparatus for 6 h. After the duration of extraction, each extract was filtered with Whatman filter paper and the solvents were removed with a rotary-evaporator. In infusion, the plant material (10 g) was steeped in boiling water (200 mL) for 15 min before being filtered. For 48 h, the mixture was lyophilized to remove water. All extracts were kept at 4 °C until analysis.

2.2. Profile of Bioactive Compounds

The extracts were dissolved in methanol (for ethyl acetate and methanol extracts) and water (for infusion). Quantification of the total phenolic and flavonoid content was performed using Folin–Ciocalteu and AlCl3 assays, respectively [20]. Gallic acid equivalents (mg GAEs/g extract) and rutin equivalents (mg REs/g extract) were used to describe the outcomes of the two tests. All experimental details are given in the Supplemental Materials.

2.3. UHPLC–MS Analysis

Compositions of the different extracts were determined using a Dionex Ultimate 3000RS UHPLC instrument(Thermo Scientific, Waltham, MA, USA). The extract was filtered through a 0.22 μm PTFE filter membrane (Labex Ltd., Budapest, Hungary) before HPLC analysis. Extracts were injected onto a Thermo Accucore C18 (100 mm × 2.1, mm i. d., 2.6 μm) column themostated at 25 °C (±1 °C). The solvents used were water (A) and methanol (B). Both were acidified with 0.1 % formic acid. The flow rate was maintained at 0.2 mL min−1. The elution gradient was isocratic 5 % B (0–3 min), a linear gradient increasing from 5% B to 100% (3–43 min), 100% B (43–61 min), a linear gradient decreasing from 100% B to 5% (61–62 min) and 5 % B (62–70 min). The column was coupled with a Thermo Q-Exactive Orbitrap mass spectrometer (Thermo Scientific, Waltham, MA, USA) equipped with electrospray ionization source. Spectra were recorded in positive- and negative-ion mode [21].

2.4. Determination of Antioxidant and Enzyme Inhibitory Effects

For antioxidant and enzyme inhibitory assays, the extracts were dissolved in methanol (for ethyl acetate and methanol extracts) and water (for infusion). Methods for measuring the extracts’ antioxidant and enzyme-inhibiting properties were previously described [22,23]. Trolox (for radical scavenging and reducing power assays) and EDTA were used as positive controls. Each of the radical scavenging activities (ABTS•+, DPPH•), as well as the reducing capacities (CUPRAC and FRAP), were reported in milligrams of Trolox equivalent (TE) per milligram of extract. Total antioxidant activity (phosphomolybdenum assay, PBD) was reported in mmol TE/g extract, while metal chelating ability (MCA) was reported as mg EDTAE/g extract. The inhibitory activities were tested against AChE, BChE, tyrosinase, amylase and glucosidase. Galanthamine (for AChE and BChE), kojic acid (for tyrosinase) and acarbose (for amylase and glucosidase) were used as standard enzyme inhibitors. The results were expressed as the equivalents of the standards (galanthamine equivalents (GALAE), kojic acid equivalents (KAE) and acarbose equivalents (ACAE). All experimental details are given in the Supplemental Materials.

2.5. Cell Line

THP-1 cells, which originate from human peripheral blood mononuclear cells, were obtained from the American Type Culture Collection (Bethesda, MD, USA). Cells were grown in a 37 °C, 5% CO2 incubator with RPMI-1640 medium containing 2 mM L-glutamine, 100 U/mL streptomycin-penicillin and 10% heat-inactivated fetal bovine serum (Sigma Aldrich, St. Louis, MO, USA). At 37 degrees Celsius for 24 h, 1 × 106 THP-1 cells were plated in 6-well culture plates and differentiated into macrophages with 100 ng/mL phorbol-12-myristate-13-acetate (PMA, St. Louis, MO, USA).

2.6. MTT Assay

Once THP-1 monocytes had been differentiated into macrophages, the effect of A. boonei extracts on the viability of the LPS-stimulated macrophages was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The extracts were solubilized in RPMI medium containing 0.1% DMSO. Twenty microliters of MTT (1 mg/mL in PBS) were added to each well after A. boonei had been present for 24 h at 25, 50 and 100 μg/mL, and the plates were incubated for 4 h under standard culture conditions. After that, 100 μL of DMSO was added to the cells. With the help of a microplate reader (MultiskanTM FC Microplate Photometer, Thermo ScientificTM, Waltham, MA, USA), the absorbance was determined to be 570 nm. The data were expressed as a percentage, with 100% corresponding to the value obtained for the solvent control.

2.7. IL-6, TNF-α and IL-1β Assays for Anti-Inflammatory Activity

In order to increase cytokine production, macrophages were treated with LPS at a final concentration of 0.1 μg/mL, and then with A. boonei extracts at 25, 50 and 100 μg/mL. The extracts were solubilized in RPMI medium containing 0.1% DMSO. Dexamethasone (Sigma Aldrich, USA) was employed as a positive control (0.04 μg/mL). A centrifuge was used to separate the supernatant from the cells after 24 h of incubation. Quantification of IL-6, IL-1β and TNF-α secretion was achieved by following the ELISA manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA). The ELISA results were normalized to the MTT values to cut down on the variation that could have resulted from differences in cell density. Cytokine concentration in the negative control (cells treated with LPS alone) was set at 100%. All data from the A. boonei extract tests were normalized by dividing them by the value obtained from the negative control.

2.8. Statistical Analysis

The results of three independent experiments were used to calculate the values for antioxidant and enzyme inhibitory assays (mean ± SD). One-way ANOVA with Tukey’s assay was used to compare the extracts’ antioxidant and enzyme-inhibiting properties. To conduct the statistical analysis, XlStat 16.0 (Addinsoft Inc., New York, NY, USA)was used.

The values are typically presented in a mean ±SEM format in cellular analysis. One-way analysis of variance was used to determine statistical significance between means, and Dunnett’s multiple comparisons test was used to further examine the data. The significance level of 0.05 was considered to be statistically significant. For the statistical analysis, we used GraphPad Prism 9.0 (San Diego, CA, USA).

3. Results and Discussion

3.1. Total Phenolic and Flavonoid Content

The total phenolic and flavonoid contents of the different extracts of A. boonei leaves and stem bark are summarised in Table 1. In general, methanol extracts showed higher phenolic contents as compared to the ethyl acetate extracts, suggesting that methanol was a better extracting solvent as compared to ethyl acetate. It was also observed that A. boonei leaves contained higher flavonoid content compared to the stem bark (Table 1). It is worth mentioning that the flavonoid content of ethyl acetate extracts of A. boonei stem bark was higher compared to the methanol extracts. The water extracts obtained by infusion also showed high phenolic content. Alkaloids, tannins, saponins, flavonoids, cardiac glycosides and ascorbic acid were previously identified in the methanol and water extracts A. boonei stem bark [14].

Table 1.

Total bioactive compounds and total antioxidant capacity (by phosphomolybdenum assay) of the studied extracts *.

Parts Extraction Methods/Solvent Total Phenolic Content
(mg GAE/g) 		Total Flavonoid Content
(mg RE/g) 		Phosphomolybdenum
(mmol TE/g)
Leaves Infusion-water 51.08 ± 0.21 b 4.18 ± 0.16 e 2.15 ± 0.08 cd
MAC-EA 30.64 ± 0.38 f 34.51 ± 0.39 c 1.78 ± 0.04 d
MAC-MeOH 48.34 ± 0.38 c 37.28 ± 0.56 b 2.55 ± 0.27 abc
MAC-EA (not stirred) 35.81 ± 0.50 e 34.81 ± 0.46 c 2.61 ± 0.15 ab
MAC-MeOH (not stirred) 53.08 ± 0.24 a 37.65 ± 0.20 b 2.78 ± 0.15 a
SE-EA 40.19 ± 0.67 d 8.14 ± 0.31 d 2.25 ± 0.09 bc
SE-MeOH 49.27 ± 0.30 c 39.10 ± 0.42 a 2.37 ± 0.13 bc
Stem bark Infusion-water 31.99 ± 0.15 f 0.54 ± 0.08 e 1.30 ± 0.02 c
MAC-EA 35.64 ± 0.62 d 3.05 ± 0.09 b 2.06 ± 0.04 a
MAC-MeOH 39.64 ± 0.03 b 1.85 ± 0.06 d 1.54 ± 0.08 bc
MAC-EA (not stirred) 34.38 ± 0.44 de 2.69 ± 0.08 bc 1.84 ± 0.13 ab
MAC- MeOH (not stirred) 45.72 ± 0.28 a 2.45 ± 0.08 c 1.91 ± 0.22 a
SE-EA 34.25 ± 0.42 e 4.07 ± 0.33 a 1.60 ± 0.09 bc
SE-MeOH 37.08 ± 0.79 c 2.76 ± 0.06 bc 1.54 ± 0.07 bc
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* Values expressed are means ± S.D. of three parallel measurements. GAE: Gallic acid equivalent; RE: Rutin equivalent; TE: Trolox equivalent. MAC: Maceration; SE: Soxhlet extraction; EA: Ethyl acetate; MeOH: Methanol. Different letters indicate significant differences in the tested extracts of each parts (p < 0.05).

3.2. Chemical Characterization

UHPLC–MS analysis led to the characterization of plant metabolites in the extracts of A. boonei. Obtained data, including identity of compounds, their molecular formula, mass fragments and retention times can be found in Table 2, Table 3, Table 4, Table 5, Table 6 and Table 7. Total ion chromatograms are given in Supplemental Materials (Figures S1–S12). Some of the characterized metabolites are well-known bioactive compounds, such as chlorogenic acid, caffeic acid, 4-coumaric acid and quercetin. These phenolic compounds have strong bioactivities, including antioxidant, antimicrobial, anti-inflammatory, neuroprotective, hypotensive and cardioprotective effects [24,25,26,27]. Quercetin derivatives, such as rutin, quercitrin and isoquercitrin, were other common compounds in the investigated extracts. These are important natural products exerting valuable therapeutic effects [28,29]. Most of the observed antioxidant effects; reducing ability, radical scavenging, enzyme inhibitory and anti-inflammatory activities, from different extracts of A. boonei could be related to these phenolic and flavonoid glycosides. In addition to the mentioned compounds, other natural substances, such as loganic acid (an iridoid), voacangine (an alkaloid) and quinic acid (a cyclitol), were also found in A. boonei. The highest number of compounds was found in Mac-MeOH- not stirred (61), and the lowest number of compounds was identified in Mac-ET- not stirred (26). To the best of our knowledge, this work is the first comprehensive phytochemical analysis on different parts and extracts of A. boonei.

Table 2.

Chemical characterization of leaves—infusion.

No. Name Formula Rt [M + H]+ [M − H]− Fragment 1 Fragment 2 Fragment 3 Fragment 4 Fragment 5 Literature
1 Unidentified dihydroxybenzoic acid derivative C22H20O12 13.58 475.08765 299.0776 153.0181 137.0232 109.0281
2 1 Chlorogenic acid (3-O-Caffeoylquinic acid) C16H18O9 14.86 355.10291 163.0390 145.0285 135.0442 117.0338 89.0388
3 3-O-Feruloylquinic acid C17H20O9 15.11 367.10291 193.0500 191.0552 173.0443 134.0361 93.0333
4 Caffeic acid C9H8O4 15.16 179.03444 135.0439 107.0487
5 Loganic acid C16H24O10 15.72 375.12913 213.0764 169.0860 151.0751 113.0230 69.0330
6 Vallesamine or isomer C20H24N2O3 15.79 341.18652 309.1599 236.1062 208.1128 194.0967 168.0807
7 Chryptochlorogenic acid (4-O-Caffeoylquinic acid) C16H18O9 16.21 355.10291 163.0390 145.0285 135.0442 117.0339 89.0390
8 4-O-(4-Coumaroyl)quinic acid cis isomer C16H18O8 16.27 337.09235 191.0557 173.0445 163.0389 119.0488 93.0330
9 Swertiamarin or isomer C16H22O10 16.73 375.12912 213.0758 195.0655 177.0549 151.0391 107.0496
10 Lochnericine or isomer C21H24N2O3 17.02 353.18652 335.1773 291.1494 166.0863 158.0966 144.0808
11 Unidentified N-formylalkaloid 1 isomer 1 C22H24N2O5 17.30 397.17635 369.1812 337.1547 299.1392 267.1128 224.0704
12 Secologanoside C16H22O11 17.32 389.10839 345.1197 209.0451 183.0657 165.0545 69.0330
13 Lochnericine or isomer C21H24N2O3 17.39 353.18652 335.1745 291.1493 166.0864 158.0966 144.0808
14 5-O-(4-Coumaroyl)quinic acid C16H18O8 17.51 337.09235 191.0554 173.0445 163.0389 119.0488 93.0331
15 Picralinal or isomer C21H22N2O4 17.65 367.16578 349.1910 339.1704 307.1441 269.1283 180.1020
16 Quercetin-O-hexoside-O-rutinoside isomer 1 C33H40O21 17.71 771.19839 609.1485 462.0805 301.0355 300.0285 299.0202
17 2 Echitamine C22H29N2O4 17.92 385.21273 367.2011 349.1919 310.1438 250.1227 220.1121 [30]
18 Quercetin-O-hexoside-O-rutinoside isomer 2 C33H40O21 17.96 771.19839 609.1474 463.0885 462.0807 301.0354 299.0201
19 Sweroside or isomer C16H22O9 18.03 359.13421 197.0811 179.0704 151.0754 127.0392 111.0809
20 Picralinal or isomer C21H22N2O4 18.04 367.16578 349.1948 339.1704 307.1442 269.1285 194.0603
21 Quebrachidine or isomer C21H24N2O3 18.05 353.18652 335.1773 324.1595 303.1491 293.1651 275.1539
22 Unidentified N-formylalkaloid 1 isomer 2 C22H24N2O5 18.11 397.17635 369.1813 337.1546 299.1393 256.0970 224.0709
23 4-O-(4-Coumaroyl)quinic acid C16H18O8 18.13 337.09235 191.0556 173.0445 163.0390 119.0489 93.0331
24 Nα-Formyl-12-methoxyechitamidine C22H26N2O5 18.14 399.19200 371.1967 339.1705 299.1406 267.1127 198.0913 [31]
25 1 4-Coumaric acid C9H8O3 18.50 163.03952 119.0488 93.0331
26 5-O-Feruloylquinic acid C17H20O9 18.51 367.10291 193.0501 191.0553 173.0445 134.0361 93.0330
27 Vallesamine or isomer C20H24N2O3 18.55 341.18652 309.1598 237.1023 209.1074 194.0960 168.0806
28 N-Formylechitamidine C21H24N2O4 18.91 369.18143 351.1710 341.1862 309.1599 202.0874 [32]
29 4-O-Feruloylquinic acid C17H20O9 19.03 367.10291 193.0501 191.0554 173.0445 134.0362 93.0330
30 5-O-(4-Coumaroyl)quinic acid cis isomer C16H18O8 19.70 337.09235 191.0554 173.0444 163.0389 119.0487 93.0331
31 Unidentified N-formylalkaloid 2 C22H24N2O4 20.33 381.18143 353.1862 339.1706 321.1599 263.1175 212.0942
32 Secologanoside methyl ester C17H24O11 20.76 403.12404 371.0987 223.0606 179.0551 165.0545 121.0281
33 Lagumicine or isomer C20H22N2O4 21.06 355.16578 337.1548 214.0863 200.0709 182.0603 154.0652
34 Akuammicine C20H22N2O2 21.47 323.17596 294.1491 291.1495 280.1342 263.1543 234.1286
35 Unidentified alkaloid isomer 1 C21H24N2O4 21.55 369.18143 337.1545 309.1619 299.1390 267.1128 224.0703
36 Echitamidine C20H24N2O3 21.87 341.18652 323.1757 309.1581 202.0863 142.0653 140.1072 [30]
37 N-Methylakuammicine C21H24N2O2 22.03 337.19161 309.1607 305.1650 294.1504 277.1700 263.1543
38 Unidentified alkaloid isomer 2 C21H24N2O4 22.06 369.18143 337.1547 309.1598 299.1392 256.0967 224.0708
39 Akuammicine isomer N-methyl derivative C21H24N2O2 22.50 337.19161 305.1649 277.1700 263.1543 248.1086 222.1277
40 Akuammicine isomer 1 C20H22N2O2 22.70 323.17596 291.1494 280.1332 263.1549 249.1385 234.1279
41 Tubotaiwine C20H24N2O2 22.88 325.19160 293.1648 265.1335 236.1440 222.1286 194.0960
42 Akuammicine isomer 2 C20H22N2O2 23.11 323.17596 291.1494 280.1335 263.1528 249.1387 234.1285
43 17-O-Acetyl-N-demethylechitamine or isomer C23H28N2O5 23.16 413.20765 395.1964 353.1862 335.1760 292.1334 232.1123
44 1 Isoquercitrin (Quercetin-3-O-glucoside) C21H20O12 23.48 463.08765 301.0357 300.0280 271.0251 255.0300 151.0022
45 1 Rutin (Quercetin-3-O-rutinoside) C27H30O16 23.56 609.14557 301.0357 300.0278 271.0251 255.0298 151.0024
46 Voacangine C22H28N2O3 24.77 369.21782 337.1911 309.1609 266.1160 252.1022 [33]
47 Akuammidine C21H24N2O3 24.95 353.18652 321.1599 310.1412 293.1650 264.1369 250.1236 [33]
48 1 Quercitrin (Quercetin-3-O-rhamnoside) C21H20O11 25.03 447.09274 301.0357 300.0280 271.0252 255.0299 151.0025
49 Dihydroakuammidine or isomer C21H26N2O3 25.84 355.20217 323.1756 295.1441 266.1541 252.1388 224.1061
50 1 Quercetin (3,3′,4′,5,7-Pentahydroxyflavone) C15H10O7 27.58 301.03484 273.0423 178.9975 151.0026 121.0283 107.0124
51 9-Hydroxyoctadecatrienoic acid C18H30O3 40.17 293.21167 275.2022 231.2111 171.1017 121.1009 59.0132
52 13-Hydroxyoctadecatrienoic acid C18H30O3 40.31 293.21167 275.2023 235.1703 223.1335 195.1386 179.1433
53 Linoleamide C18H33NO 44.49 280.26404 263.2341 245.2264 109.1016 95.0861 81.0705
54 Oleamide C18H35NO 45.75 282.27969 265.2526 247.2421 135.1172 83.0861 69.0705
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1 Confirmed by standard. 2 [M]+.

Table 3.

Chemical characterization of leaves—MAC-EA (not stirred).

No. Name Formula Rt [M + H]+ [M − H]− Fragment 1 Fragment 2 Fragment 3 Fragment 4 Fragment 5 Literature
1 Quinic acid C7H12O6 1.28 191.05557 173.0444 171.0286 127.0387 111.0437 93.0330
2 1 Chlorogenic acid (3-O-Caffeoylquinic acid) C16H18O9 14.95 355.10291 163.0390 145.0285 135.0443 117.0338 89.0389
3 Caffeic acid C9H8O4 15.18 179.03444 135.0439 107.0483
4 Loganic acid C16H24O10 15.75 375.12913 213.0764 169.0857 151.0755 113.0230 69.0330
5 Swertiamarin or isomer C16H22O10 16.78 375.12912 213.0757 195.0655 177.0547 151.0390 107.0495
6 Secologanoside C16H22O11 17.32 389.10839 345.1188 209.0453 183.0652 165.0547 69.0330
7 Unidentified N-formylalkaloid 1 isomer 1 C22H24N2O5 17.42 397.17635 369.1809 337.1545 299.1388 267.1129 224.0708
8 5-O-(4-Coumaroyl)quinic acid C16H18O8 17.44 337.09235 191.0555 173.0447 163.0388 119.0486 93.0331
9 Lochnericine or isomer C21H24N2O3 17.45 353.18652 335.1740 291.1498 166.0862 158.0964 144.0808
10 2 Echitamine C22H29N2O4 18.04 385.21273 367.2022 349.1909 310.1435 250.1225 220.1121 [30]
11 Sweroside or isomer C16H22O9 18.07 359.13421 197.0810 179.0705 151.0753 127.0392 111.0807
12 Unidentified N-formylalkaloid 1 isomer 2 C22H24N2O5 18.25 397.17635 369.1808 337.1544 299.1390 256.0969 224.0708
13 Nα-Formyl-12-methoxyechitamidine C22H26N2O5 18.31 399.19200 371.1963 339.1702 299.1392 267.1128 198.0916 [31]
14 5-O-Feruloylquinic acid C17H20O9 18.47 367.10291 193.0502 191.0555 173.0444 134.0362 93.0330
15 1 4-Coumaric acid C9H8O3 18.50 163.03952 119.0488 93.0331
16 Vallesamine or isomer C20H24N2O3 18.73 341.18652 309.1596 237.1022 209.1075 194.0965 168.0806
17 Loliolide C11H16O3 20.10 197.11777 179.1068 161.0961 135.1170 133.1014 107.0859
18 Unidentified N-formylalkaloid 2 C22H24N2O4 20.41 381.18143 353.1858 339.1705 321.1596 263.1185 212.0939
19 Secologanoside methyl ester C17H24O11 20.72 403.12404 371.0987 223.0608 179.0550 165.0548 121.0281
20 Lagumicine or isomer C20H22N2O4 21.02 355.16578 337.1545 214.0863 200.0708 182.0602 154.0652
21 Akuammicine C20H22N2O2 21.58 323.17596 294.1490 291.1494 280.1331 263.1543 234.1281
22 Unidentified alkaloid isomer 1 C21H24N2O4 21.67 369.18143 337.1547 309.1600 299.1390 267.1127 224.0703
23 Echitamidine C20H24N2O3 21.98 341.18652 323.1758 309.1598 202.0864 142.0653 140.1071 [30]
24 Unidentified alkaloid isomer 2 C21H24N2O4 22.17 369.18143 337.1547 309.1602 299.1391 256.0969 224.0710
25 Akuammicine isomer 1 C20H22N2O2 22.83 323.17596 291.1493 280.1337 263.1538 249.1389 234.1289
26 Tubotaiwine C20H24N2O2 23.02 325.19160 293.1648 265.1340 236.1435 222.1282 194.0967
27 Akuammicine isomer 2 C20H22N2O2 23.25 323.17596 291.1493 280.1341 263.1549 249.1388 234.1277
28 17-O-Acetyl-N-demethylechitamine or isomer C23H28N2O5 23.29 413.20765 395.1972 353.1858 335.1755 292.1332 232.1121
29 1 Isoquercitrin (Quercetin-3-O-glucoside) C21H20O12 23.44 463.08765 301.0358 300.0280 271.0252 255.0288 151.0025
30 1 Rutin (Quercetin-3-O-rutinoside) C27H30O16 23.52 609.14557 301.0355 300.0280 271.0251 255.0302 151.0031
31 Voacangine C22H28N2O3 24.93 369.21782 337.1909 309.1598 266.1167 252.1030 [33]
32 1 Quercitrin (Quercetin-3-O-rhamnoside) C21H20O11 25.01 447.09274 301.0359 300.0279 271.0252 255.0289 151.0030
33 Akuammidine C21H24N2O3 25.02 353.18652 321.1599 310.1441 293.1654 264.1377 250.1245 [33]
34 Dihydroakuammidine or isomer C21H26N2O3 25.92 355.20217 323.1753 295.1441 266.1550 252.1387 224.1069
35 Dihydroactinidiolide C11H16O2 27.15 181.12286 163.1117 145.1014 135.1170 121.1013 107.0859
36 9-Hydroxyoctadecatrienoic acid C18H30O3 40.14 293.21167 275.2021 231.2113 171.1016 121.1009 59.0130
37 13-Hydroxyoctadecatrienoic acid C18H30O3 40.25 293.21167 275.2021 235.1700 223.1336 195.1384 179.1437
38 Linoleamide C18H33NO 44.45 280.26404 263.2369 245.2264 109.1015 95.0860 81.0704
39 Oleamide C18H35NO 45.70 282.27969 265.2530 247.2418 135.1171 83.0861 69.0705
40 Pheophytin A C55H74N4O5 65.78 871.57375 593.2762 533.2550 460.2255
Open in a new tab

1 Confirmed by standard. 2 [M]+.

Table 4.

Chemical characterization of leaves—MAC-MeOH (not stirred).

No. Name Formula Rt [M + H]+ [M − H]− Fragment 1 Fragment 2 Fragment 3 Fragment 4 Fragment 5 Literature
1 Quinic acid C7H12O6 1.26 191.05557 173.0446 171.0292 127.0387 111.0438 93.0330
2 Unidentified dihydroxybenzoic acid derivative C22H20O12 13.64 475.08765 299.0774 153.0181 137.0232 109.0281
3 1 Chlorogenic acid (3-O-Caffeoylquinic acid) C16H18O9 14.88 355.10291 163.0390 145.0285 135.0442 117.0337 89.0390
4 Caffeic acid C9H8O4 15.19 179.03444 135.0440 107.0489
5 Loganic acid C16H24O10 15.76 375.12913 213.0764 169.0860 151.0753 113.0230 69.0330
6 Vallesamine or isomer C20H24N2O3 15.77 341.18652 309.1596 236.1069 208.1127 194.0962 168.0807
7 Swertiamarin or isomer C16H22O10 16.73 375.12912 213.0757 195.0653 177.0546 151.0390 107.0495
8 Unidentified N-formylalkaloid 1 isomer 1 C22H24N2O5 17.27 397.17635 369.1810 337.1546 299.1392 267.1127 224.0704
9 Lochnericine or isomer C21H24N2O3 17.29 353.18652 335.1751 291.1490 166.0862 158.0964 144.0807
10 Secologanoside C16H22O11 17.33 389.10839 345.1191 209.0452 183.0656 165.0546 69.0330
11 5-O-(4-Coumaroyl)quinic acid C16H18O8 17.47 337.09235 191.0555 173.0445 163.0390 119.0489 93.0331
12 Picralinal or isomer C21H22N2O4 17.59 367.16578 349.1923 339.1703 307.1440 269.1285 180.1020
13 Quercetin-O-hexoside-O-rutinoside isomer 1 C33H40O21 17.70 771.19839 609.1468 462.0807 301.0357 300.0279 299.0201
14 2 Echitamine C22H29N2O4 17.89 385.21273 367.2024 349.1920 310.1432 250.1231 220.1122 [30]
15 Picralinal or isomer C21H22N2O4 17.94 367.16578 349.1930 339.1703 307.1441 269.1285 194.0601
16 Quebrachidine or isomer C21H24N2O3 17.96 353.18652 335.1750 324.1594 303.1491 293.1651 275.1537
17 Quercetin-O-hexoside-O-rutinoside isomer 2 C33H40O21 17.99 771.19839 609.1483 463.0887 462.0813 301.0358 299.0201
18 Sweroside or isomer C16H22O9 18.02 359.13421 197.0810 179.0703 151.0754 127.0392 111.0808
19 Unidentified N-formylalkaloid 1 isomer 2 C22H24N2O5 18.06 397.17635 369.1809 337.1545 299.1390 256.0968 224.0708
20 4-O-(4-Coumaroyl)quinic acid C16H18O8 18.10 337.09235 191.0555 173.0445 163.0389 119.0488 93.0330
21 Nα-Formyl-12-methoxyechitamidine C22H26N2O5 18.12 399.19200 371.1962 339.1701 299.1392 267.1130 198.0914 [31]
22 1 4-Coumaric acid C9H8O3 18.50 163.03952 119.0488 93.0332
23 Vallesamine or isomer C20H24N2O3 18.52 341.18652 309.1597 237.1018 209.1073 194.0965 168.0808
24 5-O-Feruloylquinic acid C17H20O9 18.54 367.10291 193.0499 191.0555 173.0445 134.0362 93.0331
25 N-Formylechitamidine C21H24N2O4 18.85 369.18143 351.1705 341.1858 309.1598 202.0864 [32]
26 5-O-(4-Coumaroyl)quinic acid cis isomer C16H18O8 19.69 337.09235 191.0553 173.0446 163.0387 119.0488 93.0330
27 Loliolide C11H16O3 20.03 197.11777 179.1067 161.0961 135.1169 133.1014 107.0858
28 Unidentified N-formylalkaloid 2 C22H24N2O4 20.20 381.18143 353.1858 339.1711 321.1595 263.1182 212.0934
29 Secologanol C17H26O10 20.35 391.16043 229.1070 211.0965 193.0862 179.0704 167.0704
30 Quercecin-O-hexosylhexoside C27H30O17 20.66 625.14048 301.0359 300.0278 271.0250 255.0298 151.0024
31 Secologanoside methyl ester C17H24O11 20.73 403.12404 371.0990 223.0611 179.0550 165.0546 121.0281
32 Lagumicine or isomer C20H22N2O4 20.90 355.16578 337.1546 214.0862 200.0708 182.0602 154.0651
33 Akuammicine C20H22N2O2 21.38 323.17596 294.1488 291.1493 280.1334 263.1543 234.1279
34 Unidentified alkaloid isomer 1 C21H24N2O4 21.47 369.18143 337.1545 309.1595 299.1392 267.1129 224.0714
35 Cinchonain I isomer 1 C24H20O9 21.70 451.10291 341.0668 231.0295 217.0138 189.0186 177.0184
36 Echitamidine C20H24N2O3 21.73 341.18652 323.1762 309.1596 202.0863 142.0652 140.1070 [30]
37 N-Methylakuammicine C21H24N2O2 21.96 337.19161 309.1580 305.1648 294.1504 277.1697 263.1541
38 Unidentified alkaloid isomer 2 C21H24N2O4 22.01 369.18143 337.1545 309.1590 299.1391 256.0968 224.0708
39 Akuammicine isomer N-methyl derivative C21H24N2O2 22.49 337.19161 305.1648 277.1702 263.1542 248.1074 222.1277
40 Cinchonain I isomer 2 C24H20O9 22.69 451.10291 341.0668 231.0294 217.0138 189.0187 177.0183
41 Akuammicine isomer 1 C20H22N2O2 22.71 323.17596 291.1491 280.1332 263.1538 249.1385 234.1290
42 Tubotaiwine C20H24N2O2 22.87 325.19160 293.1647 265.1341 236.1434 222.1282 194.0964
43 Akuammicine isomer 2 C20H22N2O2 23.09 323.17596 291.1491 280.1328 263.1541 249.1385 234.1309
44 17-O-Acetyl-N-demethylechitamine or isomer C23H28N2O5 23.15 413.20765 395.1971 353.1856 335.1757 292.1333 232.1120
45 1 Isoquercitrin (Quercetin-3-O-glucoside) C21H20O12 23.45 463.08765 301.0357 300.0278 271.0249 255.0299 151.0024
46 1 Rutin (Quercetin-3-O-rutinoside) C27H30O16 23.54 609.14557 301.0357 300.0278 271.0250 255.0299 151.0024
47 Di-O-caffeoylquinic acid C25H24O12 24.65 515.11896 353.0880 335.0790 191.0553 179.0340 173.0444
48 Voacangine C22H28N2O3 24.75 369.21782 337.1907 309.1591 266.1159 252.1027 [33]
49 Akuammidine C21H24N2O3 24.91 353.18652 321.1598 310.1444 293.1647 264.1374 250.1248 [33]
50 1 Quercitrin (Quercetin-3-O-rhamnoside) C21H20O11 25.03 447.09274 301.0356 300.0278 271.0250 255.0299 151.0024
51 Dihydroakuammidine or isomer C21H26N2O3 25.79 355.20217 323.1753 295.1441 266.1559 252.1392 224.1068
52 Dimethoxy-tetrahydroxy(iso)flavone C17H14O8 26.52 345.06105 330.0384 315.0151 287.0199 271.0262 243.0297
53 1 Quercetin (3,3′,4′,5,7-Pentahydroxyflavone) C15H10O7 27.56 301.03484 273.0393 178.9981 151.0024 121.0281 107.0125
54 Dimethoxy-trihydroxy(iso)flavone C17H14O7 33.32 329.06613 314.0436 299.0200 285.0400 271.0251 243.0299
55 Dihydroxy-trimethoxy(iso)flavone isomer 1 C18H16O7 35.18 343.08178 328.0594 313.0359 285.0395 269.0471 257.0465
56 Dihydroxy-trimethoxy(iso)flavone isomer 2 C18H16O7 35.52 343.08178 328.0589 313.0358 285.0405 269.0451 257.0447
57 9-Hydroxyoctadecatrienoic acid C18H30O3 40.17 293.21167 275.2022 231.2119 171.1016 121.1009 59.0125
58 13-Hydroxyoctadecatrienoic acid C18H30O3 40.27 293.21167 275.2021 235.1699 223.1334 195.1384 179.1430
59 Linoleamide C18H33NO 44.44 280.26404 263.2371 245.2262 109.1015 95.0860 81.0705
60 Oleamide C18H35NO 45.68 282.27969 265.2525 247.2418 135.1168 83.0860 69.0705
61 Pheophytin A C55H74N4O5 65.73 871.57375 593.2762 533.2551 460.2258
Open in a new tab

1 Confirmed by standard. 2 [M]+.

Table 5.

Chemical characterization of Stem bark—infusion.

No. Name Formula Rt [M + H]+ [M − H]− Fragment 1 Fragment 2 Fragment 3 Fragment 4 Fragment 5 Literature
1 Quinic acid C7H12O6 1.25 191.05557 173.0444 171.0289 127.0387 111.0437 93.0330
2 Neochlorogenic acid (5-O-Caffeoylquinic acid) C16H18O9 10.29 355.10291 163.0389 145.0284 135.0442 117.0337 89.0390
3 3-O-(4-Coumaroyl)quinic acid C16H18O8 13.33 337.09235 191.0553 173.0444 163.0389 119.0488 93.0329
4 1 Chlorogenic acid (3-O-Caffeoylquinic acid) C16H18O9 14.90 355.10291 163.0390 145.0284 135.0442 117.0338 89.0389
5 3-O-Feruloylquinic acid C17H20O9 15.14 367.10291 193.0499 191.0556 173.0445 134.0361 93.0331
6 Loganic acid C16H24O10 15.72 375.12913 213.0763 169.0860 151.0754 113.0230 69.0330
7 Vallesamine or isomer C20H24N2O3 15.75 341.18652 309.1598 236.1071 208.1126 194.0968 168.0807
8 Chryptochlorogenic acid (4-O-Caffeoylquinic acid) C16H18O9 16.15 355.10291 163.0389 145.0285 135.0442 117.0337 89.0390
9 Swertiamarin or isomer C16H22O10 16.75 375.12912 213.0757 195.0654 177.0546 151.0390 107.0495
10 Unidentified N-formylalkaloid 1 isomer 1 C22H24N2O5 17.06 397.17635 369.1811 337.1546 299.1391 267.1130 224.0709
11 Lochnericine or isomer C21H24N2O3 17.16 353.18652 335.1754 291.1495 166.0863 158.0965 144.0808
12 Secologanoside C16H22O11 17.32 389.10839 345.1191 209.0453 183.0654 165.0546 69.0330
13 2 Echitamine C22H29N2O4 17.33 385.21273 367.2018 349.1896 310.1437 250.1225 220.1120 [30]
14 5-O-(4-Coumaroyl)quinic acid C16H18O8 17.48 337.09235 191.0553 173.0444 163.0388 119.0488 93.0330
15 Picralinal or isomer C21H22N2O4 17.94 367.16578 349.1930 339.1703 307.1441 269.1285 194.0601
16 Sweroside or isomer C16H22O9 18.02 359.13421 197.0811 179.0704 151.0754 127.0392 111.0808
17 4-O-(4-Coumaroyl)quinic acid C16H18O8 18.10 337.09235 191.0551 173.0444 163.0390 119.0488 93.0330
18 Unidentified N-formylalkaloid 1 isomer 2 C22H24N2O5 18.12 397.17635 369.1810 337.1546 299.1389 256.0966 224.0708
19 Nα-Formyl-12-methoxyechitamidine C22H26N2O5 18.17 399.19200 371.1965 339.1703 299.1392 267.1126 198.0917 [31]
20 1 4-Coumaric acid C9H8O3 18.46 163.03952 119.0488 93.0333
21 Vallesamine or isomer C20H24N2O3 18.48 341.18652 309.1596 237.1027 209.1079 194.0961 168.0806
22 5-O-Feruloylquinic acid C17H20O9 18.50 367.10291 193.0500 191.0554 173.0445 134.0362 93.0330
23 N-Formylechitamidine C21H24N2O4 18.91 369.18143 351.1702 341.1859 309.1596 202.0861 [32]
24 4-O-Feruloylquinic acid C17H20O9 19.00 367.10291 193.0500 191.0555 173.0445 134.0361 93.0330
25 Loganin C17H26O10 19.03 391.16043 229.1071 211.0966 197.0812 179.0704 109.0651
26 Loliolide C11H16O3 20.06 197.11777 179.1068 161.0960 135.1170 133.1014 107.0859
27 Venalstonine or isomer C21H24N2O2 20.26 337.19161 305.1649 294.1490 277.1698 234.1279 196.0996
28 Unidentified N-formylalkaloid 2 C22H24N2O4 20.36 381.18143 353.1859 339.1691 321.1598 263.1185 212.0945
29 Secologanoside methyl ester C17H24O11 20.72 403.12404 371.0988 223.0607 179.0547 165.0545 121.0281
30 Akuammicine C20H22N2O2 21.42 323.17596 294.1490 291.1493 280.1322 263.1541 234.1304
31 Unidentified alkaloid isomer 1 C21H24N2O4 21.66 369.18143 337.1545 309.1601 299.1392 267.1126 224.0701
32 Echitamidine C20H24N2O3 21.93 341.18652 323.1738 309.1594 202.0864 142.0653 140.1071 [30]
33 Unidentified alkaloid isomer 2 C21H24N2O4 22.13 369.18143 337.1546 309.1597 299.1391 256.0969 224.0708
34 Unidentified alkaloid 2 C24H30N2O5 22.27 427.22330 409.2123 352.1542 292.1332 250.1227 232.1121
35 7-Deoxyloganic acid C16H24O9 22.36 359.13421 197.0813 153.0909 135.0803 109.0645 89.0229
36 Akuammicine isomer 1 C20H22N2O2 22.74 323.17596 291.1494 280.1335 263.1538 249.1387 234.1281
37 Tubotaiwine C20H24N2O2 22.94 325.19160 293.1648 265.1342 236.1444 222.1281 194.0965
38 Akuammicine isomer 2 C20H22N2O2 23.21 323.17596 291.1494 280.1336 263.1546 249.1387 234.1281
39 Boonein C9H14O3 23.23 169.08647 151.0751 141.0915 125.0957 [34]
40 17-O-Acetyl-N-demethylechitamine or isomer C23H28N2O5 23.24 413.20765 395.1967 353.1859 335.1765 292.1330 232.1123
41 Di-O-caffeoylquinic acid C25H24O12 24.62 515.11896 353.0883 335.0790 191.0554 179.0341 173.0445
42 Dimethoxy-trihydroxy(iso)flavone C17H14O7 33.34 329.06613 314.0438 299.0200 285.0400 271.0247 243.0299
43 Emodin C15H10O5 39.63 269.04500 241.0501 225.0552 197.0601 210.0318 181.0649
44 9-Hydroxyoctadecatrienoic acid C18H30O3 40.14 293.21167 275.2021 231.2109 171.1016 121.1009 59.0125
45 13-Hydroxyoctadecatrienoic acid C18H30O3 40.30 293.21167 275.2020 235.1701 223.1335 195.1383 179.1429
46 Linoleamide C18H33NO 44.45 280.26404 263.2367 245.2264 109.1014 95.0859 81.0704
47 Oleamide C18H35NO 45.72 282.27969 265.2527 247.2418 135.1169 83.0860 69.0705
Open in a new tab

1 Confirmed by standard. 2 [M]+.

Table 6.

Chemical characterization of stem bark—MAC-EA (not stirred).

No. Name Formula Rt [M + H]+ [M − H]− Fragment 1 Fragment 2 Fragment 3 Fragment 4 Fragment 5 Literature
1 Quinic acid C7H12O6 1.27 191.05557 173.0447 171.0288 127.0388 111.0437 93.0330
2 1 Chlorogenic acid (3-O-Caffeoylquinic acid) C16H18O9 14.91 355.10291 163.0390 145.0285 135.0442 117.0338 89.0389
3 Loganic acid C16H24O10 15.77 375.12913 213.0762 169.0859 151.0754 113.0229 69.0330
4 Swertiamarin or isomer C16H22O10 16.73 375.12912 213.0757 195.0654 177.0548 151.0390 107.0495
5 Secologanoside C16H22O11 17.34 389.10839 345.1187 209.0451 183.0653 165.0545 69.0330
6 5-O-(4-Coumaroyl)quinic acid C16H18O8 17.46 337.09235 191.0553 173.0439 163.0383 119.0489 93.0328
7 2 Echitamine C22H29N2O4 17.83 385.21273 367.2011 349.1912 310.1437 250.1226 220.1121 [30]
8 Sweroside or isomer C16H22O9 18.05 359.13421 197.0810 179.0704 151.0755 127.0392 111.0808
9 Nα-Formyl-12-methoxyechitamidine C22H26N2O5 18.31 399.19200 371.1964 339.1703 299.1392 267.1131 198.0914 [31]
10 5-O-Feruloylquinic acid C17H20O9 18.50 367.10291 193.0498 191.0554 173.0445 134.0362 93.0330
11 1 4-Coumaric acid C9H8O3 18.52 163.03952 119.0487 93.0328
12 Vallesamine or isomer C20H24N2O3 18.58 341.18652 309.1598 237.1021 209.1074 194.0965 168.0806
13 Loganin C17H26O10 18.97 391.16043 229.1070 211.0965 197.0812 179.0704 109.0652
14 Loliolide C11H16O3 20.07 197.11777 179.1068 161.0961 135.1170 133.1013 107.0859
15 Venalstonine or isomer C21H24N2O2 20.38 337.19161 305.1649 294.1485 277.1699 234.1279 196.0998
16 Secologanoside methyl ester C17H24O11 20.73 403.12404 371.0984 223.0609 179.0549 165.0545 121.0281
17 Echitamidine C20H24N2O3 22.03 341.18652 323.1751 309.1603 202.0864 142.0653 140.1071 [30]
18 7-Deoxyloganic acid C16H24O9 22.35 359.13421 197.0813 153.0909 135.0802 109.0644 89.0228
19 Unidentified alkaloid 2 C24H30N2O5 22.37 427.22330 409.2125 352.1544 292.1333 250.1228 232.1122
20 Tubotaiwine C20H24N2O2 22.95 325.19160 293.1648 265.1346 236.1428 222.1279 194.0959
21 17-O-Acetyl-N-demethylechitamine or isomer C23H28N2O5 23.22 413.20765 395.1960 353.1850 335.1763 292.1329 232.1122
22 Di-O-caffeoylquinic acid C25H24O12 24.63 515.11896 353.0882 335.0790 191.0553 179.0339 173.0444
23 Dimethoxy-trihydroxy(iso)flavone C17H14O7 33.35 329.06613 314.0434 299.0199 285.0410 271.0249 243.0290
24 Linoleamide C18H33NO 44.47 280.26404 263.2370 245.2264 109.1015 95.0860 81.0705
25 Oleamide C18H35NO 45.71 282.27969 265.2525 247.2424 135.1170 83.0860 69.0705
26 Pheophytin A C55H74N4O5 65.51 871.57375 593.2759 533.2547 460.2255
Open in a new tab

1 Confirmed by standard. 2 [M]+.

Table 7.

Chemical characterization of stem bark—MAC-MEOH (not stirred).

No. Name Formula Rt [M + H]+ [M − H]− Fragment 1 Fragment 2 Fragment 3 Fragment 4 Fragment 5 Literature
1 Quinic acid C7H12O6 1.25 191.05557 173.0443 171.0289 127.0388 111.0435 93.0330
2 1 Chlorogenic acid (3-O-Caffeoylquinic acid) C16H18O9 14.88 355.10291 163.0389 145.0284 135.0442 117.0337 89.0390
3 Loganic acid C16H24O10 15.69 375.12913 213.0762 169.0859 151.0750 113.0229 69.0329
4 Swertiamarin or isomer C16H22O10 16.69 375.12912 213.0757 195.0653 177.0546 151.0389 107.0495
5 Unidentified N-formylalkaloid 1 isomer 1 C22H24N2O5 16.86 397.17635 369.1806 337.1543 299.1389 267.1126 224.0704
62 Echitamine C22H29N2O4 16.92 385.21273 367.2010 349.1913 310.1436 250.1224 220.1118 [30]
7 Secologanoside C16H22O11 17.33 389.10839 345.1187 209.0447 183.0656 165.0544 69.0329
8 5-O-(4-Coumaroyl)quinic acid C16H18O8 17.48 337.09235 191.0552 173.0444 163.0388 119.0487 93.0330
9 Sweroside or isomer C16H22O9 17.86 359.13421 197.0808 179.0702 151.0753 127.0391 111.0807
10 Unidentified N-formylalkaloid 1 isomer 2 C22H24N2O5 18.05 397.17635 369.1807 337.1545 299.1389 256.0966 224.0707
11 Nα-Formyl-12-methoxyechitamidine C22H26N2O5 18.11 399.19200 371.1964 339.1702 299.1392 267.1128 198.0919 [31]
12 Vallesamine or isomer C20H24N2O3 18.42 341.18652 309.1596 237.1024 209.1073 194.0963 168.0807
13 1 4-Coumaric acid C9H8O3 18.49 163.03952 119.0487 93.0331
14 5-O-Feruloylquinic acid C17H20O9 18.50 367.10291 193.0498 191.0554 173.0444 134.0360 93.0330
15 N-Formylechitamidine C21H24N2O4 18.82 369.18143 351.1700 341.1859 309.1596 202.0860 [32]
16 Loganin C17H26O10 18.89 391.16043 229.1071 211.0969 197.0800 179.0705 109.0650
17 Loliolide C11H16O3 20.04 197.11777 179.1068 161.0960 135.1170 133.1014 107.0859
18 Venalstonine or isomer C21H24N2O2 20.07 337.19161 305.1647 294.1482 277.1697 234.1274 196.0992
19 Unidentified N-formylalkaloid 2 C22H24N2O4 20.28 381.18143 353.1859 339.1705 321.1596 263.1181 212.0936
20 Secologanoside methyl ester C17H24O11 20.71 403.12404 371.0983 223.0604 179.0547 165.0545 121.0280
21 Akuammicine C20H22N2O2 21.46 323.17596 294.1487 291.1491 280.1331 263.1540 234.1284
22 Echitamidine C20H24N2O3 21.75 341.18652 323.1754 309.1594 202.0863 142.0652 140.1071 [30]
23 Unidentified alkaloid 2 C24H30N2O5 22.06 427.22330 409.2113 352.1541 292.1334 250.1222 232.1119
24 7-Deoxyloganic acid C16H24O9 22.35 359.13421 197.0812 153.0908 135.0802 109.0644 89.0228
25 Akuammicine isomer 1 C20H22N2O2 22.62 323.17596 291.1491 280.1329 263.1538 249.1384 234.1269
26 Tubotaiwine C20H24N2O2 22.81 325.19160 293.1646 265.1328 236.1436 222.1281 194.0963
27 Akuammicine isomer 2 C20H22N2O2 23.06 323.17596 291.1491 280.1329 263.1538 249.1384 234.1269
28 Boonein C9H14O3 23.12 169.08647 151.0752 141.0909 125.0959 [34]
29 17-O-Acetyl-N-demethylechitamine or isomer C23H28N2O5 23.14 413.20765 395.1974 353.1853 335.1755 292.1328 232.1122
30 Di-O-caffeoylquinic acid C25H24O12 24.62 515.11896 353.0878 335.0742 191.0553 179.0339 173.0443
31 Dimethoxy-trihydroxy(iso)flavone C17H14O7 33.34 329.06613 314.0434 299.0197 285.0404 271.0247 243.0296
32 9-Hydroxyoctadecatrienoic acid C18H30O3 40.16 293.21167 275.2019 231.2110 171.1015 121.1008 59.0125
33 13-Hydroxyoctadecatrienoic acid C18H30O3 40.26 293.21167 275.2017 235.1699 223.1332 195.1384 179.1441
34 Linoleamide C18H33NO 44.44 280.26404 263.2368 245.2262 109.1015 95.0860 81.0704
35 Oleamide C18H35NO 45.70 282.27969 265.2524 247.2424 135.1169 83.0860 69.0705
36 Pheophytin A C55H74N4O5 65.49 871.57375 593.2759 533.2549 460.2255
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1 Confirmed by standard. 2 [M]+.

3.3. Antioxidant Effect

A comprehensive study of the antioxidant activity of the A. boonei leaves and stem bark extracts obtained from infusion, maceration and Soxhlet extraction using water, ethyl acetate and methanol was carried out. Results of the antioxidant activities determined by ABTS•+, DPPH•, CUPRAC, FRAP and metal chelating are shown in Table 8. In line with the total phenolic results, the antioxidant activity of the leaves extracts was higher compared to the stem bark extracts, and higher activity was observed for the methanol extracts. The ability of the extracts to scavenge free radicals, namely, ABTS•+ and DPPH•, was determined. The water extract of A. boonei leaves obtained by infusion showed the highest ABTS•+ and DPPH• scavenging ability. It was also observed that the leaves extracts were more potent radical scavengers compared to the stem bark extracts. The presence of antioxidant compounds, such as phenolics and phenolic acids, causes the TPTZ-Fe3+ complex to be reduced to the TPTZ-Fe2+ complex, yielding a chromophore with maximum absorption at 593 nm, and the neocuproine-Cu2+ complex to be reduced to the neocuproine-Cu+ complex, yielding an orange–yellow chromophore with maximum absorption at 450 nm. The methanol extract of A. boonei leaves showed the highest reducing activity. The metal chelating potential of A. boonei leaves and stem bark extracts was evaluated and reported in Table 8. The water extract of the stem bark showed the highest chelating ability while none of the ethyl acetate extracts of A. boonei stem bark were active.

Table 8.

Antioxidant properties of the studied extracts *.

Parts Extraction Methods/Solvent DPPH•
(mg TE/g) 		ABTS•+
(mg TE/g) 		CUPRAC
(mg TE/g)		 FRAP (mg TE/g) Metal Chelating (mg EDTAE/g)
Leaves Infusion-water 74.70 ± 1.27 a 131.50 ± 2.34 a 140.57 ± 0.37 d 109.35 ± 1.48 a 13.42 ± 2.06 e
MAC-EA 13.96 ± 0.87 f 36.78 ± 1.73 f 82.79 ± 1.68 f 41.54 ± 0.43 f 31.51 ± 1.05 a
MAC-MeOH 55.14 ± 0.44 c 109.24 ± 3.07 c 146.71 ± 3.80 c 87.37 ± 1.42 d 26.07 ± 0.52 b
MAC-EA (not stirred) 18.40 ± 0.64 e 43.67 ± 2.11 e 87.72 ± 1.10 f 38.03 ± 0.86 g 24.32 ± 1.11 bc
MAC-MeOH (not stirred) 61.23 ± 1.46 b 123.70 ± 0.27 b 163.91 ± 2.20 a 100.86 ± 0.40 b 22.67 ± 0.50 cd
SE-EA 27.99 ± 1.11 d 88.67 ± 1.26 d 118.54 ± 0.86 e 61.48 ± 1.78 e 21.17 ± 0.63 d
SE-MeOH 61.32 ± 0.47 b 127.63 ± 1.65 ab 155.36 ± 1.10 b 97.05 ± 0.74 c 21.90 ± 0.41 cd
Stem bark Infusion-water 33.82 ± 0.66 b 59.94 ± 1.70 c 74.80 ± 0.56 c 55.48 ± 1.17 ab 36.71 ± 0.14 a
MAC-EA 4.54 ± 0.94 c na 76.51 ± 1.11 c 40.41 ± 0.80 c na
MAC-MeOH 35.41 ± 0.47 b 69.24 ± 2.82 b 94.89 ± 1.59 b 53.94 ± 2.18 b 22.21 ± 1.93 b
MAC-EA (not stirred) 5.88 ± 0.76 c na 74.37 ± 2.06 c 36.04 ± 0.12 d na
MAC- MeOH (not stirred) 37.57 ± 0.74 a 73.69 ± 1.17 a 105.92 ± 4.86 a 54.48 ± 1.67 b 23.77 ± 0.23 b
SE-EA 5.94 ± 1.07 c 6.04 ± 0.91 d 78.31 ± 0.72 c 36.64 ± 0.15 d na
SE-MeOH 34.74 ± 0.48 b 71.22 ± 0.17 ab 101.53 ± 1.80 a 58.03 ± 0.80 a 18.23 ± 0.16 c
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* Values expressed are means ± S.D. of three parallel measurements. TE: Trolox equivalent; EDTAE: EDTA equivalent. MAC: Maceration; SE: Soxhlet extraction; EA: Ethyl acetate; MeOH: Methanol, na: not active. Different letters indicate significant differences in the tested extracts of each parts (p < 0.05).

3.4. Enzyme Inhibitory Effects

In the present study, the inhibitory action of A. boonei leaves and stem bark extracts on cholinesterase enzymes, namely, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), targeted in the management of neurodegenerative diseases, such as Alzheimer’s disease, was studied and reported in Table 9. Both A. boonei leaves and stem bark water extracts obtained by infusion showed no activity against AChE, while inhibition was noted against BChE. It was also observed that the value for AChE inhibition ranged from 4.89–5.57 mg GALAE/g for A. boonei leaves extracts and 4.91–5.78 mg GALAE/g for stem bark extracts, showing no significant variations among the different extracts. On the other hand, variable inhibitory action was observed against BChE (Table 9). It is worth highlighting that water extract of A. boonei stem bark showed the highest inhibitory activity against BChE. In general, A. boonei leaves and stem bark extracts showed low inhibitory activity against α-amylase. In Table 9, it is noted that the ethyl acetate extracts of A. boonei leaves and both water extracts showed no inhibitory action against α-glucosidase. However, ethyl acetate and methanol extracts of A. boonei stem bark inhibited α-glucosidase. The ability of A. boonei leaves and stem bark extracts to inhibit tyrosinase activity was also assessed and presented in Table 9. Ethyl acetate and methanol extracts of both A. boonei leaves and stem bark were active inhibitors of tyrosinase. In accordance with total phenolic results, ethyl acetate extracts showed lower inhibitory action against tyrosinase compared to their corresponding methanol extracts. Poor inhibition was observed for the water extracts.

Table 9.

Enzyme inhibitory properties of the studied extracts *.

Parts Extraction Methods/Solvent AChE
(mg GALAE/g) 		BChE
(mg GALAE/g) 		Amylase
(mmol ACAE/g)		 Glucosidase
(mmol ACAE/g)		 Tyrosinase
(mg KAE/g)
Leaves Infusion-water na 6.35 ± 0.18 b 0.17 ± 0.01 d na 0.72 ± 0.07 c
MAC-EA 5.02 ± 0.34 ab 4.78 ± 0.11 cd 1.16 ± 0.08 a na 129.70 ± 2.85 ab
MAC-MeOH 4.49 ± 0.11 b 3.22 ± 0.92 e 0.91 ± 0.02 c 5.24 ± 0.09 a 132.83 ± 1.04 ab
MAC-EA (not stirred) 4.45 ± 0.67 b 8.25 ± 0.43 a 1.13 ± 0.06 a na 127.11 ± 9.18 b
MAC-MeOH (not stirred) 5.57 ± 0.49 a 6.18 ± 0.19 bc 0.97 ± 0.03 bc 5.27 ± 0.09 a 135.89 ± 0.63 ab
SE-EA 4.89 ± 0.06 ab 5.19 ± 0.58 bc 1.10 ± 0.09 ab na 130.08 ± 5.20 ab
SE-MeOH 5.04 ± 0.34 ab 3.49 ± 0.60 de 0.88 ± 0.02 c 5.04 ± 0.60 a 139.20 ± 0.48 a
Stem bark Infusion-water na 10.08 ± 0.29 a 0.20 ± 0.01 c na 0.43 ± 0.01 d
MAC-EA 5.02 ± 0.43 a 1.44 ± 0.36 d 1.01 ± 0.08 a 4.71 ± 0.01 c 121.85 ± 1.56 b
MAC-MeOH 5.34 ± 0.37 a 2.51 ± 0.28 d 0.71 ± 0.06 b 5.40 ± 0.01 a 133.51 ± 1.10 a
MAC-EA (not stirred) 4.91 ± 0.62 a 1.59 ± 0.03 d 0.98 ± 0.06 a 4.67 ± 0.02 d 123.69 ± 0.53 b
MAC- MeOH (not stirred) 5.62 ± 0.23 a 4.13 ± 0.65 c 0.80 ± 0.02 b 5.38 ± 0.01 a 135.02 ± 1.20 a
SE-EA 5.78 ± 0.18 a 6.39 ± 0.47 b 1.04 ± 0.01 a 4.83 ± 0.01 b 94.63 ± 2.53 c
SE-MeOH 5.67 ± 0.20 a 4.38 ± 0.66 c 0.78 ± 0.03 b 5.41 ± 0.01 a 134.63 ± 0.56 a
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* Values expressed are means ± S.D. of three parallel measurements. GALAE: Galatamine equivalent; KAE: Kojic acid equivalent; ACAE: Acarbose equivalent; MAC: Maceration; SE: Soxhlet extraction; EA: Ethyl acetate; MeOH: Methanol, na: not active. Different letters indicate significant differences in the tested extracts of each part (p < 0.05).

3.5. Cell Viability

The cell viability percentages can be verified by the MTT (Table 10) in the groups treated at different concentrations of Alstonia boonei extracts (25, 50 and 100 µg/mL) and the control group. Alstonia boonei extracts did not induce significant cell viability reduction at the concentration of 25 µg/mL. A decrease of the cell viability percentage of macrophage at 50 and 100 µg/mL was observed after exposure to the ethyl acetate extracts. A reduction of cell viability was observed at 100 µg/mL, but not at 50 µg/mL, after exposure to the methanol extracts. There was no significant cell viability reduction after macrophage exposure to infusions. The results indicated that all the extracts were safe up to 25 μg/mL to conduct the assay of anti-inflammatory activity.

Table 10.

Effects induced by Alstonia boonei extracts on cell viability and LPS-induced cytokine release (IL-6, TNF-α and IL-1β) in macrophages. Macrophages were exposed for 24 h to 25, 50 and 100 μg/mL of extracts. a is for p < 0.05, b is for p < 0.01, c is for p < 0.001 and d is for p < 0.0001 vs. LPS. * is for absolute values in pg/mL. Data were statistically analyzed using One-Way ANOVA followed by Dunnett’s multiple comparisons test (n = 6 replicates of 2 separate sets of experiments). Results are expressed as mean ± SEM.

Control and Extracts Sample (μg/mL) Cell Viability-MTT (%)
(Mean ± SEM)		 IL-6 Release (%)
(Mean ± SEM)		 TNF-α Release (%)
(Mean ± SEM)		 IL-1β Release (%)
(Mean ± SEM)
LPS 0.1 583.3 ± 61.3 * 916.7 ± 48.7 * 1033.3 ± 68.0 *
Dexamethasone 0.04 150.0 ± 26.4 * 189.2 ± 36.8 * 330.0 ± 88.5 *
Leaves—infusion 25 97.0 ± 1.2 85.4 ± 4.4 92.0 ± 4.7 77.8 ± 5.9
50 94.3 ± 3.1 73.5 ± 7.0 a 69.7 ± 3.2 a 61.8 ± 4.4 a
100 89.9 ± 4.7 64.0 ± 4.7 b 52.4 ± 6.9 c 53.2 ± 5.9 b
Leaves—MAC-EA (not stirred) 25 99.1 ± 2.1 91.7 ± 3.7 92.0 ± 3.0 93.3 ± 3.0
50 83.3 ± 5.9 b 87.2 ± 3.9 85.1 ± 5.1 88.9 ± 3.8
100 73.7 ± 2.7 c 73.1 ± 3.6 a 66.3 ± 5.2 b 69.5 ± 4.8 b
Leaves—MAC-MeOH (not stirred) 25 94.5 ± 2.6 90.6 ± 2.0 82.3 ± 4.2 82.2 ± 5.0
50 96.5 ± 2.4 68.4 ± 2.2 c 70.0 ± 4.6 b 64.3 ± 4.5 b
100 85.9 ± 3.7 b 62.9 ± 1.4 c 62.9 ± 4.1 c 44.5 ± 5.9 d
Stem bark—Infusion 25 98.9 ± 1.0 68.1 ± 6.7 b 76.6 ± 6.3 a 70.7 ± 7.1 a
50 96.7 ± 2.1 44.6 ± 5.4 d 50.7 ± 4.6 d 44.2 ± 5.3 d
100 94.3 ± 3.5 19.0 ± 1.7 d 31.0 ± 2.4 d 23.5 ± 3.0 d
Stem bark—MAC-EA (not stirred) 25 92.9 ± 3.6 94.8 ± 2.0 92.2 ± 1.8 98.3 ± 1.6
50 83.9 ± 3.2 a 92.4 ± 3.1 86.1 ± 4.3 87.1 ± 3.9
100 76.1 ± 4.8 c 81.5 ± 5.4 a 71.5 ± 4.2 c 64.3 ± 5.4 d
Stem bark—MAC-MeOH (not stirred) 25 98.6 ± 2.7 66.6 ± 2.6 d 66.2 ± 7.2 c 64.0 ± 6.1 d
50 92.6 ± 2.2 48.5 ± 4.7 d 44.0 ± 4.0 d 38.4 ± 4.1 d
100 89.4 ± 3.2 a 29.0 ± 4.1 d 22.9 ± 3.3 d 22.4 ± 2.8 d
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3.6. Anti-Inflammatory Activity

The levels of IL-6, TNF- and IL-1 in macrophage culture supernatants were measured using an ELISA kit, and then the anti-inflammatory effects of Alstonia boonei extracts on LPS-stimulated macrophages were studied. LPS-induced macrophages were shown to have significantly increased production of pro-inflammatory cytokines, while dexamethasone reduced it (Table 10). After cell exposition to leaves—infusion, the results demonstrated that IL-6, IL-1β and TNF-α production was significantly downregulated in LPS-induced macrophages treated with the extracts at the highest concentrations of 50 and 100 µg/mL (Table 10). On the contrary, the ethyl acetate extract of leaves from maceration reduced cytokine release induced by LPS in macrophages at the concentration of 100 µg/mL only (Table 10). Cells treated with the methanol extract of leaves from maceration showed a reducing effect on IL-6, TNF-α and IL-1β production at 50 and 100 µg/mL (Table 10). The stem bark—infusion and maceration—methanol extracts appear to be the most effective of the series in reducing the release of pro-inflammatory cytokines, since at all the concentrations used (Table 10). The ethyl acetate extract of stem bark reduced cytokine release at 100 µg/mL, whereas no effects were observed at the concentration of 10 and 50 µg/mL (Table 10).

4. Conclusions

This study provided scientific evidence that A. boonei leaves and stem bark have biological activities, specifically, antioxidant and enzyme inhibitory properties. In the same extraction methods, namely, maceration and Soxhlet, the solvents were affected to chemical composition and biological activities. Generally, the methanol extracts for both parts exhibited more antioxidant abilities when compared to ethyl acetate and water extracts. Based on the parts, the extracts of leaves were more active in the antioxidant assays. The ethyl acetate and methanol showed greater AChE, tyrosinase and amylase inhibitory effects than did infusions. In addition, the chemical composition of the extracts depended on the extraction solvents, and the methanol extracts contained more components compared to ethyl acetate and water extracts. In the UHLPC–MS analysis, the presence of bioactive compounds, such as quinic acid, caffeic acid, rutin and isoquercetin, was found. In particular, stem bark extracts showed great anti-inflammatory potential. From the results, methanol could be useful for the preparation of further applications using A. booeni at industrial scale. However, the toxic properties of methanol should not be forgotten, and ethanol could be used in these applications. Future experiments, including animal and bioavailability studies, should be conducted to corroborate the findings.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/antiox11112171/s1, Figure S1. Total ion chromatogram of Alstonia boonei leaves infusion in positive mode; Figure S2. Total ion chromatogram of Alstonia boonei leaves infusion in negative mode; Figure S3. Total ion chromatogram of Alstonia boonei leaves maceration-EA in positive mode; Figure S4. Total ion chromatogram of Alstonia boonei leaves maceration-EA in negative mode; Figure S5. Total ion chromatogram of Alstonia boonei leaves maceration-MeOH in positive mode; Figure S6. Total ion chromatogram of Alstonia boonei leaves maceration-MeOH in negative mode; Figure S7. Total ion chromatogram of Alstonia boonei stem bark infusion in positive mode; Figure S8. Total ion chromatogram of Alstonia boonei stem bark infusion in negative mode; Figure S9. Total ion chromatogram of Alstonia boonei stem bark maceration-EA in positive mode; Figure S10. Total ion chromatogram of Alstonia boonei stem bark maceration-EA in negative mode; Figure S11. Total ion chromatogram of Alstonia boonei stem bark maceration-MeOH in positive mode; Figure S12. Total ion chromatogram of Alstonia boonei stem bark maceration-MeOH in negative mode

Click here for additional data file. (352.7KB, zip)

Author Contributions

Conceptualization, A.M., G.Z., K.I.S., M.M., S.P., methodology, G.Z., K.I.S., M.M., S.P., J.J., Z.C., software, A.M., A.S., M.B.B., validation, G.Z., O.K.E., C.P.-A., M.F.M., formal analysis, G.Z., investigation, A.M., G.Z., K.I.S., M.M., S.P., A.S., resources, G.Z., K.I.S., O.K.E., data curation, A.M., G.Z., writing—original draft preparation, M.M., S.P., C.P.-A., M.F.M.; writing—review and editing, A.M., G.Z., A.S., visualization, M.M., S.P.; supervision, A.M., G.Z., M.F.M.; project administration, G.Z.; funding acquisition, A.M. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

Funding Statement

This research received no external funding.

Footnotes

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

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