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

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.


Introduction
Alstonia boonei, belonging to the Apocynaceae family, has been extensively used in traditional medicine. This ethnomedicinal plant, commonly found in tropical and subtropi-cal 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 (IC 50 : 3.17 mg/mL) and α-glucosidase (IC 50 : 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.

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.

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.

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.

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 Scientific TM , 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.

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.

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. Oneway 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).

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].

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 Tables 2-7. 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.

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-Fe 3+ complex to be reduced to the TPTZ-Fe 2+ complex, yielding a chromophore with maximum absorption at 593 nm, and the neocuproine-Cu 2+ 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.

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.

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).

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