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Article

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

by
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 and
Mohamad Fawzi Mahomoodally
8,9,10
1
Department of Pharmacy, University “G. d’Annunzio” of Chieti-Pescara, 66100 Chieti, Italy
2
Department of Biology, Faculty of Science, Selcuk University, Campus, 42250 Konya, Turkey
3
University Guglielmo Marconi, 00193 Rome, Italy
4
National Centre for Drug Research and Evaluation, Istituto Superiore di Sanità, 00161 Rome, Italy
5
Laboratoire de Botanique, UFR Biosciences, Université Félix Houphouët-Boigny, Abidjan 83111, Côte d’Ivoire
6
Agricultural and Molecular Research and Service Institute, University of Nyíregyháza, 4400 Nyíregyháza, Hungary
7
Medicinal Plants Research Center, Maragheh University of Medical Sciences, Maragheh 83111, Iran
8
Department of Health Sciences, Faculty of Medicine and Health Sciences, University of Mauritius, Réduit 230, Mauritius
9
Center for Transdisciplinary Research, Department of Pharmacology, Saveetha Dental College, Saveetha Institute of Medical and Technical Science, Chennai 600077, India
10
Centre of Excellence for Pharmaceutical Sciences, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa
*
Author to whom correspondence should be addressed.
Antioxidants 2022, 11(11), 2171; https://doi.org/10.3390/antiox11112171
Submission received: 5 October 2022 / Revised: 27 October 2022 / Accepted: 29 October 2022 / Published: 1 November 2022
Versions Notes

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.

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].

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.

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.

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.

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.

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

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.

Funding

This research received no external funding.

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.

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Table
Table 1. Total bioactive compounds and total antioxidant capacity (by phosphomolybdenum assay) of the studied extracts *.
Table 1. Total bioactive compounds and total antioxidant capacity (by phosphomolybdenum assay) of the studied extracts *.
PartsExtraction Methods/SolventTotal Phenolic Content
(mg GAE/g)		Total Flavonoid Content
(mg RE/g)		Phosphomolybdenum
(mmol TE/g)
LeavesInfusion-water51.08 ± 0.21 b4.18 ± 0.16 e2.15 ± 0.08 cd
MAC-EA30.64 ± 0.38 f34.51 ± 0.39 c1.78 ± 0.04 d
MAC-MeOH48.34 ± 0.38 c37.28 ± 0.56 b2.55 ± 0.27 abc
MAC-EA (not stirred)35.81 ± 0.50 e34.81 ± 0.46 c2.61 ± 0.15 ab
MAC-MeOH (not stirred)53.08 ± 0.24 a37.65 ± 0.20 b2.78 ± 0.15 a
SE-EA40.19 ± 0.67 d8.14 ± 0.31 d2.25 ± 0.09 bc
SE-MeOH49.27 ± 0.30 c39.10 ± 0.42 a2.37 ± 0.13 bc
Stem barkInfusion-water31.99 ± 0.15 f0.54 ± 0.08 e1.30 ± 0.02 c
MAC-EA35.64 ± 0.62 d3.05 ± 0.09 b2.06 ± 0.04 a
MAC-MeOH39.64 ± 0.03 b1.85 ± 0.06 d1.54 ± 0.08 bc
MAC-EA (not stirred)34.38 ± 0.44 de2.69 ± 0.08 bc1.84 ± 0.13 ab
MAC- MeOH (not stirred)45.72 ± 0.28 a2.45 ± 0.08 c1.91 ± 0.22 a
SE-EA34.25 ± 0.42 e4.07 ± 0.33 a1.60 ± 0.09 bc
SE-MeOH37.08 ± 0.79 c2.76 ± 0.06 bc1.54 ± 0.07 bc
* 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).
Table
Table 2. Chemical characterization of leaves—infusion.
Table 2. Chemical characterization of leaves—infusion.
No.NameFormulaRt[M + H]+[M − H]−Fragment 1Fragment 2Fragment 3Fragment 4Fragment 5Literature
1Unidentified dihydroxybenzoic acid derivativeC22H20O1213.58 475.08765299.0776153.0181137.0232109.0281
2 1Chlorogenic acid (3-O-Caffeoylquinic acid)C16H18O914.86355.10291 163.0390145.0285135.0442117.033889.0388
33-O-Feruloylquinic acidC17H20O915.11 367.10291193.0500191.0552173.0443134.036193.0333
4Caffeic acidC9H8O415.16 179.03444135.0439107.0487
5Loganic acidC16H24O1015.72 375.12913213.0764169.0860151.0751113.023069.0330
6Vallesamine or isomerC20H24N2O315.79341.18652 309.1599236.1062208.1128194.0967168.0807
7Chryptochlorogenic acid (4-O-Caffeoylquinic acid)C16H18O916.21355.10291 163.0390145.0285135.0442117.033989.0390
84-O-(4-Coumaroyl)quinic acid cis isomerC16H18O816.27 337.09235191.0557173.0445163.0389119.048893.0330
9Swertiamarin or isomerC16H22O1016.73375.12912 213.0758195.0655177.0549151.0391107.0496
10Lochnericine or isomerC21H24N2O317.02353.18652 335.1773291.1494166.0863158.0966144.0808
11Unidentified N-formylalkaloid 1 isomer 1C22H24N2O517.30397.17635 369.1812337.1547299.1392267.1128224.0704
12SecologanosideC16H22O1117.32 389.10839345.1197209.0451183.0657165.054569.0330
13Lochnericine or isomerC21H24N2O317.39353.18652 335.1745291.1493166.0864158.0966144.0808
145-O-(4-Coumaroyl)quinic acidC16H18O817.51 337.09235191.0554173.0445163.0389119.048893.0331
15Picralinal or isomerC21H22N2O417.65367.16578 349.1910339.1704307.1441269.1283180.1020
16Quercetin-O-hexoside-O-rutinoside isomer 1C33H40O2117.71 771.19839609.1485462.0805301.0355300.0285299.0202
17 2EchitamineC22H29N2O417.92385.21273 367.2011349.1919310.1438250.1227220.1121[30]
18Quercetin-O-hexoside-O-rutinoside isomer 2C33H40O2117.96 771.19839609.1474463.0885462.0807301.0354299.0201
19Sweroside or isomerC16H22O918.03359.13421 197.0811179.0704151.0754127.0392111.0809
20Picralinal or isomerC21H22N2O418.04367.16578 349.1948339.1704307.1442269.1285194.0603
21Quebrachidine or isomerC21H24N2O318.05353.18652 335.1773324.1595303.1491293.1651275.1539
22Unidentified N-formylalkaloid 1 isomer 2C22H24N2O518.11397.17635 369.1813337.1546299.1393256.0970224.0709
234-O-(4-Coumaroyl)quinic acidC16H18O818.13 337.09235191.0556173.0445163.0390119.048993.0331
24Nα-Formyl-12-methoxyechitamidineC22H26N2O518.14399.19200 371.1967339.1705299.1406267.1127198.0913[31]
25 14-Coumaric acidC9H8O318.50 163.03952119.048893.0331
265-O-Feruloylquinic acidC17H20O918.51 367.10291193.0501191.0553173.0445134.036193.0330
27Vallesamine or isomerC20H24N2O318.55341.18652 309.1598237.1023209.1074194.0960168.0806
28N-FormylechitamidineC21H24N2O418.91369.18143 351.1710341.1862309.1599202.0874 [32]
294-O-Feruloylquinic acidC17H20O919.03 367.10291193.0501191.0554173.0445134.036293.0330
305-O-(4-Coumaroyl)quinic acid cis isomerC16H18O819.70 337.09235191.0554173.0444163.0389119.048793.0331
31Unidentified N-formylalkaloid 2C22H24N2O420.33381.18143 353.1862339.1706321.1599263.1175212.0942
32Secologanoside methyl esterC17H24O1120.76 403.12404371.0987223.0606179.0551165.0545121.0281
33Lagumicine or isomerC20H22N2O421.06355.16578 337.1548214.0863200.0709182.0603154.0652
34AkuammicineC20H22N2O221.47323.17596 294.1491291.1495280.1342263.1543234.1286
35Unidentified alkaloid isomer 1C21H24N2O421.55369.18143 337.1545309.1619299.1390267.1128224.0703
36EchitamidineC20H24N2O321.87341.18652 323.1757309.1581202.0863142.0653140.1072[30]
37N-MethylakuammicineC21H24N2O222.03337.19161 309.1607305.1650294.1504277.1700263.1543
38Unidentified alkaloid isomer 2C21H24N2O422.06369.18143 337.1547309.1598299.1392256.0967224.0708
39Akuammicine isomer N-methyl derivativeC21H24N2O222.50337.19161 305.1649277.1700263.1543248.1086222.1277
40Akuammicine isomer 1C20H22N2O222.70323.17596 291.1494280.1332263.1549249.1385234.1279
41TubotaiwineC20H24N2O222.88325.19160 293.1648265.1335236.1440222.1286194.0960
42Akuammicine isomer 2C20H22N2O223.11323.17596 291.1494280.1335263.1528249.1387234.1285
4317-O-Acetyl-N-demethylechitamine or isomerC23H28N2O523.16413.20765 395.1964353.1862335.1760292.1334232.1123
44 1Isoquercitrin (Quercetin-3-O-glucoside)C21H20O1223.48 463.08765301.0357300.0280271.0251255.0300151.0022
45 1Rutin (Quercetin-3-O-rutinoside)C27H30O1623.56 609.14557301.0357300.0278271.0251255.0298151.0024
46VoacangineC22H28N2O324.77369.21782 337.1911309.1609266.1160252.1022 [33]
47AkuammidineC21H24N2O324.95353.18652 321.1599310.1412293.1650264.1369250.1236[33]
48 1Quercitrin (Quercetin-3-O-rhamnoside)C21H20O1125.03 447.09274301.0357300.0280271.0252255.0299151.0025
49Dihydroakuammidine or isomerC21H26N2O325.84355.20217 323.1756295.1441266.1541252.1388224.1061
50 1Quercetin (3,3′,4′,5,7-Pentahydroxyflavone)C15H10O727.58 301.03484273.0423178.9975151.0026121.0283107.0124
519-Hydroxyoctadecatrienoic acidC18H30O340.17 293.21167275.2022231.2111171.1017121.100959.0132
5213-Hydroxyoctadecatrienoic acidC18H30O340.31 293.21167275.2023235.1703223.1335195.1386179.1433
53LinoleamideC18H33NO44.49280.26404 263.2341245.2264109.101695.086181.0705
54OleamideC18H35NO45.75282.27969 265.2526247.2421135.117283.086169.0705
1 Confirmed by standard. 2 [M]+.
Table
Table 3. Chemical characterization of leaves—MAC-EA (not stirred).
Table 3. Chemical characterization of leaves—MAC-EA (not stirred).
No.NameFormulaRt[M + H]+[M − H]−Fragment 1Fragment 2Fragment 3Fragment 4Fragment 5Literature
1Quinic acidC7H12O61.28 191.05557173.0444171.0286127.0387111.043793.0330
2 1Chlorogenic acid (3-O-Caffeoylquinic acid)C16H18O914.95355.10291 163.0390145.0285135.0443117.033889.0389
3Caffeic acidC9H8O415.18 179.03444135.0439107.0483
4Loganic acidC16H24O1015.75 375.12913213.0764169.0857151.0755113.023069.0330
5Swertiamarin or isomerC16H22O1016.78375.12912 213.0757195.0655177.0547151.0390107.0495
6SecologanosideC16H22O1117.32 389.10839345.1188209.0453183.0652165.054769.0330
7Unidentified N-formylalkaloid 1 isomer 1C22H24N2O517.42397.17635 369.1809337.1545299.1388267.1129224.0708
85-O-(4-Coumaroyl)quinic acidC16H18O817.44 337.09235191.0555173.0447163.0388119.048693.0331
9Lochnericine or isomerC21H24N2O317.45353.18652 335.1740291.1498166.0862158.0964144.0808
10 2EchitamineC22H29N2O418.04385.21273 367.2022349.1909310.1435250.1225220.1121[30]
11Sweroside or isomerC16H22O918.07359.13421 197.0810179.0705151.0753127.0392111.0807
12Unidentified N-formylalkaloid 1 isomer 2C22H24N2O518.25397.17635 369.1808337.1544299.1390256.0969224.0708
13Nα-Formyl-12-methoxyechitamidineC22H26N2O518.31399.19200 371.1963339.1702299.1392267.1128198.0916[31]
145-O-Feruloylquinic acidC17H20O918.47 367.10291193.0502191.0555173.0444134.036293.0330
15 14-Coumaric acidC9H8O318.50 163.03952119.048893.0331
16Vallesamine or isomerC20H24N2O318.73341.18652 309.1596237.1022209.1075194.0965168.0806
17LoliolideC11H16O320.10197.11777 179.1068161.0961135.1170133.1014107.0859
18Unidentified N-formylalkaloid 2C22H24N2O420.41381.18143 353.1858339.1705321.1596263.1185212.0939
19Secologanoside methyl esterC17H24O1120.72 403.12404371.0987223.0608179.0550165.0548121.0281
20Lagumicine or isomerC20H22N2O421.02355.16578 337.1545214.0863200.0708182.0602154.0652
21AkuammicineC20H22N2O221.58323.17596 294.1490291.1494280.1331263.1543234.1281
22Unidentified alkaloid isomer 1C21H24N2O421.67369.18143 337.1547309.1600299.1390267.1127224.0703
23EchitamidineC20H24N2O321.98341.18652 323.1758309.1598202.0864142.0653140.1071[30]
24Unidentified alkaloid isomer 2C21H24N2O422.17369.18143 337.1547309.1602299.1391256.0969224.0710
25Akuammicine isomer 1C20H22N2O222.83323.17596 291.1493280.1337263.1538249.1389234.1289
26TubotaiwineC20H24N2O223.02325.19160 293.1648265.1340236.1435222.1282194.0967
27Akuammicine isomer 2C20H22N2O223.25323.17596 291.1493280.1341263.1549249.1388234.1277
2817-O-Acetyl-N-demethylechitamine or isomerC23H28N2O523.29413.20765 395.1972353.1858335.1755292.1332232.1121
29 1Isoquercitrin (Quercetin-3-O-glucoside)C21H20O1223.44 463.08765301.0358300.0280271.0252255.0288151.0025
30 1Rutin (Quercetin-3-O-rutinoside)C27H30O1623.52 609.14557301.0355300.0280271.0251255.0302151.0031
31VoacangineC22H28N2O324.93369.21782 337.1909309.1598266.1167252.1030 [33]
32 1Quercitrin (Quercetin-3-O-rhamnoside)C21H20O1125.01 447.09274301.0359300.0279271.0252255.0289151.0030
33AkuammidineC21H24N2O325.02353.18652 321.1599310.1441293.1654264.1377250.1245[33]
34Dihydroakuammidine or isomerC21H26N2O325.92355.20217 323.1753295.1441266.1550252.1387224.1069
35DihydroactinidiolideC11H16O227.15181.12286 163.1117145.1014135.1170121.1013107.0859
369-Hydroxyoctadecatrienoic acidC18H30O340.14 293.21167275.2021231.2113171.1016121.100959.0130
3713-Hydroxyoctadecatrienoic acidC18H30O340.25 293.21167275.2021235.1700223.1336195.1384179.1437
38LinoleamideC18H33NO44.45280.26404 263.2369245.2264109.101595.086081.0704
39OleamideC18H35NO45.70282.27969 265.2530247.2418135.117183.086169.0705
40Pheophytin AC55H74N4O565.78871.57375 593.2762533.2550460.2255
1 Confirmed by standard. 2 [M]+.
Table
Table 4. Chemical characterization of leaves—MAC-MeOH (not stirred).
Table 4. Chemical characterization of leaves—MAC-MeOH (not stirred).
No.NameFormulaRt[M + H]+[M − H]−Fragment 1Fragment 2Fragment 3Fragment 4Fragment 5Literature
1Quinic acidC7H12O61.26 191.05557173.0446171.0292127.0387111.043893.0330
2Unidentified dihydroxybenzoic acid derivativeC22H20O1213.64 475.08765299.0774153.0181137.0232109.0281
3 1Chlorogenic acid (3-O-Caffeoylquinic acid)C16H18O914.88355.10291 163.0390145.0285135.0442117.033789.0390
4Caffeic acidC9H8O415.19 179.03444135.0440107.0489
5Loganic acidC16H24O1015.76 375.12913213.0764169.0860151.0753113.023069.0330
6Vallesamine or isomerC20H24N2O315.77341.18652 309.1596236.1069208.1127194.0962168.0807
7Swertiamarin or isomerC16H22O1016.73375.12912 213.0757195.0653177.0546151.0390107.0495
8Unidentified N-formylalkaloid 1 isomer 1C22H24N2O517.27397.17635 369.1810337.1546299.1392267.1127224.0704
9Lochnericine or isomerC21H24N2O317.29353.18652 335.1751291.1490166.0862158.0964144.0807
10SecologanosideC16H22O1117.33 389.10839345.1191209.0452183.0656165.054669.0330
115-O-(4-Coumaroyl)quinic acidC16H18O817.47 337.09235191.0555173.0445163.0390119.048993.0331
12Picralinal or isomerC21H22N2O417.59367.16578 349.1923339.1703307.1440269.1285180.1020
13Quercetin-O-hexoside-O-rutinoside isomer 1C33H40O2117.70 771.19839609.1468462.0807301.0357300.0279299.0201
14 2EchitamineC22H29N2O417.89385.21273 367.2024349.1920310.1432250.1231220.1122[30]
15Picralinal or isomerC21H22N2O417.94367.16578 349.1930339.1703307.1441269.1285194.0601
16Quebrachidine or isomerC21H24N2O317.96353.18652 335.1750324.1594303.1491293.1651275.1537
17Quercetin-O-hexoside-O-rutinoside isomer 2C33H40O2117.99 771.19839609.1483463.0887462.0813301.0358299.0201
18Sweroside or isomerC16H22O918.02359.13421 197.0810179.0703151.0754127.0392111.0808
19Unidentified N-formylalkaloid 1 isomer 2C22H24N2O518.06397.17635 369.1809337.1545299.1390256.0968224.0708
204-O-(4-Coumaroyl)quinic acidC16H18O818.10 337.09235191.0555173.0445163.0389119.048893.0330
21Nα-Formyl-12-methoxyechitamidineC22H26N2O518.12399.19200 371.1962339.1701299.1392267.1130198.0914[31]
22 14-Coumaric acidC9H8O318.50 163.03952119.048893.0332
23Vallesamine or isomerC20H24N2O318.52341.18652 309.1597237.1018209.1073194.0965168.0808
245-O-Feruloylquinic acidC17H20O918.54 367.10291193.0499191.0555173.0445134.036293.0331
25N-FormylechitamidineC21H24N2O418.85369.18143 351.1705341.1858309.1598202.0864 [32]
265-O-(4-Coumaroyl)quinic acid cis isomerC16H18O819.69 337.09235191.0553173.0446163.0387119.048893.0330
27LoliolideC11H16O320.03197.11777 179.1067161.0961135.1169133.1014107.0858
28Unidentified N-formylalkaloid 2C22H24N2O420.20381.18143 353.1858339.1711321.1595263.1182212.0934
29SecologanolC17H26O1020.35391.16043 229.1070211.0965193.0862179.0704167.0704
30Quercecin-O-hexosylhexosideC27H30O1720.66 625.14048301.0359300.0278271.0250255.0298151.0024
31Secologanoside methyl esterC17H24O1120.73 403.12404371.0990223.0611179.0550165.0546121.0281
32Lagumicine or isomerC20H22N2O420.90355.16578 337.1546214.0862200.0708182.0602154.0651
33AkuammicineC20H22N2O221.38323.17596 294.1488291.1493280.1334263.1543234.1279
34Unidentified alkaloid isomer 1C21H24N2O421.47369.18143 337.1545309.1595299.1392267.1129224.0714
35Cinchonain I isomer 1C24H20O921.70 451.10291341.0668231.0295217.0138189.0186177.0184
36EchitamidineC20H24N2O321.73341.18652 323.1762309.1596202.0863142.0652140.1070[30]
37N-MethylakuammicineC21H24N2O221.96337.19161 309.1580305.1648294.1504277.1697263.1541
38Unidentified alkaloid isomer 2C21H24N2O422.01369.18143 337.1545309.1590299.1391256.0968224.0708
39Akuammicine isomer N-methyl derivativeC21H24N2O222.49337.19161 305.1648277.1702263.1542248.1074222.1277
40Cinchonain I isomer 2C24H20O922.69 451.10291341.0668231.0294217.0138189.0187177.0183
41Akuammicine isomer 1C20H22N2O222.71323.17596 291.1491280.1332263.1538249.1385234.1290
42TubotaiwineC20H24N2O222.87325.19160 293.1647265.1341236.1434222.1282194.0964
43Akuammicine isomer 2C20H22N2O223.09323.17596 291.1491280.1328263.1541249.1385234.1309
4417-O-Acetyl-N-demethylechitamine or isomerC23H28N2O523.15413.20765 395.1971353.1856335.1757292.1333232.1120
45 1Isoquercitrin (Quercetin-3-O-glucoside)C21H20O1223.45 463.08765301.0357300.0278271.0249255.0299151.0024
46 1Rutin (Quercetin-3-O-rutinoside)C27H30O1623.54 609.14557301.0357300.0278271.0250255.0299151.0024
47Di-O-caffeoylquinic acidC25H24O1224.65 515.11896353.0880335.0790191.0553179.0340173.0444
48VoacangineC22H28N2O324.75369.21782 337.1907309.1591266.1159252.1027 [33]
49AkuammidineC21H24N2O324.91353.18652 321.1598310.1444293.1647264.1374250.1248[33]
50 1Quercitrin (Quercetin-3-O-rhamnoside)C21H20O1125.03 447.09274301.0356300.0278271.0250255.0299151.0024
51Dihydroakuammidine or isomerC21H26N2O325.79355.20217 323.1753295.1441266.1559252.1392224.1068
52Dimethoxy-tetrahydroxy(iso)flavoneC17H14O826.52 345.06105330.0384315.0151287.0199271.0262243.0297
53 1Quercetin (3,3′,4′,5,7-Pentahydroxyflavone)C15H10O727.56 301.03484273.0393178.9981151.0024121.0281107.0125
54Dimethoxy-trihydroxy(iso)flavoneC17H14O733.32 329.06613314.0436299.0200285.0400271.0251243.0299
55Dihydroxy-trimethoxy(iso)flavone isomer 1C18H16O735.18 343.08178328.0594313.0359285.0395269.0471257.0465
56Dihydroxy-trimethoxy(iso)flavone isomer 2C18H16O735.52 343.08178328.0589313.0358285.0405269.0451257.0447
579-Hydroxyoctadecatrienoic acidC18H30O340.17 293.21167275.2022231.2119171.1016121.100959.0125
5813-Hydroxyoctadecatrienoic acidC18H30O340.27 293.21167275.2021235.1699223.1334195.1384179.1430
59LinoleamideC18H33NO44.44280.26404 263.2371245.2262109.101595.086081.0705
60OleamideC18H35NO45.68282.27969 265.2525247.2418135.116883.086069.0705
61Pheophytin AC55H74N4O565.73871.57375 593.2762533.2551460.2258
1 Confirmed by standard. 2 [M]+.
Table
Table 5. Chemical characterization of Stem bark—infusion.
Table 5. Chemical characterization of Stem bark—infusion.
No.NameFormulaRt[M + H]+[M − H]−Fragment 1Fragment 2Fragment 3Fragment 4Fragment 5Literature
1Quinic acidC7H12O61.25 191.05557173.0444171.0289127.0387111.043793.0330
2Neochlorogenic acid (5-O-Caffeoylquinic acid)C16H18O910.29355.10291 163.0389145.0284135.0442117.033789.0390
33-O-(4-Coumaroyl)quinic acidC16H18O813.33 337.09235191.0553173.0444163.0389119.048893.0329
4 1Chlorogenic acid (3-O-Caffeoylquinic acid)C16H18O914.90355.10291 163.0390145.0284135.0442117.033889.0389
53-O-Feruloylquinic acidC17H20O915.14 367.10291193.0499191.0556173.0445134.036193.0331
6Loganic acidC16H24O1015.72 375.12913213.0763169.0860151.0754113.023069.0330
7Vallesamine or isomerC20H24N2O315.75341.18652 309.1598236.1071208.1126194.0968168.0807
8Chryptochlorogenic acid (4-O-Caffeoylquinic acid)C16H18O916.15355.10291 163.0389145.0285135.0442117.033789.0390
9Swertiamarin or isomerC16H22O1016.75375.12912 213.0757195.0654177.0546151.0390107.0495
10Unidentified N-formylalkaloid 1 isomer 1C22H24N2O517.06397.17635 369.1811337.1546299.1391267.1130224.0709
11Lochnericine or isomerC21H24N2O317.16353.18652 335.1754291.1495166.0863158.0965144.0808
12SecologanosideC16H22O1117.32 389.10839345.1191209.0453183.0654165.054669.0330
13 2EchitamineC22H29N2O417.33385.21273 367.2018349.1896310.1437250.1225220.1120[30]
145-O-(4-Coumaroyl)quinic acidC16H18O817.48 337.09235191.0553173.0444163.0388119.048893.0330
15Picralinal or isomerC21H22N2O417.94367.16578 349.1930339.1703307.1441269.1285194.0601
16Sweroside or isomerC16H22O918.02359.13421 197.0811179.0704151.0754127.0392111.0808
174-O-(4-Coumaroyl)quinic acidC16H18O818.10 337.09235191.0551173.0444163.0390119.048893.0330
18Unidentified N-formylalkaloid 1 isomer 2C22H24N2O518.12397.17635 369.1810337.1546299.1389256.0966224.0708
19Nα-Formyl-12-methoxyechitamidineC22H26N2O518.17399.19200 371.1965339.1703299.1392267.1126198.0917[31]
20 14-Coumaric acidC9H8O318.46 163.03952119.048893.0333
21Vallesamine or isomerC20H24N2O318.48341.18652 309.1596237.1027209.1079194.0961168.0806
225-O-Feruloylquinic acidC17H20O918.50 367.10291193.0500191.0554173.0445134.036293.0330
23N-FormylechitamidineC21H24N2O418.91369.18143 351.1702341.1859309.1596202.0861 [32]
244-O-Feruloylquinic acidC17H20O919.00 367.10291193.0500191.0555173.0445134.036193.0330
25LoganinC17H26O1019.03391.16043 229.1071211.0966197.0812179.0704109.0651
26LoliolideC11H16O320.06197.11777 179.1068161.0960135.1170133.1014107.0859
27Venalstonine or isomerC21H24N2O220.26337.19161 305.1649294.1490277.1698234.1279196.0996
28Unidentified N-formylalkaloid 2C22H24N2O420.36381.18143 353.1859339.1691321.1598263.1185212.0945
29Secologanoside methyl esterC17H24O1120.72 403.12404371.0988223.0607179.0547165.0545121.0281
30AkuammicineC20H22N2O221.42323.17596 294.1490291.1493280.1322263.1541234.1304
31Unidentified alkaloid isomer 1C21H24N2O421.66369.18143 337.1545309.1601299.1392267.1126224.0701
32EchitamidineC20H24N2O321.93341.18652 323.1738309.1594202.0864142.0653140.1071[30]
33Unidentified alkaloid isomer 2C21H24N2O422.13369.18143 337.1546309.1597299.1391256.0969224.0708
34Unidentified alkaloid 2C24H30N2O522.27427.22330 409.2123352.1542292.1332250.1227232.1121
357-Deoxyloganic acidC16H24O922.36 359.13421197.0813153.0909135.0803109.064589.0229
36Akuammicine isomer 1C20H22N2O222.74323.17596 291.1494280.1335263.1538249.1387234.1281
37TubotaiwineC20H24N2O222.94325.19160 293.1648265.1342236.1444222.1281194.0965
38Akuammicine isomer 2C20H22N2O223.21323.17596 291.1494280.1336263.1546249.1387234.1281
39BooneinC9H14O323.23 169.08647151.0751141.0915125.0957 [34]
4017-O-Acetyl-N-demethylechitamine or isomerC23H28N2O523.24413.20765 395.1967353.1859335.1765292.1330232.1123
41Di-O-caffeoylquinic acidC25H24O1224.62 515.11896353.0883335.0790191.0554179.0341173.0445
42Dimethoxy-trihydroxy(iso)flavoneC17H14O733.34 329.06613314.0438299.0200285.0400271.0247243.0299
43EmodinC15H10O539.63 269.04500241.0501225.0552197.0601210.0318181.0649
449-Hydroxyoctadecatrienoic acidC18H30O340.14 293.21167275.2021231.2109171.1016121.100959.0125
4513-Hydroxyoctadecatrienoic acidC18H30O340.30 293.21167275.2020235.1701223.1335195.1383179.1429
46LinoleamideC18H33NO44.45280.26404 263.2367245.2264109.101495.085981.0704
47OleamideC18H35NO45.72282.27969 265.2527247.2418135.116983.086069.0705
1 Confirmed by standard. 2 [M]+.
Table
Table 6. Chemical characterization of stem bark—MAC-EA (not stirred).
Table 6. Chemical characterization of stem bark—MAC-EA (not stirred).
No.NameFormulaRt[M + H]+[M − H]−Fragment 1Fragment 2Fragment 3Fragment 4Fragment 5Literature
1Quinic acidC7H12O61.27 191.05557173.0447171.0288127.0388111.043793.0330
2 1Chlorogenic acid (3-O-Caffeoylquinic acid)C16H18O914.91355.10291 163.0390145.0285135.0442117.033889.0389
3Loganic acidC16H24O1015.77 375.12913213.0762169.0859151.0754113.022969.0330
4Swertiamarin or isomerC16H22O1016.73375.12912 213.0757195.0654177.0548151.0390107.0495
5SecologanosideC16H22O1117.34 389.10839345.1187209.0451183.0653165.054569.0330
65-O-(4-Coumaroyl)quinic acidC16H18O817.46 337.09235191.0553173.0439163.0383119.048993.0328
7 2EchitamineC22H29N2O417.83385.21273 367.2011349.1912310.1437250.1226220.1121[30]
8Sweroside or isomerC16H22O918.05359.13421 197.0810179.0704151.0755127.0392111.0808
9Nα-Formyl-12-methoxyechitamidineC22H26N2O518.31399.19200 371.1964339.1703299.1392267.1131198.0914[31]
105-O-Feruloylquinic acidC17H20O918.50 367.10291193.0498191.0554173.0445134.036293.0330
11 14-Coumaric acidC9H8O318.52 163.03952119.048793.0328
12Vallesamine or isomerC20H24N2O318.58341.18652 309.1598237.1021209.1074194.0965168.0806
13LoganinC17H26O1018.97391.16043 229.1070211.0965197.0812179.0704109.0652
14LoliolideC11H16O320.07197.11777 179.1068161.0961135.1170133.1013107.0859
15Venalstonine or isomerC21H24N2O220.38337.19161 305.1649294.1485277.1699234.1279196.0998
16Secologanoside methyl esterC17H24O1120.73 403.12404371.0984223.0609179.0549165.0545121.0281
17EchitamidineC20H24N2O322.03341.18652 323.1751309.1603202.0864142.0653140.1071[30]
187-Deoxyloganic acidC16H24O922.35 359.13421197.0813153.0909135.0802109.064489.0228
19Unidentified alkaloid 2C24H30N2O522.37427.22330 409.2125352.1544292.1333250.1228232.1122
20TubotaiwineC20H24N2O222.95325.19160 293.1648265.1346236.1428222.1279194.0959
2117-O-Acetyl-N-demethylechitamine or isomerC23H28N2O523.22413.20765 395.1960353.1850335.1763292.1329232.1122
22Di-O-caffeoylquinic acidC25H24O1224.63 515.11896353.0882335.0790191.0553179.0339173.0444
23Dimethoxy-trihydroxy(iso)flavoneC17H14O733.35 329.06613314.0434299.0199285.0410271.0249243.0290
24LinoleamideC18H33NO44.47280.26404 263.2370245.2264109.101595.086081.0705
25OleamideC18H35NO45.71282.27969 265.2525247.2424135.117083.086069.0705
26Pheophytin AC55H74N4O565.51871.57375 593.2759533.2547460.2255
1 Confirmed by standard. 2 [M]+.
Table
Table 7. Chemical characterization of stem bark—MAC-MEOH (not stirred).
Table 7. Chemical characterization of stem bark—MAC-MEOH (not stirred).
No.NameFormulaRt[M + H]+[M − H]−Fragment 1Fragment 2Fragment 3Fragment 4Fragment 5Literature
1Quinic acidC7H12O61.25 191.05557173.0443171.0289127.0388111.043593.0330
2 1Chlorogenic acid (3-O-Caffeoylquinic acid)C16H18O914.88355.10291 163.0389145.0284135.0442117.033789.0390
3Loganic acidC16H24O1015.69 375.12913213.0762169.0859151.0750113.022969.0329
4Swertiamarin or isomerC16H22O1016.69375.12912 213.0757195.0653177.0546151.0389107.0495
5Unidentified N-formylalkaloid 1 isomer 1C22H24N2O516.86397.17635 369.1806337.1543299.1389267.1126224.0704
62EchitamineC22H29N2O416.92385.21273 367.2010349.1913310.1436250.1224220.1118[30]
7SecologanosideC16H22O1117.33 389.10839345.1187209.0447183.0656165.054469.0329
85-O-(4-Coumaroyl)quinic acidC16H18O817.48 337.09235191.0552173.0444163.0388119.048793.0330
9Sweroside or isomerC16H22O917.86359.13421 197.0808179.0702151.0753127.0391111.0807
10Unidentified N-formylalkaloid 1 isomer 2C22H24N2O518.05397.17635 369.1807337.1545299.1389256.0966224.0707
11Nα-Formyl-12-methoxyechitamidineC22H26N2O518.11399.19200 371.1964339.1702299.1392267.1128198.0919[31]
12Vallesamine or isomerC20H24N2O318.42341.18652 309.1596237.1024209.1073194.0963168.0807
13 14-Coumaric acidC9H8O318.49 163.03952119.048793.0331
145-O-Feruloylquinic acidC17H20O918.50 367.10291193.0498191.0554173.0444134.036093.0330
15N-FormylechitamidineC21H24N2O418.82369.18143 351.1700341.1859309.1596202.0860 [32]
16LoganinC17H26O1018.89391.16043 229.1071211.0969197.0800179.0705109.0650
17LoliolideC11H16O320.04197.11777 179.1068161.0960135.1170133.1014107.0859
18Venalstonine or isomerC21H24N2O220.07337.19161 305.1647294.1482277.1697234.1274196.0992
19Unidentified N-formylalkaloid 2C22H24N2O420.28381.18143 353.1859339.1705321.1596263.1181212.0936
20Secologanoside methyl esterC17H24O1120.71 403.12404371.0983223.0604179.0547165.0545121.0280
21AkuammicineC20H22N2O221.46323.17596 294.1487291.1491280.1331263.1540234.1284
22EchitamidineC20H24N2O321.75341.18652 323.1754309.1594202.0863142.0652140.1071[30]
23Unidentified alkaloid 2C24H30N2O522.06427.22330 409.2113352.1541292.1334250.1222232.1119
247-Deoxyloganic acidC16H24O922.35 359.13421197.0812153.0908135.0802109.064489.0228
25Akuammicine isomer 1C20H22N2O222.62323.17596 291.1491280.1329263.1538249.1384234.1269
26TubotaiwineC20H24N2O222.81325.19160 293.1646265.1328236.1436222.1281194.0963
27Akuammicine isomer 2C20H22N2O223.06323.17596 291.1491280.1329263.1538249.1384234.1269
28BooneinC9H14O323.12 169.08647151.0752141.0909125.0959 [34]
2917-O-Acetyl-N-demethylechitamine or isomerC23H28N2O523.14413.20765 395.1974353.1853335.1755292.1328232.1122
30Di-O-caffeoylquinic acidC25H24O1224.62 515.11896353.0878335.0742191.0553179.0339173.0443
31Dimethoxy-trihydroxy(iso)flavoneC17H14O733.34 329.06613314.0434299.0197285.0404271.0247243.0296
329-Hydroxyoctadecatrienoic acidC18H30O340.16 293.21167275.2019231.2110171.1015121.100859.0125
3313-Hydroxyoctadecatrienoic acidC18H30O340.26 293.21167275.2017235.1699223.1332195.1384179.1441
34LinoleamideC18H33NO44.44280.26404 263.2368245.2262109.101595.086081.0704
35OleamideC18H35NO45.70282.27969 265.2524247.2424135.116983.086069.0705
36Pheophytin AC55H74N4O565.49871.57375 593.2759533.2549460.2255
1 Confirmed by standard. 2 [M]+.
Table
Table 8. Antioxidant properties of the studied extracts *.
Table 8. Antioxidant properties of the studied extracts *.
PartsExtraction Methods/SolventDPPH•
(mg TE/g)		ABTS•+
(mg TE/g)		CUPRAC
(mg TE/g)		FRAP (mg TE/g)Metal Chelating (mg EDTAE/g)
LeavesInfusion-water74.70 ± 1.27 a131.50 ± 2.34 a140.57 ± 0.37 d109.35 ± 1.48 a13.42 ± 2.06 e
MAC-EA13.96 ± 0.87 f36.78 ± 1.73 f82.79 ± 1.68 f41.54 ± 0.43 f31.51 ± 1.05 a
MAC-MeOH55.14 ± 0.44 c109.24 ± 3.07 c146.71 ± 3.80 c87.37 ± 1.42 d26.07 ± 0.52 b
MAC-EA (not stirred)18.40 ± 0.64 e43.67 ± 2.11 e87.72 ± 1.10 f38.03 ± 0.86 g24.32 ± 1.11 bc
MAC-MeOH (not stirred)61.23 ± 1.46 b123.70 ± 0.27 b163.91 ± 2.20 a100.86 ± 0.40 b22.67 ± 0.50 cd
SE-EA27.99 ± 1.11 d88.67 ± 1.26 d118.54 ± 0.86 e61.48 ± 1.78 e21.17 ± 0.63 d
SE-MeOH61.32 ± 0.47 b127.63 ± 1.65 ab155.36 ± 1.10 b97.05 ± 0.74 c21.90 ± 0.41 cd
Stem barkInfusion-water33.82 ± 0.66 b59.94 ± 1.70 c74.80 ± 0.56 c55.48 ± 1.17 ab36.71 ± 0.14 a
MAC-EA4.54 ± 0.94 cna76.51 ± 1.11 c40.41 ± 0.80 cna
MAC-MeOH35.41 ± 0.47 b69.24 ± 2.82 b94.89 ± 1.59 b53.94 ± 2.18 b22.21 ± 1.93 b
MAC-EA (not stirred)5.88 ± 0.76 cna74.37 ± 2.06 c36.04 ± 0.12 dna
MAC- MeOH (not stirred)37.57 ± 0.74 a73.69 ± 1.17 a105.92 ± 4.86 a54.48 ± 1.67 b23.77 ± 0.23 b
SE-EA5.94 ± 1.07 c6.04 ± 0.91 d78.31 ± 0.72 c36.64 ± 0.15 dna
SE-MeOH34.74 ± 0.48 b71.22 ± 0.17 ab101.53 ± 1.80 a58.03 ± 0.80 a18.23 ± 0.16 c
* 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).
Table
Table 9. Enzyme inhibitory properties of the studied extracts *.
Table 9. Enzyme inhibitory properties of the studied extracts *.
PartsExtraction Methods/SolventAChE
(mg GALAE/g)		BChE
(mg GALAE/g)		Amylase
(mmol ACAE/g)		Glucosidase
(mmol ACAE/g)		Tyrosinase
(mg KAE/g)
LeavesInfusion-waterna6.35 ± 0.18 b0.17 ± 0.01 dna0.72 ± 0.07 c
MAC-EA5.02 ± 0.34 ab4.78 ± 0.11 cd1.16 ± 0.08 ana129.70 ± 2.85 ab
MAC-MeOH4.49 ± 0.11 b3.22 ± 0.92 e0.91 ± 0.02 c5.24 ± 0.09 a132.83 ± 1.04 ab
MAC-EA (not stirred)4.45 ± 0.67 b8.25 ± 0.43 a1.13 ± 0.06 ana127.11 ± 9.18 b
MAC-MeOH (not stirred)5.57 ± 0.49 a6.18 ± 0.19 bc0.97 ± 0.03 bc5.27 ± 0.09 a135.89 ± 0.63 ab
SE-EA4.89 ± 0.06 ab5.19 ± 0.58 bc1.10 ± 0.09 abna130.08 ± 5.20 ab
SE-MeOH5.04 ± 0.34 ab3.49 ± 0.60 de0.88 ± 0.02 c5.04 ± 0.60 a139.20 ± 0.48 a
Stem barkInfusion-waterna10.08 ± 0.29 a0.20 ± 0.01 cna0.43 ± 0.01 d
MAC-EA5.02 ± 0.43 a1.44 ± 0.36 d1.01 ± 0.08 a4.71 ± 0.01 c121.85 ± 1.56 b
MAC-MeOH5.34 ± 0.37 a2.51 ± 0.28 d0.71 ± 0.06 b5.40 ± 0.01 a133.51 ± 1.10 a
MAC-EA (not stirred)4.91 ± 0.62 a1.59 ± 0.03 d0.98 ± 0.06 a4.67 ± 0.02 d123.69 ± 0.53 b
MAC- MeOH (not stirred)5.62 ± 0.23 a4.13 ± 0.65 c0.80 ± 0.02 b5.38 ± 0.01 a135.02 ± 1.20 a
SE-EA5.78 ± 0.18 a6.39 ± 0.47 b1.04 ± 0.01 a4.83 ± 0.01 b94.63 ± 2.53 c
SE-MeOH5.67 ± 0.20 a4.38 ± 0.66 c0.78 ± 0.03 b5.41 ± 0.01 a134.63 ± 0.56 a
* 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).
Table
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.
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)
LPS0.1 583.3 ± 61.3 *916.7 ± 48.7 *1033.3 ± 68.0 *
Dexamethasone0.04 150.0 ± 26.4 *189.2 ± 36.8 *330.0 ± 88.5 *
Leaves—infusion2597.0 ± 1.285.4 ± 4.492.0 ± 4.777.8 ± 5.9
5094.3 ± 3.173.5 ± 7.0 a69.7 ± 3.2 a61.8 ± 4.4 a
10089.9 ± 4.764.0 ± 4.7 b52.4 ± 6.9 c53.2 ± 5.9 b
Leaves—MAC-EA (not stirred)2599.1 ± 2.191.7 ± 3.792.0 ± 3.093.3 ± 3.0
5083.3 ± 5.9 b87.2 ± 3.985.1 ± 5.188.9 ± 3.8
10073.7 ± 2.7 c73.1 ± 3.6 a66.3 ± 5.2 b69.5 ± 4.8 b
Leaves—MAC-MeOH (not stirred)2594.5 ± 2.690.6 ± 2.082.3 ± 4.282.2 ± 5.0
5096.5 ± 2.468.4 ± 2.2 c70.0 ± 4.6 b64.3 ± 4.5 b
10085.9 ± 3.7 b62.9 ± 1.4 c62.9 ± 4.1 c44.5 ± 5.9 d
Stem bark—Infusion2598.9 ± 1.068.1 ± 6.7 b76.6 ± 6.3 a70.7 ± 7.1 a
5096.7 ± 2.144.6 ± 5.4 d50.7 ± 4.6 d44.2 ± 5.3 d
10094.3 ± 3.519.0 ± 1.7 d31.0 ± 2.4 d23.5 ± 3.0 d
Stem bark—MAC-EA (not stirred)2592.9 ± 3.694.8 ± 2.092.2 ± 1.898.3 ± 1.6
5083.9 ± 3.2 a92.4 ± 3.186.1 ± 4.387.1 ± 3.9
10076.1 ± 4.8 c81.5 ± 5.4 a71.5 ± 4.2 c64.3 ± 5.4 d
Stem bark—MAC-MeOH (not stirred)2598.6 ± 2.766.6 ± 2.6 d66.2 ± 7.2 c64.0 ± 6.1 d
5092.6 ± 2.248.5 ± 4.7 d44.0 ± 4.0 d38.4 ± 4.1 d
10089.4 ± 3.2 a29.0 ± 4.1 d22.9 ± 3.3 d22.4 ± 2.8 d
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Mollica, A.; Zengin, G.; Sinan, K.I.; Marletta, M.; Pieretti, S.; Stefanucci, A.; Etienne, O.K.; Jekő, J.; Cziáky, Z.; Bahadori, M.B.; et al. A Study on Chemical Characterization and Biological Abilities of Alstonia boonei Extracts Obtained by Different Techniques. Antioxidants 2022, 11, 2171. https://doi.org/10.3390/antiox11112171

AMA Style

Mollica A, Zengin G, Sinan KI, Marletta M, Pieretti S, Stefanucci A, Etienne OK, Jekő J, Cziáky Z, Bahadori MB, et al. A Study on Chemical Characterization and Biological Abilities of Alstonia boonei Extracts Obtained by Different Techniques. Antioxidants. 2022; 11(11):2171. https://doi.org/10.3390/antiox11112171

Chicago/Turabian Style

Mollica, Adriano, Gokhan Zengin, Kouadio Ibrahime Sinan, Marcella Marletta, Stefano Pieretti, Azzurra Stefanucci, Ouattara Katinan Etienne, József Jekő, Zoltán Cziáky, Mir Babak Bahadori, and et al. 2022. "A Study on Chemical Characterization and Biological Abilities of Alstonia boonei Extracts Obtained by Different Techniques" Antioxidants 11, no. 11: 2171. https://doi.org/10.3390/antiox11112171

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