Secondary Metabolites from Anthonotha cladantha (Harms) J.Léonard

Introduction

The genus Anthonotha (Fabaceae) comprises 17 species exclusively native to Cameroon and Democratic Republic of Congo. It is represented by 10 species, including Anthonotha macrophylla P. Beauv, and Anthonotha cladantha (Harms) J.Léonard.¹ The latter is a tree between 35 to 45 m tall and 120 cm wide. The bark peels off in flakes of different shapes with heavy branches sinuous. Branches are densely rusty villous-velutinous and glabrescent. Seeds are irregular in shape, subtriangular in outline, and laterally compressed.²

Various plants in the genus Anthonotha are used for diverse purposes. For example, the leaves of Anthonotha macrophylla P. Beauv are used in folk medicine with claims of their effectiveness in treating pain, boils, malaria, worm infestation, gonorrhea, dysentery, yellow fever, and diarrhea.^3,4 However, there is limited scientific evidence on most of such usage. The infusions of leaves and stem bark are consumed in case of jaundice and malaria.^1,5,6 Boiled product of a poultice of leaves are rubbed to relieve pain and promote pus formation.⁷ The leaves cure dysentery and snake bites while decoction alleviates toothache.¹ The bark has been claimed to help treat venereal diseases and act as vermifuge.⁸ The roots are used for intestinal-related discomfort.^9,10 Gum extracted from the bark has analgesic properties.⁴

The aqueous leaf extract of A. macrophylla P. Beauv has been screened, and we observed the presence of alkaloids, tannins, saponins, flavonoids, phenolics, and cardiac glycosides. Phlobatannins, anthraquinones, steroids, and terpenes were also screened but not detected.⁵ The GC-MS analysis of oil seeds extracts from A. macrophylla P. Beauv revealed the presence of saturated and unsaturated fatty acids and sterol, including campesterol, stigmasterol, and γ-sitosterol⁴⁾. Leaves ethanolic extract from A. macrophylla demonstrated antibacterial activity against some pathogenic bacteria such as Staphylococcus aureus, Escherichia coli, Salmonella Typhi, Klebsiella pneumonia, Streptococcus pyogenes, and Bacillus subtilis.

Toxicological investigation of leaf extracts showed no significant alterations in hematological and biochemical indices and the vital organs. The LD50 in rats was more than 5000 mg/kg.³ Aqueous extract of A. macrophylla P. Beauv leaves revealed aphrodisiac activity in female Wistar rats.¹¹ A. macrophylla P. Beauv induced antiestrogenic effects in vitro and in vivo at all the tested concentrations inhibited estradiol-induced MCF-7 cell proliferation.¹² A literature survey revealed that no phytochemical studies had been carried out on A. cladantha (Harms) J.Léonard. The chemotaxonomic and ethnopharmacological virtues from the Anthonotha genus prompted us to carry out phytochemical studies on A. cladantha (Harms) J.Léonard. In this paper, we report the isolation and characterization of twelve secondary metabolites (1–12). Moreover, 2,3-dihydroxypropyl hexacosanoate (1), which was obtained in a sufficient amount (23.2 mg), was acetylated to get its hitherto unreported diacetate 1a. Both 1 and 1a have been subjected to biological screening.

Experimental

General experimental procedures – The optical rotations were measured using a JASCO DIP-3600 digital polarimeter (JASCO, Tokyo, Japan) in methanol at 23°C. Ultraviolet spectra were recorded on a Hitachi UV 3200 spectrophotometer in MeOH. Infrared spectra were recorded on a JASCO 302-A spectrophotometer. EI-MS and ESI-MS were recorded on a Finnigan MAT 95 spectrometer (70 eV) with perfluorokerosene as a reference substance for HR–EI-MS. The ¹H NMR spectra were recorded at 400 and 500 MHz while ¹³C NMR at 100 and 125 MHz, respectively, on Bruker AMX 500 NMR spectrometers, including ¹H-¹H COSY, HMQC, HMBC, and NOESY experiments. Chemical shifts are reported in δ (ppm) using TMS as the internal standard and coupling constants (J) were measured in Hz. Column chromatography was carried out on silica gel (70‒230 mesh, Merck). Thin layer chromatography (TLC) was performed on Merck precoated silica gel 60 F₂₅₄ aluminum sheets, and spots were detected using a ceric sulfate spray reagent.

Plant materials – The stem bark (2.18 kg) and roots (1.18 kg) of A. cladantha J.Léonard were collected in December 2018 at Dibombari, Littoral region of Cameroon. Their identification was made at the National Herbarium of Cameroon, where a specimen was deposited (voucher number 37110 HNC).

Extraction and isolation – The air-dried and powdered roots (1.18 kg) and stem bark (2.18 kg) of A. cladantha (Harms) J.Léonard were separately extracted with MeOH at room temperature for 48 h, followed by the concentration of each extract under reduced pressure to yield brown viscous extract (52.3 g from roots) and green viscous extract (102.4 g from stem bark), respectively. Then 5 g of each extract was kept for biological assays. The remaining crude extract from stem bark was subjected to column chromatography over silica gel using a gradient of EtOAc in n-hexane and EtOAc leading to four major fractions, S₁ to S₄. The fraction S1 (n-hexane‒EtOAc, 37:3) was subjected to silica-gel column chromatography using the same solvent system to afford 94 fractions of 200 mL each from which lupeol (6) (8.7 mg) was obtained as a white powder. The fraction S₂ (n-hexane‒EtOAc, 17:3) comprised of 104 fractions, provided stigmasterol (11) (7.3 mg). Hederagenine (2) (8.2 mg) was obtained from the fraction S₃ (n-hexane‒EtOAc, 7:3), while the fraction S4 (EtOAc) was further purified by column chromatography to yield stigmasterol glucoside (10.2 mg) as a white amorphous powder.

The extract from the roots (52.3 g) was chromatographed over silica gel with n-hexane‒CH₂Cl₂‒EtOAc‒MeOH gradients resulting in six sub-fractions. The sub-fraction A1 was further chromatographed and eluted with nhexane‒CH₂Cl₂ (37:3) to afford triacontanoic acid (8) (5.2 mg) and heptacosan-2-one (7) (7.4 mg). The sub-fraction A2, which was eluted with n-hexane–CH₂Cl₂ (4:1), led to lupeol (6) (10.1 mg) and cycloeucalenol (3) (3.4 mg). Stigmast-4-en-3-one (9) (3.9 mg) and β-sitosterol (10) (3.3 mg) were obtained from sub-fraction A3 eluted with n-hexane–CH₂Cl₂ (3:2) while betulinic acid (5) (8.1 mg) was isolated from sub-fraction A4 with n-hexane–CH₂Cl₂ (1:4). The 2α-hydroxylupeol (4) (5.7 mg) was obtained from sub-fraction A5 [n-hexane–EtOAc (9:1)] and the sub-fraction A6 [n-hexane–EtOAc (4:1)] afforded 2,3-dihydroxypropyl hexacosanoate (1) (23.2 mg).

2,3-Dihydroxypropyl hexacosanoate (1) – White powder; ¹H NMR (Pyridine-d₅, 400 MHz): 4.72 (2H, dd, J = 4.56, 11.04 Hz, H-3'a), 4.68 (2H, dd, J = 6.36, 11.08 Hz, H-3'b), 4.44 (1H, m, H-2'), 4.13 (2H, t, J = 5.44 Hz, H-1'), 2.35 (2H, t, J = 7.44 Hz, H-2), 1.64 (2H, q, J = 7.48 Hz, H-3), 0.86 (3H, t, J = 6.28 Hz, H-25). ¹³C NMR (DMSOd₆, 100 MHz): 70.92 (C-2'), 66.75 (C-1'), 64.29 (C-3'), 34.41 (C-2), 32.09 (C-3), 25.29 (C-23), 22.9 (C-24), 14.25 (C-25); EI-MS m/z: 470.43 [M]⁺.

3-(Hexacosanoyloxy)propane-1,2-diyl diacetate (1a) – White powder; [α]²⁵ +8 (c = 1.0, CHCl₃); ¹H NMR (Pyridine-d₅, 400 MHz): 5.25 (1H, q, J = 4.48 Hz, H-2'), 4.30 (2H, t, J = 5.80 Hz, H-1'), 4.15 (2H, d, J = 3.76, H-3), 2.32 (2H, t, J = 7.48 Hz, H-2), 2.08 (6H, s, CH₃), 1.58 (2H, q, J = 6.92 Hz, H-3), 0.88 (3H, t, J = 6.48 Hz, H-25); ESI-MS m/z: 577.45 [M+Na]⁺.

Hederagenine (2) – White amorphous powder; ¹H NMR (CDCl₃, 400 MHz): 5.17 (1H, s, H-12), 3.06 (3H, s, H-23), 3.01 (1H, m, H-3), 2.50 (1H, s, H-18), 1.80 (2H, m, H-11), 1.70 (2H, m, H-19b), 1.70 (2H, m, H-15), 1.60 (2H, m, H-21b), 1.53 (1H, m, H-9), 1.53 (1H, m, H-1b), 1.50 (2H, m, H-6), 1.48 (2H, m, H-2), 1.40 (2H, m, H-21a), 1.25 (2H, m, H-7b), 1.10 (3H, s, H-27), 0.98 (2H, m, H-19a), 0.95 (1H, s, H-1a), 0.95 (2H, s, H-7a), 0.90 (3H, s, H-30), 0.86 (2H, s, H-24), 0.80 (3H, s, H-29), 0.75 (3H, s, H-26), 0.70 (1H, s, H-5), 0.70 (3H, s, H-25); ¹³C NMR (CDCl₃, 100 MHz): 180.2 (C-28), 144.9 (C-13), 122.5 (C-12), 78.1 (C-3), 55.9 (C-5), 73.9 (C-23), 48.2 (C-9), 47.2 (C-17), 42.2 (C-14), 41.4 (C-19),41.4 (C-18), 39.8 (C-8), 39.4 (C-4), 38.9 (C-1), 37.4 (C-10), 36.6 (C-20), 33.3 (C-7), 32.7 (C-22), 29.1 (C-21), 28.8 (C-30), 28.2 (C-15), 28.1 (C-2), 26.2 (C-27), 23.8 (C-11), 23.8 (C-16), 19.8 (C-29), 18.8 (C-6), 17.4 (C-26), 16.5 (C-24), 15.5 (C-25); EI-MS m/z: 472.73 [M]⁺.

Cycloeucalenol (3) – White powder; ¹H NMR (CDCl₃, 400 MHz): 4.73 (s, H-28), 4.64 (s, H-28), 3.20 (m, H-3), 0.38 (d, H-19), 0.15 (d, H-19); EI-MS m/z: 426.7 [M]⁺.

2-α-Hydroxylupeol (4) – White amorphous powder; ¹H NMR (DMSO-d₆, 400 MHz): 4.65 (2H, s, H-29b), 4.57 (2H, s, H-29a), 3.15 (1H, dd , J = 4.3, 4.1 Hz, H-2), 3.07–3.10 (1H, m, H-3), 2.95–3.00 (1H, m, H-19), 2.35–2.38 (2H, t, J = 4.08 Hz, H-15), 1.83–1.91 (2H, m, H-21), 1.67 (3H, s, H-30), 1.64 (2H, m, H-1a), 1.50–1.62 (8H, m, H-6, 13, 18), 1.39 (2H, m, H-1b), 1.23–1.37 (7H, m, H-7, 9, 16, 22), 1.06–1.17 (4H, m, H-11, 12), 1.02 (3H, s, H-27), 0.95 (3H, s, H-26), 0.92 (3H, s, H-25), 0.81 (3H, s, H-24), 0.77 (3H, s, H-23), 0.75 (3H, s, H-28), 0.61–0.62 (1H, t, J = 3.52 Hz, H-5); ¹³C NMR (DMSO-d₆, 100 MHz): 150.0 (C-20), 109.0 (C-29), 79.1 (C-3), 68.5 (C-2), 55.3 (C-5), 50.4 (C-9), 48.6 (C-17), 48.1 (C-18), 48.0 (C-19), 44.8 (C-1), 42.8 (C-14), 40.2 (C-8), 40.0 (C-22), 38.8 (C-4), 37.9 (C-13), 37.7 (C-10), 35.6 (C-16), 33.8 (C-7), 28.5 (C-21), 28.1 (C-23), 27.4 (C-15), 25.1 (C-12), 21.1 (C-11), 19.5 (C-30), 18.3 (C-6), 18.0 (C-28), 16.3 (C-25), 15.8 (C-26), 15.3 (C-24), 14.5 (C-27); EI-MS m/z: 442.73 [M]⁺.

Betulinic acid (5) – White amorphous powder; ¹H NMR (CDCl₃, 400 MHz): 4.66 (2H, s, H-29b), 4.59 (2H, s, H-29a), 3.50–3.56 (2H, m, H-3, 19), 2.37–2.43 (3H, m, H-13,15), 1.75 (3H, s, H-30), 1.48–1.57 (7H, m, H-2, 18, 21, 22), 1.34–1.45 (6H, t, J = 6.92 Hz, H-6, 7, 16), 1.15–1.26 (5H, m, H-9, 11, 12), 1.11 (3H, s, H-23), 1.09 (3H, s, H-26), 1.05 (3H, s, H-27), 1.02 (3H, s, H-24), 0.97 (3H, s, H-25), 0.72–0.78 (3H, m, H-1,5); ¹³C NMR (CDCl₃, 100 MHz): 180.8 (C-28), 150.2 (C-20), 108.5 (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), 18.6 (C-6), 21.1 (C-11), 19.1 (C-30), 16.8 (C-26), 16.3 (C-24), 16.5 (C-25), 15.8 (C-27); EI-MS m/z: 456.3 [M]⁺.

Lupeol (6) – White fiber; ¹H NMR (DMSO-d₆, 400 MHz): 4.65 (2H, s, H-29b), 4.57 (2H, s, H-29a), 3.07–3.10 (1H, m, H-3), 2.95–3.00 (1H, m, H-19), 2.35–2.38 (2H, t, J = 4.08 Hz, H-15), 1.83–1.91 (2H, m, H-21), 1.67 (3H, s, H-30), 1.50–1.62 (8H, m, H-1, 2, 6, 13, 18), 1.23–1.37 (7H, m, H-7,9,16,22), 1.06–1.17 (4H, m, H-11, 12), 1.02 (3H, s, H-27), 0.95 (3H, s, H-26), 0.92 (3H, s, H-25), 0.81 (3H, s, H-24), 0.77 (3H, s, H-23), 0.75 (3H, s, H-28), 0.61–0.62 (1H, t, J = 3.52 Hz, H-5); ¹³C NMR (DMSOd₆, 100 MHz): 150.0 (C-20), 109.0 (C-29), 79.1 (C-3), 55.3 (C-5), 50.4 (C-9), 48.3 (C-17), 48.1 (C-18), 48.0 (C-19), 42.8 (C-14), 40.2 (C-8), 40.0 (C-22), 38.8 (C-4), 38.0 (C-1), 37.9 (C-13), 37.7 (C-10), 35.9 (C-16), 33.9 (C-7), 28.5 (C-21), 28.1 (C-23), 27.5 (C-2), 27.4 (C-15), 25.1 (C-12), 21.1 (C-11), 19.5 (C-30), 18.3 (C-6), 18.0 (C-28), 16.3 (C-25), 15.9 (C-26), 15.3 (C-24), 14.5 (C-27); EI-MS m/z: 426.38 [M]⁺.

Heptacosan-2-one (7) – ¹H NMR (CDCl₃, 400 MHz): 2.39 (2H, t, J = 5.96 Hz, H-3), 2.19 (3H, s, H-1), 1.23-1.53 (2H, H-4, H-5), 0.86 (3H, t, J = 5.68 Hz, H-23); EIMS m/z: 394.17 [M]⁺.

Triacontanoic acid (8) – ¹H NMR (CDCl₃, 400 MHz): 3.61 (2H, t, J = 5.31 Hz, H-2), 1.23–1.51 (2H, H-3), 0.87 (3H, t, J = 5.44 Hz, H-28); EI-MS m/z: 452.96 [M]⁺.

Stigmast-4-en-3-one (9) – ¹H NMR (CDCl₃, 400 MHz): 5.72 (1H, s, H-4), 1.18 (3H, s, H-19), 0.92 (3H, d, J = 6.5 Hz, H-21), 0.85 (3H, m, H-29), 0.84 (3H, d, J = 6.8 Hz, H-26), 0.82 (3H, d, J = 6.8 Hz, H-27), 0.71 (3H, s, H-18); ¹³C NMR (CDCl₃,100 MHz): 199.7 (C-3), 171.8 (C-5), 123.7 (C-4), 55.9 (C-17), 55.8 (C-14), 53.8 (C-9), 45.8 (C-24), 42.3 (C-13), 38.6 (C-10), 36.1 (C-20), 35.6 (C-1), 35.6 (C-8), 33.9 (C-2), 33.8 (C-6), 32.9 (C-7), 32.0 (C-22), 29.1 (C-25), 28.2 (C-16), 26.0 (C-23), 24.1 (C-15), 23.0 (C-28), 21.1 (C-11), 19.8 (C-26), 19.0 (C27), 18.7 (C-21), 17.3 (C-19), 12.2 (C-18), 11.9 (C-29); EI-MS m/z: 412.69 [M]⁺.

Fig. 1.

The structures of isolated compounds 1‒12 from A. cladantha.

β-Sitosterol (10) – White powder; ¹H NMR (CDCl₃, 500 MHz) : 5.34 (1H, t, J = 6.4, H-6), 3.51 (1H, dd, J = 4.5, 4.2 Hz, H-3), 1.23 (3H, s, H-19), 1.15 (3H, s, H-18), 1.02 (3H, d, J = 6.5 Hz, H-26), 0.92 (3H, d, J = 6.3 Hz, H-27), 0.92 (3H, m, H-29), 0.86 (3H, d, J = 6.3 Hz, H-21); ¹³C NMR (CDCl₃, 125 MHz) : 141.1 (C-5), 121.6 (C-6), 72.2 (C-3), 57.9 (C-14), 56.6 (C-17), 50.4 (C-9), 46.4 (C-24), 42.7 (C-13), 42.6 (C-4), 38.9 (C-12), 37.7 (C-1), 36.8 (C-10), 36.5 (C-20), 34.5 (C-22), 32.3 (C-7), 32.4 (C-8), 32.1 (C-2), 29.7 (C-25), 28.7 (C-16), 26.5 (C-23), 24.4 (C-15), 23.7 (C-28), 21.4 (C-11), 20.3 (C-26), 19.7 (C-27), 19.5 (C-19), 19.2 (C-21), 12.5 (C-29), 12.4 (C-18); EI-MS m/z: 414.70 [M]⁺.

Stigmasterol (11) – White powder; ¹H NMR (CDCl₃, 500 MHz): 5.36 (1H, t, J = 6.2, H-6), 5.14 (1H, m, H-22), 4.98 (1H, m, H-23), 3.53 (1H, dd, J = 4.3, 4.1 Hz, H-3), 1.03 (3H, s, H-19), 0.93 (3H, s, H-18), 0.87 (1H, t, J = 7.2 Hz, H-24), 0.84 (3H, d, J = 6.6 Hz, H-26), 0.82 (3H, t, J = 6.5 Hz, H-29), 0.81 (3H, d, J = 6.6 Hz, H-27), 0.71 (3H, d, J = 6.3 Hz, H-21); ¹³C NMR (CDCl₃, 125 MHz): 140.8 (C-5), 138.3 (C-22), 129.3 (C-23), 121.7 (C-6), 71.8 (C-3), 56.9 (C-14, C-17), 51.3 (C-24), 50.2 (C-9), 42.4 (C-4, C-13), 40.5 (C-20), 39.8 (C-12), 37.3 (C-1), 36.6 (C-10), 32.0 (C-7), 31.9 (C-8), 31.7 (C-2), 31.2 (C-25), 28.9 (C-16), 25.4 (C-28), 24.4 (C-15), 21.2 (C-26, C-27), 21.1 (C-11, C-21), 19.4 (C-19), 12.2 (C-18,C-29); EIMS m/z: 412.69 [M]⁺.

Stigmasterol-3-O-β-D-glucopyranoside (12) – White amorphous powder; ¹H NMR (Pyridine-d₅, 500 MHz) : 5.32 (2H, m, H-6), 5.16 (1H, dd, J = 12.1, 8.2 Hz, H-22), 5.03 (1H, dd, J = 12.1, 8.2 Hz, H-23), 4.22 (1H, d, J = 6.5 Hz, H-1'), 3.67 (1H, m, H-6'b), 3.47 (1H, m, H-3), 3.44 (1H, m, H-6'a), 3.12 (1H, d, J = 7.5 Hz, H-3'), 3.10 (1H, m, H-5'), 3.06 (1H, m, H-4'), 2.90 (1H, t, J = 7.0 Hz, H-2'), 2.12 (1H, m, H-20), 1.93 (2H, m, H-4), 1.80 (4H, m, H-15, H-16), 1.80 (2H, m, H-7), 1.63 (1H, m, H-27), 1.50 (6H, m, H-8, H-9, H-11, H-12), 1.50 (4H, m, H-24, H-26), 1.39 (2H, m, H-2), 1.24 (2H, m, H-1), 1.24 (1H, m, H-17), 1.15 (2H, m, H-25), 1.15 (1H, m, H-14), 0.99 (3H, d, J = 6.9 Hz, H-29), 0.95 (3H, s, H-19), 0.90 (3H, d, J = 6.4 Hz, H-21), 0.82 (3H, s, H-18), 0.76 (3H, m, H-28); ¹³C NMR (Pyridine-d₅, 125 MHz): 141.0 (C-5), 138.8 (C-22), 129.6 (C-23), 121.9 (C-6), 102.6 (C-1'), 78.6 (C-3), 78.4 (C-3'), 78.2 (C-5'), 75.4 (C-2'), 71.8 (C-4'), 63.0 (C-6'), 57.0 (C-14), 56.2 (C-17), 51.5 (C-24), 50.4 (C-9), 42.4 (C-4, C-13), 40.8 (C-20), 39.4 (C-12), 37.5 (C-1), 37.0 (C-10), 32.2 (C-2, C-7), 32.1 (C-8, C-25), 29.3 (C-16), 25.7 (C-28), 24.6 (C-15), 21.3 (C-21), 19.5 (C-11, C-26), 19.3 (C-27), 19.2 (C-19), 12.5 (C-29), 12.2 (C-18); EI-MS m/z: 474.42 [M]⁺.

Acetylation of compound 1 – 2,3-Dihydroxypropyl hexacosanoate (11.5 mg) was dissolved in equal volumes (1 mL each) of pyridine and acetic anhydride. After 10 h under agitation at room temperature, the reaction was quenched with water, and the medium was partitioned between CH₂Cl₂ and H₂O. The organic layer was freed of solvent to afford an oily residue that was chromatographed over silica gel and eluted with n-hexane-EtOAc (1:19) to furnish 3-(hexacosanoyloxy)propane-1,2-diyl diacetate (1a, 8.00 mg) as a white powder.

Determination of DPPH radical scavenging activity – The free radical scavenging activity was measured by 1,1-diphenyl-2-picryl-hydrazil (DPPH) using the method described by Gulcin in 2005.¹³ The solution of DPPH of 0.3 mM was prepared in ethanol. 5 μL of each sample of different concentrations (62.5 μg ‒ 500 μg) were mixed with 95 μL of DPPH solution in ethanol. The mixture was dispersed in 96 healthy plates and incubated at 37°C for 30 min. Butylhydroxyanisole (BHA) was used as standard. The formula Ac calculated the percentage of DPPH scavenging activity Ac – As / Ac × 100 where Ac is the absorbance of control (DMSO treated) and the sample’s absorbance. The absorbance was measured at 515 nm by a microtiter plate reader (Spectramax plus 384 Molecular Device, USA), and percent radical scavenging activity was determined compared with the methanol-treated control.

Urease inhibition assay – Reaction mixtures comprising 25 μL of enzyme (Jack bean urease) solution and 55 μL of buffers containing 100 mM urea were incubated with 5 μL of test compounds (1 mM concentration) at 30°C for 15 min in 96-well plates. Urease activity was determined by measuring ammonia production using the indophenol, the method described by Weatherburn.¹⁴ Briefly, 45 μL each of phenol reagent (1% w/v phenol, 0.005% w/v sodium nitroprusside) and 70 μL of alkali reagent (0.5% w/v NaOH and 0.1% active chloride NaOCl) were added to each well. The increasing absorbance at 630 nm was measured after 50min using a microplate reader (Molecular Device, USA). All reactions were performed in triplicate in a final volume of 200 μL. The results (change in absorbance per min) were processed using SoftMax Pro software (Molecular Device, USA). All the assays were performed at pH 8.2 (0.01 M K₂HPO₄·3H₂O, 1 mM EDTA, and 0.01 M LiCl₂). Percentage inhibition was calculated from the formula 100 – (OD_testwell / OD_control) × 100. Thiourea was used as the standard inhibitor of urease.

α-Glucosidase inhibition assay – The enzyme inhibition assay was based on the breakdown of the substrate to produce a colored product, followed by measuring the absorbance throughout time.^15,16 Briefly, α-glucosidase (Sigma, type III, from yeast) was dissolved in buffer A (0.1 mol/L potassium phosphate, 3.2 mmol/L-MgCl₂, pH = 6.8) (0.1 unit/ml), p-nitrophenyl-α-D-glucopyranoside dissolved in buffer A at 6 mmol/L was used as substrates. The 102 μL buffer B (0.5 mol/L potassium phosphate, 16 mmol/L-MgCl₂, pH = 6.8), 120 μL sample solution (0.6 mg/mL in dimethylsulfoxide), 282 μL water and 200 μL substrate were mixed. This mixture was incubated in a water bath at 37°C for 5 min, and then 200 μL of enzyme solution was added and mixed. The enzyme reaction was carried out at 37°C for 30 min, and then 1.2 mL 0.4 mol/L glycine buffer (pH = 10.4) was added to complete the reaction. Enzymatic activity was quantified by measuring the absorbance at 410 nm. The lipids extracted from algae showed various colors due to pigments, so the background absorption of every sample should be considered. 1-Deoxynojirimycin hydrochloride was used as the positive control. The formula Ac calculated the percentage of inhibition – As / Ac × 100, Ac being the absorbance of the control and As the absorbance of the sample.

Antibacterial activity – Antibacterial activity was determined by the method of disk diffusion.^17,18 The McFarland Index was prepared, and 0.5 standards was used for the testing method.¹⁹ Briefly, an inoculum of the test organism was cultured into 5 mL of Luria-Bertani (LB) broth. Using sterile cotton swabs, the LB agar plates were inoculated to make an evenly microbial lawn. 1 mM solutions were prepared of test samples in methanol. Paper disks of 4 mm diameter were placed on the inoculated plates, and 10 μL sample was loaded on each disk and left to dry. Methanol was used as negative control and antibiotic disks of streptomycin as a positive control. The plates were incubated at 37°C for 24 h. The samples were run in triplicates. The positive test samples showing multiple inhibition zones were then examined for minimal inhibitory concentration (MIC). The stock solution of 100 μL of 1 mM concentration of each test sample was prepared in DMSO/methanol. Then two-fold dilutions were made. LB agar plates streaked with test organism and borrowed 4 mm agar well marked with dilution factor were then loaded with one mL of the serially diluted test sample. The plates were incubated at 37°C for 24 h. The test was conducted in triplicates.

Results and Discussion

Structure elucidation – The phytochemical study of the crude methanolic extracts of the roots and stem bark of A. cladantha (Harms) J.Léonard led to the isolation of twelve compounds characterized with the help of their physical and spectral data as 2,3-dihydroxypropyl hexacosanoate (1),²⁰ hederagenine (2),²¹ cycloeucalenol (3),²² 2α-hydroxylupeol (4),²³ betulinic acid (5),²⁴ lupeol (6),²⁴ heptacosan-2-one (7),²⁵ triacontanoic acid (8),²⁶ stigmast-4-en-3-one (9),²⁷ β-sitosterol (10),²⁸ stigmasterol (11),²⁹⁾ and stigmasterol glucopyranoside (12).³⁰ Acetylation of 2,3-dihydroxypropyl hexacosanoate (1) yielded compound 1a as a white powder, [α]²⁵ +8 (c = 1.0, CHCl₃). Its HR-EI-MS showed the molecular ion peak M⁺. at m/z 554.4524 (calcd. for C₃₃H₆₂O₆, 554.4522) consistent with the molecular formula C₃₃H₆₂O₆, suggesting the acetylation of two hydroxyl groups. This was further confirmed with its ¹H NMR spectrum, which showed signals of two additional methyl groups at δH 2.05 (6H, s, COCH₃), an oxymethine at δH 5.25 (1H, q, J = 4.48 Hz) and an oxymethylene at δH 4.30 (1H, q, J = 5.8 Hz). Furthermore, three ester carbonyls at δC 170.1, 170.2, and 173.2 on its decoupled broadband ¹³C NMR spectrum were in good agreement with the diacetylation of the glycerol moiety of compound 1 and hence the structure of 1a was confirmed as 2,3-diacetyl-2,3-dihydroxypropyl hexacosanoate. Its ¹³C NMR spectrum displayed additional resonances, including signals of three new oxygenated carbons at δC 62.4, 62.7, and 68.7, and methyl signals of the acetate group between δC 22–28.

Antioxidant property – The extracts and some fractions along with compounds 1 and 1a were subjected to DPPH radical scavenging assay. The stem bark extract and its EtOAc fractions were dormant, whereas the fraction of CH₂Cl₂–EtOAc (1:1) showed moderate activity (IC₅₀ 69.2 ± 0.99 μM). Compound 1a showed weak activity (80.3 ± 0.83 μM) while 1 was inactive. The absence of phenolic compounds can explain these results. The potency in antioxidant activity is due to the availability of the phenolic hydroxyl groups in their core structures-the more available phenolic hydroxyl groups, the higher activity.

Urease inhibition activity – The extract of stem bark, various fractions, and compounds 1 and 1a were also screened for their urease inhibitory activity. The stem bark extract and its fractions displayed significant inhibition activity close to the reference thiourea (22.4 ± 0.24 μM), but compounds 1 and 1a were inactive (Table 1). According to the literature, 2,3-dihydroxypropyl hexacosanoate has been isolated from the stem bark of Piptadeniastrum africanum by Amadou Dawi 2017,³¹ displaying feeble urease activity. This result could be supported by the fact that the urease inhibition ability of medical plants is attributed to their large classes of phytoconstituents, including phenolic compounds, saponins, cardiac glycosides, and gallocatechin derivatives.³²

Table 1.

IC50 values in μM of extract, fractions, compound 1 and 1a in antioxidant assay and enzyme inhibition studies

α-Glucosidase activity – The crude extract of stem bark, some fractions, and compound 1 exhibited significant activity in the range of 21.1 to 39.2 μM compared to the reference (1-deoxynojirimycin, IC₅₀ = 3.9 ± 0.71 μM). The ethyl acetate fraction was the most potent, with an IC₅₀ value of 21.1 ± 0.76 μM, less than the reference but more potent than acarbose (IC₅₀ = 273.12 μM) prescribed for the treatment of diabetes.¹⁹ The activity is followed by the crude extract of stem bark with the IC₅₀ value of 29.6 ± 0.73. Compound 1 showed moderate activity with IC₅₀ 39.2 ± 0.22 μM, while compound 1a was inactive. These results suggested that the ethyl acetate fraction and the crude extract might be substantial in the formulation of ameliorated traditional medicine for treating diabetes and other diseases related to the inhibition of α-glucosidase.

Antibacterial activity – The antibacterial activity of the crude extracts, their fractions, compounds 1 and 1a were tested against seven bacterial strains, including Pseudomonas aeruginosa, Escherichia coli, and Salmonella Typhi, Proteus ssp., Klebsiella pneumonia, Staphylococcus aureus, and Saccharomyces cerevisiae. Antibacterial activity was determined by the method of disk diffusion.^33,34 Unfortunately, none of these showed any activity. Several fatty acids from various classes have been reported in the literature as not active or slightly active against some bacterial strains. For instance, Estaban Plata synthesized 3-((2,3-diacetoxypropanoyl)oxy)propane-1,2-diyl diacetate,³⁵ a dimeric glycerol ester type closed to 3-(hexacosanoyloxy)propane-1,2-diyl diacetate (1a) and compound 1, which was not active against several bacterial strains. Yan Ri-ming isolated 2,3-dihydroxypropyl stearate and 2,3-dihydroxypropylhexadecanoate,³⁶ which showed weak antibacterial activity against some strains. Knowing that antibacterial activities are practical from phenolics and alkaloid compounds, we can partially conclude that A. cladantha’s crude extracts are less rich in antibacterial molecules.

Chemotaxonomic significance – The present study led to the isolation of twelve known secondary metabolites comprising a cycloartane cycloeucalenol (3), three lupane-type triterpenes including 2α-hydroxylupeol (4), betulinic acid (5), lupeol (6), four steroids stigmast-4-en-3-one (9), β-sitosterol (10), stigmasterol (11), and stigmasterol-3-O-β-D-glucopyranoside (12), an oleanane-type triterpene namely hederagenine (2), and three fatty-acid derivatives including 2,3-dihydroxypropyl hexacosanoate (1), heptacosan-2-one (7), and triacontanoic acid (8). Their isolation from this species is not surprising since the phytochemical studies on different species of the genus Anthonotha, including A. macrophylla P. Beauv, revealed the presence of tannins, triterpenoids, saponins, sterols, and fatty acid.³⁷ However, all these compounds are herein reported for the first time from the genus Anthonotha. Moreover, it is the first report of the isolation of heptacosan-2-one (7) from the family Fabaceae. β-Sitosterol glucoside has been previously reported from A. macrophylla P. Beauv¹ and can be considered chemotaxonomic markers of the genus Anthonotha. β-Sitosterol and stigmasterol have also been isolated from Entada africana³⁸ and Machaerium brasiliense Vogel,³⁹ indicating a close relationship between the genera Anthonotha, Machaerium, and Entada. However, further investigation is required to support these observations. 2,3-Dihydroxypropyl hexacosanoate (1) was isolated from the stem bark of Pterocarpus erinaceus.⁴⁰ Hederagenine (2) was reported from Pterodon emarginatus Vogel⁴¹ and Medicago sativa.⁴² Lupeol (6) was reported from Desmodium oojeinensis,⁴³ Diplotropis ferruginea,⁴⁴ and Pterocarpus erinaceus.⁴⁵ Stigmast-4-en-3-one (9) has been previously isolated from Uraria picta,⁴⁶ Cassia singueana,⁴⁷ and Parkia speciose.⁴⁸ Triacontanoic acid (8) has been reported from Astragalus criticus,⁴⁹ Pterocarpus erinaceus,⁴⁵ and Alhagi pseudalhagi.⁵⁰ Betulinic acid was isolated from Desmodium oojeinensis,⁴³ Bauhinia ungulate,⁵¹ and Ormosia robusta.⁵² Cycloeucalenol was earlier reported from Uraria crinite,⁵³ Senna spectabilis,⁵⁴ and Acrocarpus fraxinifolius.⁵⁵ These findings indicated the close phylogenetic relationship between all these genera. However, further shreds of evidence are required to support this affirmation.

Conclusion

The phytochemical investigations of the stem bark and roots of the Cameroonian medicinal plant A. cladantha led to the isolation and characterization of twelve secondary metabolites reported for the first time from the genus Anthonotha. At the same time, compound 7 has been isolated for the first time from the family Fabaceae. The extract, various fractions, compounds 1 and 1a were subjected to pharmacological screening. The most significant finding was the α-glucosidase inhibitory activity of the extracts, fractions, and compound 1 with IC₅₀ 39.2 ± 0.22 μM. The ethyl acetate fraction deserves further attention for the development of new potent hypoglycemic drugs, in the other hand the acetylation of compound 1 decrease the inhibition of the α-glucosidase, confirms the importance of hydroxyl groups in the α-glucosidase activity. In addition, further semisynthetic research monitored by bioassays will be needed to clarify the relation structure activity of the compound.

Acknowledgments

The authors are deeply indebted to the TWAS-ICCBS 2019 program Fellowship for the opportunity to carry out the above research. We want to thank ICCBS, Karachi, Pakistan, for the excellent training received by BLVL during his stay from 15^th November 2019 to 14^th September 2020.

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