Chemical constituents of the leaves of Campylospermum elongatum

1 Introduction

Campylospermum elongatum (Oliv.) Tiegh, a plant of the Ochnaceae family, is represented as shrubs growing up to 2.5 m high under dense secondary forests, well exposed to sunlight [1]. In Cameroun, this plant is found in the eastern and southern regions where natives of the Baka pigmy tribe use the leaves and alcohol to prepare medicinal portions destined to remedy many health problems such as palpitations, heart pain, and stomach disorders. According to our knowledge, no previous phytochemical work has been reported on this species.

2 Results and discussion

In the continuation of the phytochemical investigations of our local plants used in folk medicine, we have isolated and characterized from the leaves of C. elongatum 11 secondary metabolites (Figure 1). The structures of these compounds were established from a complete spectroscopic study (IR, UV, RMN, and MS), as well as by comparing the obtained data with literature. These include the biflavonoids: robustaflavone 1; 4′-O-methyl robustaflavone 2; 4′,4″′-di-O-methyl robustaflavone 3; 7,4′,4″-tri-O-methyl robustaflavone 4; 4′,7″-di-O-methyl robustaflavone 5; amentoflavone 6; 7-O-methyl amentoflavone 7; 7,7″-di-O-methyl amentoflavone 8; 7,4′,7″-tri-O-methyl amentoflavone 9; and a cyano-glycoside, lithospermoside 10 [2], [3], [4], [5], [6], [7], [8], [9]. All were earlier reported as secondary metabolites from other sources with important biological activities [10], [11], [12], [13], [14], [15].

Figure 1:

Structures of compounds 1–11.

Equally was obtained compound 11 as an amorphous pale yellow solid for which the molecular formula C₂₂H₂₂O₁₀ was assigned in conformity with its high- resolution mass spectrum in which the [M+H]⁺ peak appeared at m/z 447.1282. This compound is a flavonoid glycoside since it gave a dark coloration with aqueous FeCl₃ solution, a brick red coloration with Mg turnings in the presence of concentrated HCl [16] and a violet coloration with Molish reagent [17].

The IR spectrum was consistent with that of a flavonoid glycoside as it displayed important absorption bands for hydroxyl and phenol functions (3318 and 3120 cm⁻¹), the chromanone carbonyl (1642 cm⁻¹), the conjugated double bond (1622 cm⁻¹), and aromatic rings (1601 and 1596 cm⁻¹) [18].

The UV spectrum of compound 11 confirmed the implication of the flavone motif as it displayed two intense bands at 257 and 349 nm attributed to the benzoyl and the cinnamoyl chromophores, respectively [19]. Evidence of the absence of free OH groups at positions 4′ and 7 was obtained when the benzoyl band was stable with methanol as solvent and the cinnamoyl band varied very little with addition of NaOAc. On the other hand, the presence of a free hydroxyl group at the position 5 of the flavone moiety was confirmed by the bathochromic shift of 16 nm of the benzoyl band with the addition of HCl.

The ¹H NMR (1D and 2D) spectra of compound 11 confirmed the presence of the flavone motif as the characteristic signals of protons corresponding to the four different proton systems on the flavone nucleus were identified (Table 1). These included a proton singlet at δ_H 6.76 (1H, s, H-3); an AB system of two meta coupled protons on ring A at δ_H 6.44 (1H, d, 2.1 Hz, H-6) and at δ_H 6.82 (1H, d, 2.1 Hz, H-8); an AA′BB′ system of four protons on a para disubstituted benzene ring B at δ_H 7.90 (2H, d, 9.0 Hz, H-2′/H-6′) and at δ_H 6.90 (2H, d, 9.0 Hz, H-3′/H-5′). In addition, two singlet signals observed at δ_H 3.86 ppm (3H, s, CH₃-O-4′) and at δ_H 12.96 (1H, s, OH-5) were accounted for by an O-methyl substituent on ring B and a much deshielded phenol proton peri to the chromanone carbonyl function. The connections displayed in the NOESY spectrum between the protons of methoxy group at δ_H 3.86 and the two aromatic protons at δ_H 6.90 (H-3′/H-5′, ring B), suggested the implication of 4′-O-methylapigenin as aglycone in the structure of 11.

In addition, the signals of a system of seven sugar protons observed at δ_H 5.34 (1H, d, 7.8 Hz, H-1″), 3.58 (1H, dd, 7.8 and 9.1 Hz, H-2″), 3.34 (1H, dd, 3.0 and 9.1 Hz, H-3″), 3.64 (1H, dd, 3.0 and 3.3 Hz, H-4″), 3.31 (1H, ddd, 3.3, 5.6 and 6.1 Hz, H-5″), 3.37 (1H, dd, 6.1 and 10.3 Hz, H-6″a) and 3.45 (1H, dd, 5.6 and 10.3 Hz, H-6″b) correspond to those reported for galactose [21].

These structural elements were confirmed when after the acid hydrolysis of 11, the spectral data of the aglycone was very similar to those described for 4′-O-methylapigenin [20] while those of the sugar were identical to the values obtained for a commercial sample of d-galactose.

The ¹³C NMR spectrum of 11 ( Table 1) displayed signals indicating the presence of all the 20 two carbon atoms given in the molecular formula among which were identified seven quaternary carbon atoms, 10 methines, a methyl, and a methylene groups. In its HSQC spectrum, the signal of the anomeric carbon C-1″ of the sugar appeared at δ_C 102.2, that of the methylene carbon at δ_C 60.1, while those of the other sugar carbons appear at δ_C 71.4, 77.6, 68.4, and δ_C 76.2. All these values correspond to those of the carbon atoms in galactose [20]. Signals of protonated carbon atoms on the aglycone include that of the methoxy carbon at δ_C 56.6, the carbons C-3, C-6, and C-8 of the chromanone moiety that appear, respectively, at δ_C 102.6, 98.7, and 94.1. HMBC correlations confirm that the signals at δ_C 182.2, 163.8 and at 161.5 are those of the carbonyl C-4, and the carbon atoms C-7 and C-4′, respectively.

The position of the glycosidic bond was deduced from the connections observed in the HMBC spectrum of 11 which included a correlation between the anomeric proton of the sugar H-1″ (δ_H 5.34) and the carbon C-7 of the aglycone at δ_C 163.8. This suggests that the glycosidic bond is between the anomeric carbon C-1′ (δ_C 102.2) and the oxygen on the aglycone carbon C-7 (δ_C 163.8). This was confirmed by the NOESY spectrum of 11 that displayed cross peaks between the anomeric proton (δ_H 5.34) and the aglycone protons H-6 (δ_H 6.44) and H-8 (δ_H 6.82), thus establishing its structure as 4′-O-methyl-7-O-β-d-galactosylapigenin. This glycoside had earlier been characterized as an active anti-HIV agent in Chrysanthemum morifolium [22], but the given spectroscopic information is incomplete with several overlapping peaks and missing coupling constants. Our report gives both the complete assignments of all the chemical shifts of the protons and carbon atoms in the molecule of 4′-O-methyl-7-O-β-d-galactosylapigenin and all the proton–proton coupling constants thus completing this earlier reported data.

3 Materials and methods