Glyphaea brevis (Spreng.) Monach (Malvaceae) is a small tree distributed in a tropical region, from Guinea-bisssau, Ouganda, RDC to Congo Brazzaville [1], traditionally known as fondron or koula-gboè (forest okro) in Ivory Coast. The flowers of this plant are used in revitalizing sexual impotency [2]. The leaves and the flowers are used to treat sterility [3]. The leaves are also used as a remedy for sexually, liver, eyes, pulmonary and stomach diseases, inflammations, wound healing, palpitations, abortive, anti-emetic and analgesic [1] [4]. The stem bark of the root is used to treat diarrhea, dysentery and epilepsy [5]. Dakam et al. described the inhibitory effects of aqueous and hydroalcoholic extracts of the leaves of G. brevis on pancreatic α-Amylase activity [6], while Ojelabi et al. (2012) showed the roles of the ethanolic extract of the leaves of G. brevis in the protection of the liver and as antioxidant against cadmium-mediated oxidative stress [7]. Chemical investigations of the leaves of G. brevis have led to the isolation of sitosteryl-3-O-β-D-glucopyranoside, n-hexacosanol, n-dotriacontanol, oleanolic acid, echinocystic acid and meso-erythritol which showed both antibacterial and antifongic activity [8]. Ekuadzi et al. (2014) reported the isolation of epicatechin and its dimer procyanidin B2 from the stem bark of G. brevis [9].

Our previous phytochemical investigation of the stem bark of the roots of G. brevis resulted in the identification and structural elucidation of the potent glucosidase inhibitors phenyalkyl iminosugars [10] (Gossan et al., 2014). In a recent study, the results of Olugbodi et al. (2022) indicated that Glyphaeaside C-enriched extract of Glyphaea brevis leaf enhanced the fertility by the quality of semen and improved the functional capabilities of spermatozoa [11].

In our continuous search for interesting bioactive compounds we examined the n-butanol extract of the leaves of G. brevis.

This paper describes the isolation and structural elucidation of one undescribed and four known flavonoids. Their structures were elucidated by spectroscopic techniques including MS, 1D and 2D NMR spectroscopy and by comparison with published data. All the compounds were isolated for the first time from Glyphaea brevis. The antioxidant activity of compounds 1 - 5, using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay was tested.

The n-BuOH fraction extract of the leaves of G. brevis was subjected to a series of chromatographic separations to yield five flavonoids 1 - 5 (Figure 1). Their structures were elucidated based on the combination of various NMR (¹H and ¹³C NMR, COSY, HSQC, HMBC, and NOESY) spectroscopic and mass spectrometric (HR-EI-MS) techniques.

The known compounds 2-5 were readily identified from their spectral data and by comparison with reported data in the literature, as apigenin 7-O-β-D-glucopyranosyl-(1 → 2)-β-D-glucuronopyranoside (2) [13], acacetin (3), acacetin-7-O-β-D-glucuronide (4) [14] and genkwanin-5-O-primeveroside (5) [15].

Compound 1 was obtained as an amorphous yellow powder. The ESIMS spectra of 1 revealed [M + Na]⁺ (positive ion mode) ion peak at m/z 645, suggesting the molecular formula C₂₈H₃₀O₁₆.

The ¹H and ¹³C-NMR spectra of 1 were closely similar to that of 2, except for the presence of OCH₃-4' in the flavone moiety of 1 (Table 1). Thus, the ¹H NMR spectrum of 1 showed an isolated proton at δ 6.97 (1H, s), a methoxyl group at δ 3.88, a meta-coupled protons at δ 6.52 and 6.92 (each 1H, d, J = 2.3 Hz) assigned to H-6 and H-8, respectively, and an ortho-coupled AA'BB'-type protons at δ 8.07 and 7.15 (each 2H, d, J = 8.7 Hz) assignable to H-2', 6' and H-3', 5' respectively. The above spectral evidence and comparison with literature data readily allowed to recognize a typical acacetin framework in 1 [14]. The spectrum further displayed signals at δ_H 5.40 (1H, d, J = 7.2 Hz, H''-1), 3.58 (1H, m, H''-2), 3.57 (1H, m, H''-3), 3.49 (1H, t, J = 9.4 Hz, H''-4), 4.11 (1H, d, J = 9.4 Hz, H''-5). These proton shifts in correlation with downfield signals at δ_C 98.0, 82.2, 74.9,

70.8, 75.0 and 170.0 in the ¹³C-NMR, HSQC and HMBC spectra, demonstrated the presence of an O-β-D-glucuronic acid moiety [13]. The glucose part of 1 was identified by the presence of six carbons signals at δ_C 104.6, 74.6, 76.2, 69.6, 76.9, 60.5 (Table 1) in the ¹³C spectrum and one anomeric proton doublet at 4.49 (1H, d,

^aNot determined: IC₅₀ value > 200 µg/mL. ^bUsed as a positive control.

J = 7.8 Hz, H'''-1) in the ¹H NMR spectrum. The coupling constant of H'''-1 (J = 7.8 Hz) indicated the β-D configuration of the terminal β-glucopyranose unit. The location of the glucuronic acid unit at C-7 of acacetin moiety was confirmed by the correlation between H''-1 and C-7 in the HMBC spectrum. The attachment of the glucosyl unit at C''-2 of acacetin glucuronide moiety was confirmed by the upfield shift of C''-2 as well as by the cross-peak between C''-2 and H'''-1 in the HMBC spectrum. Accordingly, 1 was characterized as acacetin-7-O-β-D-glucopyranosyl-(1 → 2)-β-D-glucuronopyranoside.

All isolated compounds 1 - 5 were tested for their antioxidant activities in the DPPH radical scavenging assay and showed no activity for 1, 2, 4 and 5 (Table 2). These results confirmed those of previous studies, which showed weak or negative effects against free radical scavenging activities in compounds 2, 3, 4 and 5 possessing acacetin and apigenin nucleus [13] [16] [17] [18]. This very weak activity may be attributed to the smaller number of phenolic groups or the blocked 4-hydroxy group in the B-ring [19]. Furthermore, the presence of the sugar groups on acacetin (1 and 4) and apigenin (2 and 5) could also be attributed to the inactivity. Very frequently, the antioxidant activities of natural phenolic compounds, such as flavonoid glycosides depend on the number or sequence of sugars [19]. Shimoda et al. (2011) showed the decrease of DPPH free-radical scavenging activity of daidzein in its glucosylation form β-glucosides and β-primeveroside [20].

The phytochemical investigation of the leaves of Glyphaea brevis resulted in the identification of a new flavonoid acacetin-7-O-β-D-glucopyranosyl-(1 → 2)-glucuronopyranoside (1) together with apigenin-7-O-β-D-glucopyranosyl-(1 → 2)-glucuronopyranoside (2) acacetin (3), acacetin-7-O-glucuronide (4) and genkwanin 5-O-primveroside (5). All the compounds were isolated for the first time from Glyphaea brevis. The antioxidant activity of compound 1 using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay showed no antioxidant activity against DPPH radicals.

The authors are grateful to CNRS, Conseil Régional Champagne Ardenne, Conseil Général de la Marne (University Reims Champagne-Ardenne), and to the PlANET CPER project and Ministry of Higher Education and Research (MESR) Government of Ivory Coast for financial support.

The authors declare that they have no conflicts of interest.

Yao-Kouassi, P.A., Gossan, D.A.P., Magid, A.A., Martinez, A., Sayagh, C. and Voutquenne-Nazabadioko, L. (2022) Flavonoid Glycosides from the Leaves of Glyphaea brevis. Open Journal of Applied Sciences, 12, 1296-1303. https://doi.org/10.4236/ojapps.2022.127089