The collection of plant material was in accordance with the previously described methodology [2] .

Extraction was performed according to the previously described method [2] . In the frame of the present study, a portion of the EtOAc extract (28 g) was subjected to silica gel (0.200 - 0.500 mm) column chromatography (φ 80 mm × 600 mm) using n-Hexane-EtOAc (100:0→0:100) followed by EtOAc-MeOH (100:0→90:10) gradient elution. A total of 77 fractions of 250 mL each were collected and subsequently combined on the basis of their TLC profiles, resulting in the formation of six distinct fractions labelled G (1 - 5), H (6 - 11), I (12 - 43), J (44 - 50), K (51 - 74) and L (75 - 77). Fraction H (2.5 g) was purified by silica gel (0.063-0.200 mm) column chromatography (φ 20 mm × 600 mm) using n-Hexane-EtOAc (95:5) as eluent, resulting in the isolation of compound 1 (1.5 g). Purification of fraction J (3.1 g) on a Sephadex LH-20 column chromatography (φ 20 mm × 400 mm) afforded compounds 2 (10.4 mg) and 3 (35.2 mg). Compounds 4 (22.3 mg) and 5 (500.0 mg) precipitated in fractions I (0.95 mg) and L (1.70 g) respectively and were purified by recrystallisation using ethyl acetate.

Column chromatography (CC) was performed with Merck silica gel 60 and gel permeation with Sephadex LH-20. Thin layer chromatography (TLC) was performed on pre-coated silica gel GF₂₅₄ plates with detection by spraying 10% H₂SO₄ followed by heating at 100˚C or by visual inspection under UV lamps at 254 and 365 nm.

One-dimensional proton (¹H) and carbon (¹³C) nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance III 600 spectrometer equipped with a cryoprobe, with ¹H operating at 600 megahertz (MHz) and ¹³C at 150 MHz. Two-dimensional nuclear magnetic resonance (2D NMR) experiments were performed using standard Bruker microprograms (Xwin-NMR version 2.1 software TopSpin 3.2). The chemical shifts (δ) are reported in parts per million (ppm), using the residual solvent signals as a secondary reference with respect to TMS (δ = 0), and the values of the coupling constants (J) are given in Hertz (Hz).

To obtain the HR-TOFESIMS spectra, an electrospray source was used with a Micromass Q-TOF micro instrument (Manchester, UK). Samples were introduced by direct infusion into a solution of methanol at a rate of 5 mL per minute.

The preparation of Jones reagent was conducted in accordance with the methodology previously described in the literature [15] . In a 100 mL beaker, 1 g of chromium trioxide, 25 mL of distilled water and 8 mL of sulfuric acid (H₂SO₄) were added. After careful stirring for one minute, the Jones reagent was successfully obtained. A solution of compound 1 (500.0 mg, 1.096 mmol) in methylene chloride (30 mL) was transferred to a 100 mL flask. Subsequently, 30 mL of Jones reagent was added. The resulting mixture was heated under reflux at 30˚C for a period of 30 minutes. After cooling the reaction mixture, 40 mL of 5% sodium bicarbonate (NaHCO₃) was added to neutralise the medium. The mixture was transferred to a separating funnel, and 50 mL of distilled water and 70 mL of ethyl acetate were added to it. Following the separation of the two phases, the organic phase was subjected to evaporation under reduced pressure using a rotavapor, resulting in the production of a residue amounting to 513.7 mg. The purification of this residue was conducted on a silica gel (0.063 - 0.200 mm) column chromatography (φ 10 mm × 200 mm) with n-Hexane-EtOAc (97:3) as eluent, leading to the isolation of compound 1a (480.3 mg, 96.51 %).

To a 100 mL flask containing 1a (200 mg, 0.471 mmol) dissolved in methylene chloride (15 mL), 50 mL of hydrogen peroxide and 20 mL of formic acid were added. The mixture was allowed to stand at room temperature (25˚C) with magnetic stirring. The reaction was monitored on a TLC plate. After 5 h, the reaction mixture was transferred to a separating funnel to which, 20 mL of distilled water and 100 mL of methylene chloride (CH₂Cl₂) were added. After stirring and decantation, the organic phase was recovered and concentrated with a rotavapor to give 217.1 mg of a mixture. This mixture was purified on a silica gel (0.063 - 0.200 mm) column chromatography (φ 10 mm × 200 mm) with n-Hexane-EtOAc (95:5) to give compound 1b (196.5 mg, 91.36%).

The microorganisms used to determine antimicrobial activities were selected based on their relevance as human pathogens and included three bacterial strains (Escherichia coli S2, Staphylococcus aureus ATCC 25923 and Shigella flexneri SDINT) and three yeast strains (Candida tropicalis, Candida albicans ATCC 9002 and Cryptococcus neoformans IP95026) from the collection of the Research Unit of Microbiology and Antimicrobial Substances of the Dschang University. The bacterial and fungal species were cultured at 37˚C and maintained on nutrient agar (NA, Conda, Madrid, Spain) and Sabouraud dextrose agar (SDA, Conda) slant, respectively.

Minimum inhibitory concentration (MIC) values were determined using the broth microdilution method previously described by the Clinical and Laboratory Standards Institute [16] [17] , with some modifications regarding the final concentration of samples and inoculum solutions. In fact, each sample was dissolved in dimethyl sulphoxide (DMSO) diluted to 10% (v/v). The solution was then added to Mueller-Hinton Broth (MHB) for bacteria or Sabouraud Dextrose Broth (SDB) for yeasts to give a final concentration of 8192 μg/mL (instead of 4000 μg/mL). This was serially diluted to a concentration range of 0.125 to 4096 μg/mL. 100 μL of each concentration was then added to each well (96-well microplate) containing 95 μL of MHB or SBD and 5 μL of inoculum to give final concentrations ranging from 0.0625 to 2048 μg/mL. The inoculum was standardized to 2.5 × 10⁵ cells/mL for yeasts and 10⁶ CFU/mL for bacteria (instead of 1.5 × 10⁶ CFU/mL) using a JENWAY 6105 UV/Vis spectrophotometer. The final concentration of DMSO in each well was < 1% (preliminary analyses with 1% (v/v) DMSO did not inhibit the growth of the test organisms). The negative control well consisted of 195 μL MHB or BSD and 5 μL standard inoculum. The plates were covered with sterile lids, shaken to mix the contents of the wells and incubated at 37˚C for 24 h (for bacteria) or 48 h (for yeasts). The MIC values of the samples were determined by adding 50 μL of a purple solution of p-iodonitrotetrazolium at 0.2 mg/mL followed by incubation at 37˚C for 30 minutes. Viable microorganisms reduced the yellow dye to pink. MIC values were defined as the lowest sample concentrations that prevented this colour change, indicating complete inhibition of microbial growth. To determine the MMC values, a portion of the liquid (5 μL) from each well that showed no microbial growth was plated on Mueller Hinton Agar (MHA) or Sabouraud Dextrose agar (SDA) and incubated at 37˚C for 24 hours (for bacteria) or 37˚C for 48 hours (for yeasts). The lowest concentrations that gave no growth after this subculture were used as MMC values. Amphotericin B for yeasts and Ciprofloxacin for bacteria were used as positive controls. All the experiments were performed in triplicate. The experimental results were expressed as the mean ± Standard Deviation (SD). Differences between groups were considered significant when p < 0.05. All analyses were performed using the Statistical Package for Social Sciences (SPSS, version 12.0) software.

The structural characterization of natural compounds: Lupeol (1), Oleanolic acid (2), Chrysoeriol (3), N-methyl-isonicotinamide (4) and β-sitosterol 3-O-β-D-glucopyranoside (5) ( Figure 1 ) as well as semi-synthetic derivatives: Lupenone (1a) and (20R)-formyloxy-29-nor-lupan-3-one (1b) ( Figure 2 ) were carried out using a combination of ¹H, NMR, ¹³C NMR, COSY, HSQC and HMBC experiments. Spin systems were identified through the COSY and HSQC experiments. Subsequently these spin systems and the quaternary carbons were connected by correlations found in the HMBC spectra. The NMR data for compounds 1, 2, 3, 4, 5 and 1a ( Table 1 , Table 2 and Table 3 ) were in perfect agreement with the literature data [18] - [22] .

Compound 1b was obtained as a white powder. The yield of synthesis (91.36%) suggests a successful conversion of compound 1a to compound 1b. However, the loss of 8.64% was probably due to the purification process. The positive HR-TOFESIMS of this compound showed a sodium adduct peak at m/z 479.3508 [M + Na]⁺ (calculated for C₃₀H₄₈O₃Na, 479.3501). These MS data and NMR spectra were consistent with a molecular formula of C₃₀H₄₈O₃ for compound 1b. The difference between 1b and 1a was observed at the double bond Δ^20-29, which underwent oxidative cleavage with the loss of a carbon atom. This structural modification was reflected in the ¹H NMR spectrum, with the disappearance of the signals at δ_H 4.70 (H-29a, d; 1.6) and 4.59 (H-29b, d, 1.6). This is due to the double bond in compound 1a, as well as the appearance of an oxymethine proton signal at δ_H 5.30 (H-20, m) and that of a formyl group at δ_H 8.14 (H-1′; s). This structural modification was corroborated by the ¹³C NMR spectrum, which revealed the disappearance of the double bond signals at δ_C: 109.4 (C-29) and 150.8 (C-20) present in compound 1a. Additionally, two signals at δ_C: 161.5 (C-1′) and 72.4 (C-20) were observed. These correspond to the carbon signals of a formyloxy group and an oxymethine, respectively. The position of the formyloxy group was determined by the HMBC experiment, which revealed a correlation between the proton at δ_H 5.30 (H-20) and the carbonyl of the formyloxy group at δ_C 161.5 (C-1′). The absolute configuration R of the C-20 stereocentre was determined on the basis of previous work [23] [24] . This work indicated that, the (20S) and (20R) isomers showed differences in the chemical shifts of C-19, C-20, C-29 and C-30, particularly C-30. Accordingly, compound 1b with C-30 at 20.0 has a 20R configuration. Based on the above evidence, the structure of 1b was determined to be (20R)-formyloxy-29-nor-lupan-3-one, a novel semi-synthetic lupeol derivative. Scheme 1 shows the plausible mechanism by which derivative 1b would have formed.

The evaluation of antimicrobial activities shows that the semi-synthetic derivatives (1a and 1b) and the starting material (1) exhibited different minimum inhibitory concentrations (MICs) against the microbial strains tested ( Table 4 ).

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.