General instrumentation

The detailed information about the general instrumentation was given in the supplementary information (Appendix A).

Plant material

The stem bark and leaves of Guibourtia ehie (A. Chev) J. Leonard were collected in December 2018 at Dibombari locality (GPS coordinates: Latitude 4°12′00″N, Longitude 9°39′00″E, Elevation: 14 m), Littoral Region, Cameroon. The identification of the plant was made by Mr. Victor Nana based on morphological comparison of its leaves and branches with the previous plant material in the database of the National Herbarium of Cameroon, where a specimen was kept under the voucher number 43216 HNC.

Extraction and isolation

MeOH extraction of G. ehie stem bark (3.16 kg) and leaves (860.0 g) yielded brown viscous extract (80.2 g) and green viscous extract (30.4 g), respectively, at room temperature for 48 h, after which the filtrates were concentrated under reduced pressure. Each extract (~5 g) was kept for biological assays, and the remaining crude extract was independently subjected to silica gel column chromatography with a gradient of EtOAc in n-hexane, followed by a gradient of methanol in EtOAc. The stem bark extract (75.2 g) was fractionated over silica gel to obtain seven main fractions labelled from S₁ to S₇. The fraction S₁ (n-hexane-EtOAc, 37:3) gave a total of 104 subfractions and afforded three pure compounds identified as a mixture of β-sitosterol (11) and stigmasterol (12) (5.6 mg) (from subfractions 5-24) and friedelan-3-one (7) (5.7 mg) (from subfractions 86-97). By applying the same method, the second fraction S₂ (n-hexane‒EtOAc 17:3) gave a total of 94 subfractions of 200 mL each from which taraxerol (6) (6.3 mg) (from subfractions 13-21) and lupeol (5) (8.3 mg) (from subfractions 57-68) have precipitated as white powders. Furthermore, 2,6-dimethoxylbenzoquinone (4) (4.2 mg) was obtained from the third fraction S₃ (n-hexane‒EtOAc, 4:1) while the last fraction S₇ (EtOAc) was further purified on silica gel column chromatography to yield 113 subfractions from which stigmasterol-3-O-β-D-glucopyranoside (13) (10.2 mg) (from subfractions 1-18) and ellagic acid (1) (7.2 mg) (from subfractions 67-82) as white amorphous and yellowish powders, respectively.

The leaves extract (25.4 g) was fractionated over a silica gel column using the gradient hexane‒DCM‒EtOAc‒MeOH, resulting in six series. The third series, S₃ (hexane‒DCM, 1:4), was a combination of 108 fractions (100 mL, each) and afforded lanosterol (8) (3.4 mg) (from fractions 34-41). The fourth series S₄ (DCM‒EtOAc, 1:1) resulted from the combination of 142 fractions and after purification process yield scopoletin (9) (3.4 mg) (from fractions 5-12), pilloin (10) (2.8 mg) (from fractions 34-53) and 3,3′-di-O-methylellagic acid 4′-O-β-D-xylopyranoside (2) (24.5 mg) (from fractions 122-135) while the last series S₆ (EtOAc‒MeOH, 49:1) with a total of 68 fractions yielded rhaponticin (3) (6.4 mg) (from fractions 34-41).

Acetylation of compound 2

3,3′-di-O-methylellagic acid 4′-O-β-D-xylopyranoside (2) (15.0 mg) was dissolved in pyridine (1.0 mL), and 1.0 mL of acetic anhydride was added. After 10 hours under agitation at room temperature, the reaction was quenched with water, and the medium was partitioned between CH₂Cl₂ and H₂O. The organic layer was reduced to dryness using a rotavapor to afford an oily extract that was further purified over silica gel eluting with hexane-AcOEt (1:19) to yield 3,3′-di-O-methylellagic acid 4′-O-β-D-(4,2′′,4′′-triacetyl)-xylopyranoside (2a) (8.0 mg) as a yellow powder.

Spectroscopic data of reported compound 2a

3,3′-di-O-methylellagic acid 4′-O-β-D-(4,2′′,4′′-triacetyl)-xylopyranoside (2a): C₂₇H₂₄O₁₅, Yellow powder (MeOH); HR-ESI-MS (m/z): 611.1007 [M+Na]⁺ (calcd for C₂₇H₂₄O₁₅Na⁺, 611.1013); [α]²⁵_D = +20 (c = 1.0, CHCl₃); UV (MeOH) λ_max (log e) 260 (3.43), 292 (3.51), 358 (3.40) nm; IR (KBr) υ_max 3500 (OH), 1748 (C=O), 1630 (C=C), 1062 (C-O) cm^-1; ¹H NMR (600 MHz, CDCl₃) δ: 7.90 (1H, s, H-5), 7.80 (1H, s, H-5′), 5.26 (1H, d, J = 5.2 Hz, H-1′′), 5.24 (2H, m, H-5′′), 5.02 (1H, dd, J = 6.9, 9.7 Hz, H-2′′), 4.40 (3H, s, 3′-OCH₃), 4.26 (1H, dd, J = 4.5, 9.9 Hz, H-3′′), 4.20 (3H, s, 3-OCH₃), 3.62 (1H, dd, J = 6.2, 10.1 Hz, H-4′′), 2.11 (3H, s, 4′′-OCOCH₃), 2.10 (6H, s, 4-OCOCH₃ and 2′′-OCOCH₃); ¹³C-NMR (150 MHz, CDCl₃) δ: 169.9 (4′′-OCOCH₃), 169.8 (2′′-OCOCH₃), 169.4 (4-OCOCH₃), 158.3 (C-7), 158.1 (C-7′), 150.8 (C-4, C-4′), 143.5 (C-3), 141.5 (C-2), 139.8 (C-2′), 138.9 (C-3′), 115.5 (C-1), 114.1 (C-5′), 113.2 (C-6), 112.4 (C-6′), 111.9 (C-1′), 111.7 (C-5), 99.5 (C-1′′), 69.9 (C-3′′), 69.6 (C-2′′), 68.1 (C-4′′), 62.2 (3-OCH₃), 62.2 (3′-OCH₃), 61.9 (C-5′′), 20.8 (4-OCOCH₃ and 2′′-OCOCH₃), 20.7 (4′′-OCOCH₃).

DPPH radical scavenging activity

As described by Gülcin, Alici, and Cesur (2005), the free radical scavenging activity was evaluated using 1,1-diphenyl-2-picrylhydrazil (DPPH). The detailed procedures were given in the supplementary information (Appendix A).

Urease inhibition assay

The urease inhibition assay was measured using the method described by (Lodhi et al., 2014). The detailed procedures were given in the supplementary information (Appendix A).

Alpha-glucosidase inhibition assay

The alpha-glucosidase inhibition assay was measured using the method described by (Atsumi, Miwa, Kimata, & Ikawa, 1990) and (Kurihara et al., 1994). The detailed procedures were given in the supplementary information (Appendix A).

Phytochemical study

Using purification techniques, the chemical study of the methanolic leaves and stem bark extracts of Guibourtia ehie led to the isolation of thirteen compounds (Figure 1). Their structures were established as ellagic acid (1) (Nkainsa et al., 2020), 3,3′-di-O-methylellagic acid 4′-O-β-D-xylopyranoside (2) (Moharram, Marzouk, Ahmed, & Aboutabl, 2003; Ngoumfo et al., 2008), rhaponticin (3) (Park, Na, Jae, Ju, & Hyun, 2018), 2,6-dimethoxylbenzoquinone (4) (Harasawa & Tagashira, 1994), lupeol (5) (Ahmed, 2019), taraxerol (6) (Midori et al., 1999), friedelan-3-one (7) (Xie, Mingxiang, Dingyong, & Runlin, 2013), lanosterol (8) (Ishii et al., 2014), scopoletin (9) (Napiroon et al., 2018), pilloin (10) (Tsai et al., 2018) and three common steroids β-sitosterol (Ododo, Choudhury, & Dekebo, 2016), stigmasterol (Ibrahim et al., 2015) and its glucoside stigmasterol-3-O-β-D-glucopyranoside (Faizi, Ali, Saleem, Irfanullah, & Bibi, 2001) by comparing their 1D-NMR and ESI- or EI-MS with those reported in the literature (Figure 1). The isolated steroids were directly identified by comparison of their Rf on TLC profiles with those of the reference compounds available in the laboratory.

In looking for significant activity, 3′-di-O-methylellagic acid 4′-O-β-D-xylopyranoside (2) obtained in sufficient amount was chemically modified by acetylation reaction to afford compound 2a, a yellow powder with an optical rotation α²⁵_D = +20 (c = 1.0, CHCl₃). It's (+)-HR-ESI-MS showed the sodium adduct ion [M+Na]⁺ at m/z 611.1007 (calcd. for C₂₇H₂₄O₁₅Na⁺, 611.1013) consistent with the molecular formula C₂₇H₂₄O₁₅ suggesting the acetylation of three hydroxyl groups of compound 2 (Figure 1). This observation was further confirmed with the ¹H NMR spectrum of 2a (Table 1 , Figure 3S Appendix A ), which showed in the upfield region, the signals of three additional methyl groups deshielded by an ester carbonyl at δ 2.11 (3H, s, 4′′-COCH₃), 2.10 (3H, s, 2′′-COCH₃) and 2.10 (3H, s, 4-COCH₃) compared to that of 2 (Figure 3S Appendix A ). In addition, the ¹³C, Dept-135 and Dept-90 NMR spectra (Figure 4S-6S Appendix A , Table 1) displayed extra resonances, including signals of three carbonyl groups at δ 169.4, 169.8 and 169.9. The careful analysis of the HSQC spectrum (Figure 8S Appendix A ) allowed us to establish the correlations between protons at δ 5.02 (1H, dd, J = 6.9, 9.7 Hz, H-2′′), δ 3.62 (1H, dd, J = 6.2, 10.1 Hz, H-4′′) and the carbonyl groups at δ 169.8 (2′′-COCH₃) and δ 169.9 (4′′-COCH₃), respectively. These observations suggested that the initial hydroxyl groups at C-2′′ and C-4′′ in the sugar moiety of 2 have been acetylated in 2a. The third acetoxy group was attached to hydroxyl at C-4 based on HMBC cross-peaks between the aromatic proton at δ 7.90 (1H, s, H-5) with the ester carbonyl group at δ 169.4 (4-COCH₃) in long-range correlation (Figure 9S Appendix A ). Thus, the structure of compound 2a was determined as 3,3′-di-O-methylellagic acid 4′-O-β-D-(4,2′′,4′′-triacetyl)-xylopyranoside as shown in (Figure 1).

Figure 1

Chemical structures of compounds 1‒13 isolated from G. ehie

Antioxidant property

The extracts, fractions and the major compounds obtained were submitted for antioxidant activity in the DPPH (Table 1). Briefly, the stem bark and leaves extracts and leaf fractions showed weak activity with effectiveness in the range of IC₅₀ value from 61.2 to 89.1 μM. As expected for the phenolic constituents, ellagic acid (1) displayed a strong potency with an IC₅₀ value of 36.42 μM more active than the standard butylhydroxyanisole (BHA) (IC₅₀ = 44.2 ± 0.2 μM) while its derivatives 3,3′-di-O-methylellagic acid 4′-O-β-D-xylopyranoside (2) and 3,3′-di-O-methylellagic acid 4′-O-β-D-(4,2′′,4′′-triacetyl)-xylopyranoside (2a) were not active. Another phenolic rhaponticin (3), was slightly active (IC₅₀ = 85.3 ± 0.4 μM) whereas 2,6-dimethoxylbenzoquinone (4) was not active. Taking together, we can partially conclude based on our results that the potency in antioxidant activity for the phenolic compounds, especially the ellagic acid derivatives, increases with the availability of the phenolic hydroxyl groups in their core structures. The more the phenolic compound is substituted on its hydroxyl functions, the less it is active. Previous investigations reported that ellagic acid (1) demonstrated a high DPPH radical scavenging, lipid peroxidation inhibition, and a high reactivity towards HO•, oxygen, and nitrogen species. Furthermore, it significantly increases the activities of SOD (superoxide dismutase), CAT (catalase) and GPX (glutathione peroxidase) in V79-4 cells (Galano, Marquez, & Pérez-González, 2014; Han, Lee, & Kim, 2006; Priyadarsini, Khopde, Kumar, & Mohan, 2002; Tošovi & Bren, 2020). Similarly, rhaponticin (3) significantly reduced oxidative stress by decreasing the level of malondialdehyde and increasing the activity of SOD, CAT and GPX in diabetic rats (Shi et al., 2020). Lastly, the triterpene taraxerone (6) exhibited a weak activity with an IC₅₀ of 89.2 ± 0.5 μM.

Urease inhibition activity

The evaluation of extracts, fractions and pure compounds for their urease inhibitory activity showed that except the DCM/AcOEt (1:1) fraction of leaves extract which had an activity close to the reference thiourea 25.2 ± 0.8 μM and 22.4 ± 0.2 μM, respectively, the stem bark and leaves crude extracts, compounds 3,3′-di-O-methylellagic acid 4′-O-β-D-(4,2′′,4′′-triacetyl)-xylopyranoside (2a) and rhaponticin (3) were slightly active with IC₅₀ values in the range of 75.3 to 89.2 μM. The other tested compounds 2,6-dimethoxylbenzoquinone (4), lupeol (5), friedelan-3-one (7) and lanosterol (8), were inactive (Table 1). Our result can be supported by the literature, which reports 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 (Modolo, Souza, Horta, Araujo, & Fátima, 2015)

Alpha-Glucosidase inhibition activity

In the α-glucosidase inhibitory assay, the crude extracts and leaves fractions of G. ehie exhibited promising results with intense activities in the range of 10.3 to 18.2 μM, which were less active than the reference (1-deoxynojirimycin, IC₅₀ = 3.9 ± 0.7 μM). 2,6-dimethoxylbenzoquinone (4) was the most potent among the tested compounds, followed by rhaponticin (3) and taraxerol (6) with IC₅₀ values of 25.5 ± 0.2, 35.5 ± 0.1 and 43.4 ± 0.3 μM, respectively. Several compounds from various classes have been reported in the literature as alpha-glucosidase inhibitors. For instance, Atta-Ur-Rahman, Zareen, Choudhary, Akhtar, and Khan (2008) isolated cichoridiol, a taraxane-type triterpenoid close to taraxerl (6), displaying a significant alpha-glucosidase inhibitory activity. These results suggested that the extracts of G. ehie might be of great importance in the formulation of ameliorated traditional medicine for the treatment of diabetes and other diseases related to the inhibition of α-glucosidase, but further analyses are necessary to support this partial conclusion. Furthermore, rhaponticin (3), 2,6-dimethoxybenzoquinone (4), and taraxerol (6) deserve further investigations for the development of new antidiabetic drugs.

Chemotaxonomic significance

This study led to the isolation of thirteen compounds, including ellagic acid (1) and its derivative 3,3′-di-O-methylellagic acid 4′-O-β-D-xylopyranoside (2), rhaponticin (3), 2,6-dimethoxybenzoquinone (4), lupeol (5), taraxerol (6), friedelan-3-one (7), lanosterol (8), scopoletin (9), pilloin (10), as well as the common steroids β-sitosterol (11), stigmasterol (12) and stigmasterol-3-O-β-D-glucopyranoside (13). The isolation of these secondary metabolites is not uncommon since the report on previous phytochemical investigations of some Guibourtia species like Guibourtia ehie, G. coleosperma, and G. tesmanii demonstrated the presence of tannins, triterpenoids, saponins and sterols (Dushimemaria, Iwanette, & Davis, 2017). All of these compounds have been reported from G. ehie for the first time, allowing us to gain a better understanding of this species' chemistry. The stilbene glycoside rhaponticin (3) was reported from G. tessmannii (Kathryn, Greg, Steven, Jaime, & Neal, 2005). Compound 3 was also isolated from a Leguminosae plant Trigonella foenum-graecum (Idris, Anuradha, & Khushtar, 2021), indicating a close relationship between genus Guibourtia and Trigonella; however, further investigation must be done to support these observations. Lanosterol (8) was identified using gas chromatography-mass spectrometry from G. colosperma (Preez, Louw, & Mumbengegwi, 2020). Lupeol (5), taraxerol (6) and friedelan-3-one (7) are isolated for the first time from the Guibourtia genus but were previously detected in other genera from Leguminosae (Fabaceae) family. Indeed, lupeol (5) was reported from Dalbergia hainanensis (Zhang, Wu, & Yu, 2003), Cassia abbreviate (Mutasa & Kahn, 1995), Caesalpinia sappan (Oswal & Garg, 1993), Caesalpinia pulcherrima (Chiang, Chiang, Liu, & Lin, 2003) while friedelan-3-one (7) was reported from Caesalpinia digyna (Srinivasan, Chandrasekar, & Nanjan, 2011), Pterocarpus santalinoides (Ichiko, Terrumun, John, & John, 2016), and Pterocarpus erinaceus (Ouedraogo et al., 2011). Taraxerol (6) was isolated from Dalbergia hainanensis (Zhang et al., 2003) and D. spinosa (Anjaneyulu, Rao, Lakshmana, & Sreedhar, 2005). These findings indicated the close phylogenetic relationship between the genera Guibourtia, Cassia, Milletia, Dalbergia and Caesalpinia belonging to the same plant family. Finally, 3,3’-Di-O-methylellagicacid-4’-O-β-D-xylopyranoside (2), scopoletin (9) and 2,6-dimethoxybenzoquinone (4) can also be found in some species across the Leguminosae (Fabaceae) family. Therefore, compound 2 was reported from Acacia farnesiana (Hussein, Afifi, & El-Mousallami, 2002), while compound 4 was previously obtained from Senna alata (synonym of Cassia alata) (Chimi et al., 2021), and compound 9 was already obtained from twigs and leaves of Caesalpinia spinosa (He et al., 2015). Additionally, pilloin (10) was identified by GS-MS from Dalbergia melanoxylon (Yin, Huang, Zhang, Liu, & Ma, 2018). Hence, this evidence further supports the taxonomy of the plant species G. ehie and enriches its chemistry.