1.   Introduction

Alstonia boonei De Wild, commonly known as "God's tree" or "Onyame dua," holds a significant place in the cultural and medicinal traditions of West Africa [1]. Revered in many forest communities, the plant is considered sacred, with its parts often reserved for therapeutic purposes rather than culinary use [1]. This cultural reverence underscores its importance as a cornerstone of traditional medicine, where it has been employed for a wide range of treatments, including antimalarial, antidiabetic, antimicrobial, aphrodisiac, and antipyretic applications [1]. Despite its extensive medicinal applications, the bioactivity of A. boonei exhibits specificity, as it lacks fungicidal effects against certain fungal isolates responsible for crown rot disease in bananas [2]. This selective efficacy suggests that its bioactive compounds may target specific pathogens or physiological conditions more effectively. Such specificity warrants a closer examination of its phytochemical constituents, which are responsible for its diverse pharmacological properties [3, 4]. The medicinal value of A. boonei is attributed to its rich phytochemical profile, encompassing alkaloids, tannins, saponins, flavonoids, cardiac glycosides, steroids, phenols, and terpenoids [3, 4]. Many bioactive compounds, including echitamine, echitamidine, akuammidine, Nα-formylechitamidine, β-amyrin, lupeol, β-amyrone, lupeol acetate, β-sitosterol and β-amyrin acetate (Fig 1) have been previously isolated from Alstonia boonei., have been isolated from various parts of the plant [1].

Figure 1. Previous compound Isolations from Alstonia boonei

These compounds play crucial roles in its therapeutic effects, offering potential pathways for targeted drug development. The extraction method and choice of solvent significantly influence the phytochemical composition and, consequently, the biological activity of A. boonei extracts. Methanol extracts, for example, have been shown to yield higher levels of phenolic compounds compared to ethyl acetate extracts. Advanced analytical techniques, such as Ultra-High-Performance Liquid Chromatography-Mass Spectrometry (UHPLC-MS), have identified key compounds in methanol extracts, including phenolic acids (caffeic, chlorogenic, and ferulic acids), flavonoids (rutin and isoquercetin), and flavonolignans (Cinchonain isomers). Notably, a novel compound, 2-methyl-3-propylbutane-1,4-diol (MPBD), was recently isolated from the methanol fraction of the stem bark extract, underscoring the plant’s potential for yielding unique bioactive agents [5, 6]. While the therapeutic applications of A. boonei are well-documented, its fungicidal properties, particularly against wood-degrading fungi, remain underexplored. The lack of comprehensive studies in this area represents a critical research gap. Investigating the bioactive compounds of A. boonei and their potential antifungal activity could not only expand its pharmacological utility but also provide novel solutions for managing wood-degrading fungi.

2.   Materials and methods

2.1 Plant collection and preparation

The stem bark of Alstonia boonei was harvested from a standing tree in Edo State, southern Nigeria, where this species is prevalent and traditionally used in medicine for treating various ailments. The collected stem bark was air-dried under shade for three weeks at an ambient temperature of 27-34 ℃, with controlled humidity conditions to preserve its phytochemical integrity. Once dried, the material was pulverized using a wooden mortar and pestle to a fine powder, ensuring uniform particle size for optimal extraction efficiency.

Fig. 2 depicts the leaves and stem of A. boonei as photographed during the study and Scheme 1 denotes the total work procedures.

Figure 2. Leaves and stem of A. boonei

Scheme 1. Isolation protocol for triterpenes and steroids isolated from A. boonei

2.2 Extraction of A. boonei stem bark

Methanol, ethyl acetate, and n-hexane solvents were purified by distillation to remove impurities.  Pulverized A. boonei stem bark (1 Kg) was macerated in n-hexane (2 L) for 24 hours (w/v ratio). The resulting extract was filtered using Whatman No. 1 filter paper and transferred into labeled glass bottles. The filtrate was concentrated using a rotary evaporator to obtain the dried crude extract. This was repeated for the ethyl acetate and methanol extracts.

2.3 Column chromatography procedure

The ethyl acetate crude extract (5.3 g) of A. boonei stem bark was subjected to column chromatography. The column was prepared by packing a glass column with silica gel 60-120 mesh (Merck) to a height of 60 cm, with a cotton wool layer at the neck. Ethyl acetate (5%) in n-hexane was used to prepare the slurry. The introduced slurry was left for eight hours to ensure proper packing and preparation for effective separation, Scheme 2 illustrates the basic isolation protocol. The column was eluted with solvent mixtures of n-hexane and ethyl acetate, varying the ratios from 95%:5% to 0%:100%; a stepwise increase in ethyl acetate to achieve increasing polarity. The collected fractions were air-dried in a fumehood and weighed with a precision balance. Fractions ABO 39, ABO 41, ABO 43, ABO 45, ABP 68, ABP 70, ABP 74, ABP 76, ABP 87, ABP 89, ABP 91, ABP 93, ABP 105, ABP 107 and ABP 109 were further analyzed using Nuclear Magnetic Resonance (NMR) spectroscopic analysis, while fractions ABO-42, ABO-44, ABO-69, and ABO-71 were reserved for antifungal testing.

Scheme 2. Work summary

2.4 Antifungal screening of test fungi

The antifungal activity of A. boonei stem bark fractions and crude extract were evaluated against selected wood-degrading fungi, including Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Coniophora puteana, Fibroporia vaillantii, Fomitopsis pinicola, Fusarium sp., Rhizopus spp., Sclerotium rolfsii, Trichoderma sp., and Serpula lacrymans. ketoconazole and sparfloxacin served as standard antifungal controls. The disk diffusion method by Chand et al. [7] was adopted to screen the extracts and fractions for antifungal activity over seven days by observing zones of fungal growth inhibition.

2.5 Determination of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC)

The broth dilution method, based on the protocol described by Chand et al. [7], was also employed to determine the MIC and MFC of the extract and fractions. Petri dishes with the lowest concentration of extracts or fractions that showed no colony growth were recorded as the MIC, while those without colony regrowth after subculturing were noted as the MFC.

3.   Results

3.1 Compounds isolated from A. boonei stem bark

Table 1 presents an array of compounds isolated from A. boonei stem bark. The compounds: 24-Methylenecycloartenol (1), β-amyrin (2), cycloeucalenol (3), stigmasterol (4), β-sitosterol (5), betulinic acid (6), α-amyrin acetate (7), and lupeol acetate (8) range from cycloartane triterpenes, lupane triterpenes, ursane, oleanane and sterols (Scheme 2).

Table 1. Compounds isolated from A. boonei

24-Methylenecycloartenol (1) (Fig. 3) (ABP 68; ABP 70): White powder. ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 4.74 (1H, m, H-31a), 4.63 (1H, m, H-31b) )[olefinic cycloartanes signals], 3.20 (1H, ddd, J = 14.1, 11.0, 4.8 Hz, H-3) [oxymethine proton], 2.37 (1H, td, J = 11.0, 5.7 Hz, H-23b), 2.26 (1H, m, H-23a), 1.76 (1H, td, J = 13.7, 4.5 Hz, H-17), 1.68 (1H, s, H-20), 1.13 (2H, s, H-18, H-19), 0.99 (2H, s, H-25, H-26), 0.96 (3H, s, H-28), 0.93 (2H, s, H-27, H-30), 0.87 (3H, s, H-29), 0.83 (2H, s, H-22, H-21), 0.79 (3H, s, H-16), 0.76 (1H, s, H-15), 0.55 (1H, d, J = 4.3 Hz, H-19a), 0.33 (1H, d, J = 4.2 Hz, H-19b) [non-equivalent high-field, cycloartane cyclopropyl protons] [37, 38].

Figure 3. 24-Methylenecycloartenol (ABP 68; ABP 70)

β-Amyrin (2) (Fig. 4) (ABP 68; ABP 70; ABP 74): White powder. ¹H NMR (400 MHz, CDCl₃): δH 5.18 (t, 1H, J = 3.7 Hz, H-12) [olefinic], 3.21 (m, 1H, H-3)) [oxymethine proton], 0.96 (s, 3H, H-29), 0.87 (s, 3H, H-30), 0.79 (s, 3H, H-24). Comparison of It’s characteristic signals with the literature [30] allowed for unambiguous characterisation of the compound as β-amyrin (Fig. 4).

Figure 4. β-Amyrin (ABP 68; BP70; ABP 74)

Cycloeucalenol (3)  (Fig. 5a) (ABP76): While crystal. 1H NMR (400 MHz, CDCl3) δ 4.72 (s, 0H), 4.66 (s, 0.4H) [olefinic cycloartanes signals], 3.21 (td, J = 9.3, 7.7, 4.5 Hz, 1H) [oxymethine proton], 1.25 (s, 2H), 1.14 (s, 1H), 1.04 – 1.01 (m, 2H), 1.00 (s, 1H), 0.97 (s, 3H), 0.94 (s, 2H), 0.89 (d, J = 2.2 Hz, 1H), 0.87 (s, 3H), 0.83 (s, 1H), 0.79 (s, 2H), 0.39 (d, J = 4.1 Hz, 0.4H), 0.14 (d, J = 4.1 Hz, 0.4H) [non-equivalent high-field, cycloartane cyclopropyl protons]. Characteristic signals and literature comparisons confirmed the compound as cycloeucalenol [31,38] .

Figure 5a. Cycloeucalenol (ABP76)

Stigmasterol (4) (Fig. 5b) (ABP 89, 91, 93): White powder. ¹H NMR (CDCl₃, 400 MHz): δ 5.35 (1H, t, J = 5.2 Hz, H-6), 5.15 (1H, dd, J = 15.2, 8.5 Hz, H-22), 5.02 (1H, dd, J = 15.2, 8.5 Hz, H-23), 3.53 (1H, tt, J = 10.8, 4.8 Hz, H-3), 2.26 (1H, dd, J = 18.2, 4.5 Hz, H-4), 1.64 (1H, q, J = 7.7 Hz, H-7), 1.25 (18H, s, H-28), 1.01 (3H, s, H-19), 0.92 (2H, d, J = 6.2 Hz, H-26), 0.83 (2H, d, J = 7.9 Hz, H-27), 0.81 (1H, s, H-18), 0.69 (3H, d, J = 7.4 Hz, H-29). Comparison of its pertinent signals with those of stigmasterol in the literature confirmed the compound to be stigmasterol [32].

Figure 5b. Stigmasterol (ABP 89, 91, 93)

β-Sitosterol (5) (Fig. 6) (ABP87, 89, 91, 93): White powder. ¹H NMR (CDCl₃, 400 MHz): δ 5.35 (1H, d, J = 5.2 Hz, H-6),  3.53 (1H, tt, J = 10.8, 4.8 Hz, H-3),  2.26 (1H, dd, J = 18.2, 4.5 Hz, H-4β),     1.64 (1H, q, J = 7.7 Hz, H-11),   1.25 (18H, s, overlapping CH₂ signals, likely from the steroid side chain),     1.01 (3H, s, H-19),     0.92 (2H, d, J = 6.2 Hz, H-26),     0.83 (2H, d, J = 7.9 Hz, H-27),     0.81 (1H, s, H-18),    0.69 (3H, d, J = 7.4 Hz, H-21)[ the coupling constant (J = 7.4 Hz) is somewhat large for a typical methyl doublet, due to overlap of stigmasterol signals and baseline distortions]. A comparison of its pertinent signals with those of β-sitosterol in the literature confirmed the compound to be stigmasterol [32].

Figure 6. β-Sitosterol (5) (ABP87, 89, 91, 93)

Betulinic acid (6) (Fig. 7) (ABP 105, 107, 109): White amorphous powder. ¹H NMR (CDCl₃, 400 MHz): δ 4.79 (1H, m, H-29b), 4.73 (1H, m, H-29a), 3.19 (1H, br s, H-3), 2.98 (1H, br s, H-19), 2.35–2.27 (2H, m, H-13, H-15), 1.76 (3H, s, H-30), 1.75–1.67 (6H, m, H-2, H-18, H-21, H-22), 1.63–1.59 (5H, m, H-6, H-7, H-16), 1.52–1.43 (4H, m, H-9, H-11, H-12), 1.34 (3H, s, H-23), 1.29 (3H, s, H-26), 1.27 (3H, s, H-27), 1.25 (3H, s, H-24), 1.03 (3H, s, H-25), 0.97 (3H, s, H-28), 0.72–0.69 (3H, m, H-1, H-5). ¹³C NMR (CDCl₃, 101 MHz): δ 180.8 (C-28), 150.2 (C-20), 109.8 (C-29), 78.5 (C-3), 55.6 (C-5), 50.1 (C-9), 49.5 (C-19), 48.1 (C-18), 47.4 (C-17), 42.2 (C-14), 40.4 (C-8), 39.1 (C-4), 39.0 (C-1), 38.8 (C-13), 38.4 (C-10), 38.3 (C-22), 33.8 (C-7), 32.3 (C-16), 30.3 (C-21), 29.9 (C-15), 27.9 (C-23), 27.4 (C-2), 24.7 (C-12), 21.1 (C-11), 19.1 (C-30), 16.8 (C-26), 16.3 (C-24), 16.5 (C-25), 15.8 (C-27). The characteristic signals and literature comparison allowed for unambiguous characterisation of the compound as betulinic acid [33-35].

Figure 7. Betulinic acid (ABP 105, 107, 109)

α-Amyrin acetate (7) (Fig. 8) (ABO 39, 41, 43): Creamy white gummy powder. ¹H NMR (400 MHz, CDCl₃):
δ 5.12 (1H, t, J = 3.7 Hz, H-12), 4.51 (1H, dd, J = 10.1, 6.3 Hz, H-3), 2.28 (2H, dt, J = 11.3, 7.6 Hz, H-2), 2.04 (2H, s, H-24), 1.13 (3H, s, H-26), 1.11 (3H, s, H-27), 1.07 (2H, s, H-25), 1.01 (2H, s, H-23), 0.98 (2H, s, H-28), 0.92 (3H, s, H-30), 0.91 (1H, s, H-29), 0.89 (3H, s, H-18), 0.80 (3H, s, H-28), 0.79 (4H, d, J = 4.2 Hz, H-29), 0.69 (0.25H, d, J = 7.6 Hz, probably form minor isomer). The characteristic signals and literature comparison allowed for unambiguous characterisation of the compound as α-amyrin acetate [36].

Figure 8. α-Amyrin acetate (7). (ABO 39, 41, 43)

Lupeol acetate (8) (Fig. 9). (ABO 43, 45): Creamy white gummy powder. ¹H NMR (400 MHz, CDCl₃): δ 4.67 (1H, d, J = 2.5 Hz, H-29a), 4.55 (1H, t, J = 2.0 Hz, H-29b), 4.53–4.41 (1H, m, H-3), 2.35 (1H, td, J = 11.0, 5.9 Hz), 2.0 (3H, s, CH₃COO), 1.90 (1H, td, J = 8.0, 3.7 Hz), 1.66 (3H, s, Me), 1.01 (2H, s, Me), 0.92 (2H, s, Me), 0.86 (1H, s, Me), 0.85 (1H, s, Me), 0.84 (3H, s, Me), 0.83 (3H, s, Me), 0.82 (1H, s, Me), 0.78 (1H, s, Me), 0.77 (3H, s, Me). The characteristic signals and literature comparison allowed for unambiguous characterisation of the compound as lupeol acetate  [37].

Figure 9. Lupeol acetate (8). (ABO 43, 45)

3.2 Antifungal activities and zone of inhibition of A. boonei stem bark fractions on treated wood fungi

Results of antifungal activities of A. boonei stem bark fractions (ABO-42, ABO-44, ABO-69, and ABO-71) were evaluated against wood fungal pathogens, comparing their efficacy to standard antifungal agents sparfloxacin and keteconazole are presented in Table 2. The results demonstrated that Aspergillus flavus, Coniophora puteana, Fibroporia vaillantii, and Sclerotium rolfsii were sensitive to the fractions, with ZoI values ranging from 24 mm to 30 mm. Aspergillus fumigatus exhibited sensitivity only to ABO-69 and ABO-71, while Aspergillus niger, Fusarium sp., Rhizopus spp., Trichoderma sp., and Serpula lacrymans showed resistance to all fractions. The standard antifungal Keteconazole demonstrated effectiveness against Aspergillus flavus and Aspergillus fumigatus, with ZoI values of 30 mm and 25 mm, respectively.

Table 2. Antifungal activities and Zone of inhibition of A. boonei fractions on treated fungi

3.3 MIC and MFC of A. boonei fractions on treated fungi

Table 3 presents results on the antifungal efficacy (MIC and MFC) of A. boonei stem bark fractions (ABO-42, ABO-44, ABO-69, ABO-71) against wood fungi. Results indicated that Aspergillus flavus, Coniophora puteana, Fibroporia vaillantii, Fomitopsis pinicola, and Sclerotium rolfsii were susceptible to all fractions with MICs ranging from 50 to 100 µg/mL and MFCs ranging from 100 to 200 µg/mL. In contrast, Aspergillus niger, Fusarium sp, Rhizopus spp, Trichoderma sp, and Serpula lacrymans were resistant to all fractions tested, showing no inhibition or fungicidal activity. The fraction ABO-69 exhibited the lowest MIC values (50 µg/mL) against Coniophora puteana and Fibroporia vaillantii, indicating its active potential as a potent antifungal agent.

Table 3. MIC and Minimum MFC of A. boonei stem bark fractions on treated fungi

3.4 Fungicidal activities and zone of inhibition of stem bark A. boonei methanol crude extracts against test fungi

The results of the fungicidal activity of stem bark methanol crude extract of A. boonei against eleven fungal strains, comparing the results with standard antibiotics sparfloxacin and keteconazole are presented in Table 4. A. boonei methanol stem bark crude extract demonstrated sensitivity (S) with a zone of inhibition (ZoI) ranging from 18 to 21 mm against seven fungal strains, including Aspergillus flavus (20 mm), Aspergillus niger (19 mm), and Rhizopus spp (21 mm). However, it showed resistance (R) against Aspergillus fumigatus, Coniophora puteana, Fomitopsis pinicola, Tricoderma sp, and Serpula lacrymans. In comparison, Keteconazole displayed higher efficacy with ZoI values up to 32 mm, while Sparfloxacin was effective only against Coniophora puteana and Rhizopus spp.

Table 4. Fungicidal activities and zone of inhibition of stem bark A. boonei methanol crude extracts against the test fungi

3.5 Fungicidal activities and zone of inhibition of stem bark A. boonei methanol stem bark crude extracts against test fungi

Results of antifungal activity (MIC and MFC) of A. boonei methanol stem bark crude extracts against eleven wood fungal strains are presented in Table 5. The results revealed that the extracts were effective against Aspergillus flavus, Aspergillus niger, Fibroporia vaillantii, Fusarium sp, Rhizopus spp, and Sclerotium rolfsii with MIC value of 2.5 mg/mL and consistent MFC values of 10 mg/mL. However, the extracts showed resistance against Aspergillus fumigatus, Coniophora puteana, Fomitopsis pinicola, Trichoderma sp, and Serpula lacrymans, indicating no inhibitory or fungicidal activity.

Table 5. Minimum inhibitory concentration and minimum fungicidal concentration of A. boonei methanol extracts against the test fungi

3.6 Pictorial presentation of cultured wood fungi and the effect of A. boonei stem bark fractions and extract by zone of inhibition

Figs. 10 - 13 show Zones of inhibition of A. boonei stem bark fractions on test fungi. Whereas Fig. 14 shows the Zones of inhibition of A. boonei stem bark methanol extract on test fungi.

Figure 10.  Zone of inhibition of A. boonei stem bark fraction (ABO71) of test fungi

Key: 1. Aspergillus flavus; 2. Aspergillus fumigatus; 3. Aspergillus nigre; 4. Coniophora puteana; 5. Fibroporia vaillantii; 6. Fomitopsis pinicola; 7. Fusarium sp; 7. Rhizopus spp; 8. Sclerotium rolfsii; 9. Tricoderma sp; 10. Serpula lacrymans

Figure 11.  Zone of inhibition of A. boonei stem bark fraction (ABO69) of test fungi

Key: 1. Aspergillus flavus; 2. Aspergillus fumigatus; 3. Aspergillus nigre; 4. Coniophora puteana; 5. Fibroporia vaillantii; 6. Fomitopsis pinicola; 7. Fusarium sp; 7. Rhizopus spp; 8. Sclerotium rolfsii; 9. Tricoderma sp; 10. Serpula lacrymans

Figure 12.  Zone of inhibition of A. boonei stem bark fraction (ABO44) of test fungi

Key: 1. Aspergillus flavus; 2. Aspergillus fumigatus; 3. Aspergillus nigre; 4. Coniophora puteana; 5. Fibroporia vaillantii; 6. Fomitopsis pinicola; 7. Fusarium sp; 7. Rhizopus spp; 8. Sclerotium rolfsii; 9. Tricoderma sp; 10. Serpula lacrymans

Figure 13.  Zone of inhibition of A. boonei stem bark fraction (ABO42) of test fungi

Key: 1. Aspergillus flavus; 2. Aspergillus fumigatus; 3. Aspergillus nigre; 4. Coniophora puteana; 5. Fibroporia vaillantii; 6. Fomitopsis pinicola; 7. Fusarium sp; 7. Rhizopus spp; 8. Sclerotium rolfsii; 9. Tricoderma sp; 10. Serpula lacrymans

Figure 14.  Zone of inhibition of A. boonei stem bark methanol crude extract of test fungi

Key: 1. Aspergillus flavus; 2. Aspergillus fumigatus; 3. Aspergillus nigre; 4. Coniophora puteana; 5. Fibroporia vaillantii; 6. Fomitopsis pinicola; 7. Fusarium sp; 7. Rhizopus spp; 8. Sclerotium rolfsii; 9. Tricoderma sp; 10. Serpula lacrymans

4.   Discussion

This study characterized 24-methylcycloartenol, α-amyrin acetate, β-amyrin, stigmasterol, β-sitosterol and phytosterol fractions from the stem bark of A. boonei. Bouvier‐Navé et al. [8] and Nair [9] reported 24-methylcycloartenol from Ficus krishnae which exerts antidiabetic activity and was also found together in many vegetable oils [10, 11]. Stigmasterol, β-sitosterol, and betulinic acid are naturally occurring compounds found in various plants, each exhibiting distinct biological activities. Stigmasterol and β-sitosterol are phytosterols with structural similarities to cholesterol, while betulinic acid is a pentacyclic triterpenoid. Stigmasterol has been identified by Griebel and Zeier [12] as a significant metabolic product in plants during pathogen attacks, such as in the Arabidopsis thaliana -Pseudomonas syringae interaction, where its accumulation is linked to increased plant susceptibility to pathogens. Conversely, β-sitosterol is the most prevalent phytosterol in plants and has been synthesized in pure form to study its oxides' biological impacts [13]. Betulinic acid, isolated from various plant extracts, has been reported for its therapeutic potential, including anti-inflammatory and antitumor activities [14, 15].

While both stigmasterol and β-sitosterol are implicated in plant defense mechanisms, their effects on lipid membranes and metabolic processes differ. Griebel and Zeier [12] noted that stigmasterol may promote disease susceptibility in plants by favoring pathogen multiplication, whereas Yoshida and Niki; Hąc-Wydro et al. [16, 17] reported that β-sitosterol has been shown to stabilize lipid membranes and act as an antioxidant. Chaturvedula et al. [12] identified betulinic acid as an inhibitor of DNA polymerase β-lyase activity, highlighting its potential in therapeutic applications.

The results on the effect of A. boonei stem bark fractions on test fungi demonstrated that A. flavus, C. puteana, F. vaillantii, and S. rolfsii were sensitive to the fractions, with zones of inhibition (ZoI) ranging from 24 mm to 30 mm. The values of ZOI obtained in this study were very active test fungi, which agrees with the findings of Guevara [18] who reported ZoI values above 19 mm as being very active. A. fumigatus exhibited sensitivity only to ABO-69 and ABO-71.  Further analysis of the MIC and MFC of the A. boonei fractions revealed that A. flavus, C. puteana, F. vaillantii, F. pinicola, and S. rolfsii were susceptible to all fractions, with MIC values ranging from 50 to 100 µg/mL and MFC values from 100 to 200 µg/mL. The fraction ABO-69 exhibited the lowest MIC value of 50 µg/mL against C. puteana and F. vaillantii, indicating its active potential as a potent antifungal agent.

These findings are consistent with previous studies that have reported the antimicrobial and antifungal properties of A. boonei. For instance, a study by Opoku and Akoto [19] found that ethanol and aqueous extracts of A. boonei root exhibited significant antimicrobial activity against various bacterial and fungal strains, including Candida albicans. Another study by Okoye and Okoye [20] isolated antioxidant and antimicrobial flavonoid glycosides from A. boonei leaves, further supporting the plant's potential as a source of bioactive compounds with therapeutic applications. Moreover, studies by Mollica et al. [21] highlighted the traditional use of A. boonei in treating various ailments, including malaria, fever, and ulcers. The current study provides scientific validation for these traditional uses by demonstrating the antifungal efficacy of A. boonei stem bark fractions against wood fungal pathogens.

In terms of MIC and MBC, A. boonei was effective against a subset of fungi with an MIC of 2.5 mg/mL and MBC of 5 mg/mL, reinforcing its potential but limited antifungal application. This selective efficacy aligns with other findings where plant extracts exhibit significant activity against certain pathogens but lack broad-spectrum effectiveness [22, 23]. The study on A. boonei methanol stem bark crude extracts demonstrates notable fungicidal activity against several fungal strains, including A. flavus and Rhizopus spp., with a Zone of Inhibition (ZoI) ranging from 18 to 21 mm. Despite this, the extract was less effective than Keteconazole, a standard antifungal, which showed ZoI values up to 32 mm. This suggests that while A. boonei exhibits some antifungal properties, it is not as potent as conventional antifungal treatments. Fagbohun [24] reported that methanolic crude extracts of the stem bark of A. boonei were tested for antifungal activity at concentrations of 50, 100, 150 and 200 mg/mL against Aspergillus flavus, Absidia corymbifera, and Aspergillus niger. According to Osagie [25], the methanol leaf extract of A. boonei demonstrated antifungal properties. The ZoIs recorded by Aruhanga [26] for three compounds isolated from the stem bark of Alstonia boonei were 12.6 mm, 12.3 mm, and 9.0 mm, while the crude extract showed a ZoI of 13.1 mm against Candida albicans and Aspergillus fumigatus.

The MIC and MFC results further reinforce this finding. A. boonei was effective at MIC values between 2.5 to 25 mg/mL and MFC of 10 mg/mL against specific strains like Aspergillus niger and Fusarium sp., but it was ineffective against others, such as A. fumigatus and Trichoderma sp., which displayed resistance. These findings are consistent with other studies on plant-based antifungals, which often show selective efficacy but are generally less broad-spectrum compared to synthetic antifungal agents like Keteconazole [27-29]. Osagie [25] reported a concentration (IC₅₀) value of 64.47 µg/mL for radical scavenging activity from methanol leaf extract of A. boonei.

5.   Conclusions

Motivated by the ethnobotany of Alstonia boonie, this investigation has successfully led to the isolation and characterisation of eight compounds: 24-Methylenecycloartenol, β-Amyrin, Cycloeucalenol, Stigmasterol, β-Sitosterol, Betulinic acid, α-Amyrin acetate, and Lupeol acetate. from the stem bark of the plant. A. boonei stem bark fractions on test fungi demonstrated that Aspergillus flavus, Coniophora puteana, Fibroporia vaillantii, and Sclerotium rolfsii were sensitive to the fractions, with ZOIs ranging from 25 - 29 mm. Evidence from the current research shows that solvent extracts of A. boonei stem bark have notable antifungal activity. These fractions and extracts hold promise as a novel tool in the fight against drug resistance, a significant worldwide concern. The results of this research provide scientific backing for the traditional application of A. boonei by the indigenous community of Okhuesan, Edo State, Nigeria, to combat microbial infections.

Authors’ contributions

Designed, coordinated the study and proof read the work, D.O.E; C.E.; Proof read the work, carried out the extraction and antifungal activities and wrote the report, M.T.A.

Acknowledgements

We would like to thank Prof. J.O. Igoli for acquiring the spectra at Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde Glasgow, United Kingdom.

Funding

This research received no specific grant from any funding agency in the public, commercial or non-profit sectors.

Availability of data and materials

All data will be made available on request according to the journal policy.

Conflicts of interest

The authors confirm that there is no conflict of interest to declare.

Reference