The roots of Gouania longipetala were collected in the BESO classified forest at AKOUPE (Latitude: 6, 3546˚ or 6˚21'17" NORTH; Longitude: −3, 6921˚ or 3˚41'32" WEST) in the East region of Côte d’Ivoire, in February 2021. The plant was identified at the floristic center of University Felix Houphouët Boigny (Abidjan, Côte d’Ivoire) under the herbarium number UCJ 014570 in comparison with the herbarium collected by AKE ASSI on 06/04/1966.

The harvested roots were air-dried for 3 weeks at ambient laboratory temperature and protected from sunlight. Then samples were ground using an electric grinder and weighed (4.5 kg).

One kg of powdered roots was soaked in a mixture of methanol and distilled water (80:20) for 48 hours. Afterward, the mixture was filtered with Whatman No. 1 filter paper. The hydromethanolic extract was then evaporated using a Heidolph rotary evaporator (4000 series), which was set to 40˚C under reduced pressure to obtain the crude sample. The resulting crude extract was oven-dried at 40˚C and weighed (146.1 g). Subsequently, 76 g of the crude extract was dissolved in 500 mL of distilled water and underwent a series of liquid-liquid extractions using solvents with increasing polarity: dichloromethane (3 × 500 mL), ethyl acetate (3 × 500 mL). The remaining residue was then dried. Following the various extractions, we obtained 1.15 g of dichloromethane extract, 5 g of ethyl acetate extract and 62 g of aqueous extract.

Fractions were successively purified using open-colon chromatography. Analytical TLC was performed on precoated silica-gel 60 F254 Merck and spots were observed under UV light at 254 and 365 nm or visualized by spraying the dried plates with sulfuric vanilin, followed by heating. Silica gel 60 (63 - 200 mesh, Merck) was used for column chromatography. NMR experiments were carried out in DMSO-d6 on Bruker Advance DRX III 500 instruments. HR-ESI-MS experiments were performed using a Micromass Q-TOF micro instrument.

The ethyl acetate fraction (5 g) was fractionated on a silica gel system with the hexane/dichloromethane/ethyl acetate/methanol mixture as eluent. This fractionation led to 5 fractions presented in Table 1 .

Subsequently, fraction 1 was purified on column chromatography on silica using mobile phase hexane/dichloromethane (80/20) gradient to afford Compound 1 (4.7 mg). Fraction 3 was also purified on column chromatography on silica gel using mobile phase dichloromethane/ethyl acetate (90/10) to afford Compound 2 (3.5 mg) and Compound 3 (5.6 mg).

The 13C-NMR spectrum of Compound 1 revealed 30 carbon signals, including seven methyl, eleven methylene, six methine, and six quaternary carbons, corresponding to the terpenoid with a lupane skeleton. Notably, a carbon atom attached to the OH group at the C-3 position was observed at 76.2 ppm. The ¹H NMR spectrum of Compound 1 shows a doublet at 4.69 ppm (J = 2.6 Hz) integrating a proton attributable to proton H-29b, triplet at 4.55 ppm (J = 2.1 Hz) integrating a proton attributable to proton H-29a, a triplet doublet at 2.97 ppm (J = 10.4 and 5.4 Hz) integrating a proton attributable to proton H-3 and six singletons corresponding to angular methyls: C-23 (0.87 ppm), C-24 (0.65 ppm), C-25 (0.77 ppm), C-26 (0.98 ppm), C-27 (0.91 ppm) and C-28 (0.76 ppm). The total assignment of proton and carbon was confirmed by COSY and HMBC correlations.

Compound 1 was identified as lupeol [11] [12] . Compound 2 structure was deduced from Compound 1 and was identified as betulin [13] - [15] . The 1H-NMR spectrum of Compound 3 shows the same characteristics as that of lupeol. The difference is the presence of two multiplets at 3.08 ppm and 3.51 ppm, each integrating a proton attributable to the hydroxylated carbon C-28 (57.4 ppm).

Their structural elucidations were carried out by HR-ESI-MS, 1D and 2D-NMR analysis and their spectroscopic data corroborated with those in the literature. The structure of Compound 3 is deduced from Compounds 1 and 2.

Compound 3 appears as white amorphous crystals. Its structure is deduced from that of Compounds 1 and 2. Indeed, the analysis of the ¹H, ¹³C ( Table 2 ) and HSQC NMR spectra shows similarities between the two compounds and suggests that Compound 3 is also a lupane-type triterpene. In the ¹H NMR spectrum, we observe a broad singlet at 4.58 ppm and a doublet at 4.69 ppm (J = 1.45 Hz) corresponding to the exocyclic ethylenic protons H-29a and H-29b, respectively. There is also a triplet of doublets at 3.01 ppm (J = 10.5, 2.73 Hz) integrating for one proton H-19, a multiplet at 2.29 ppm integrating for one proton H-13, and five singlets corresponding to the five angular methyl groups. Three of these signals integrate for 3 protons each at 0.99 ppm (H-23), 0.89 ppm (H-2), and 1.64 ppm (H-30), and one signals at 0.95 ppm integrate for six protons corresponding to methyl groups 25 and 26.

Furthermore, the HMBC spectrum shows correlation peaks with the carbons C-30 (18.43 ppm) and H-19 (3.00 ppm).

The ¹³C NMR spectrum of Compound 3 shows signals at 204.4 (C-3), 176.3 (C-27), and 177.1 (C-28) ppm ( Table 2 ). These high chemical shift values suggest that these carbons bear a ketone and an acid functional groups, respectively. Indeed, the HMBC spectrum reveals correlations between the signal at 204.4 ppm and the protons H-2a (1.66 ppm), H-2b (1.79 ppm), H-1 (2.62 ppm) and H-5 (0.96 ppm). These different correlations allow for the assignment of carbon C-3. Carbon C-28 (177.1 ppm) is attributed through the HMBC spectrum, by its correlations with protons H-16a (1.25 ppm), H-16b (2.22 ppm), H-18 (1.51 ppm), and H-22b (1.82 ppm). Carbon C-27 (176.3 ppm) is also attributed to its HMBC correlations with proton H-13 (2.29 m), H-15a (1.24 m) and H-15b (1.89 m). The important COSY and HMBC correlations are reported in Figure 2 . Compound 3 was assigned to the molecular formula C₃₀H₄₃O₆ from its negative HR-ESI-MS, which exhibited a molecular ion peak [M-H]⁻ at m/z 499.3062 and was identified as 1α-hydroxy-lup-20(29)-en-3-oxo-27, 28-dioic acid.

Lupeol (1) : ¹³C NMR (125 MHz, DMSO); δ_C (ppm): 37.8 (C-1), 26.6 (C-2), 76.2 (C-3), 38.98 (C-4), 54.4 (C-5), 17.44 (C-6), 33.3 (C-7), 39.8 (C-8), 49.3 (C-9), 36.2 (C-10), 19.9 (C-11), 24.2 (C-12), 37.1 (C-13), 41.9 (C-14), 26.5 (C-15), 34.6 (C-16), 42.0 (C-17), 47.3 (C-18), 46.9 (C-19), 149.9 (C-20), 28.7 (C-21), 38.9 (C-22), 27.6 (C-23), 15.3 (C-24), 15.4 (C-25), 15.2 (C-26), 13.84 (C-27), 17.3 (C-28), 109. (C-29), 18.5 (C-30).

¹H NMR (500 MHz, DMSO), δ_H (ppm) : 0.82 (m, H-1a), 1.56 (m, H-1b), 1.43 (m, H-2), 2.96 (m, H-3), 0.63 (m, H-5), 1.34 (m, H-6a), 1.46 (m, H-6b), 1.33 (m, H-7), 1.24 (m, H-9), ), 1.36 (m, H-11), 1.04 (m, H-12a), 1.63 (m, H-12b), 1.62 (m, H-13), 0.96 (m, H-15a), 1.63 (m, H-15b), 1.35 (m, H-16a), 1.44 (m, H-16b), 1.32 (m, H-18), 2.37 (m, H-19), 1.26 (m, H-21a), 1.86 (m, H-21b), 1.18 (m, H-22a), 1.34 (m, H-22b), 0.87 (s, H-23), 0.65 (s, H-24), 0.77 (s, H-25), 0.98 (s, H-26), 0.91 (s, H-27), 0.76 (s, H-28), 4.54 (brs, H-29a), 4.68 (d, J = 2.49 Hz, H-29b), 1.64 (s, H-30).

Betulin (2): ¹³C NMR (125 MHz, DMSO), δ_C (ppm) : 37.7 (C-1), 26.7 (C-2), 76.3 (C-3), 38.01 (C-4), 54.3 (C-5), 17.5 (C-6), 33.3 (C-7), 39.9 (C-8), 49.3 (C-9), 36.2 (C-10), 19.8 (C-11), 24.3 (C-12), 36.3 (C-13), 41.7 (C-14), 26.2 (C-15), 28.5 (C-16), 46.9 (C-17), 47.6 (C-18), 46.8 (C-19),149.90 (C-20), 28.8 (C-21), 33.3 (C-22), 27.6 (C-23), 15.3 (C-24), 15.4 (C-25), 15.2 (C-26), 14.1(C-27), 57.4 (C-28), 109.1 (C-29), 18.3 (C-30).

¹H NMR (500 MHz, DMSO), δ_H (ppm): 0.83 (m, H-1a), 1.56 (m, H-1b), 1.44 (m, H-2), 2.97 (m, H-3), 0.63 (m, H-5), 1.33 (m, H-6a), 1.45 (m, H-6b), 1.32 (m, H-7), 1.24 (m, H-9), 1.15 (m, H-11a), 1.31 (m, H-11b), 0.97 (m, H-12a), 1.58 (m, H-12b), 1.61 (m, H-13), 0.91 (m, H-15a), 1.62 (m, H-15b), 1.22 (m, H-16a), 1.87 (m, H-16b), 1.48 (m, H-18), 2.38 (m, H-19), 1.27 (m, H-21a), 1.86 (m, H-21b), 1.86 (m, H-22), 0.87 (s, H-23), 0.65 (s, H-24), 0.76 (s, H-25), 0.96 (s, H-26), 0.91 (s, H-27), 3.08 (m, H-28a), 3.51 (m, H-28b), 4.53 (brs, H-29a), 4.66 (brs, H-29b), 1.63 (s, H-30).