Formiside and seco-formiside: lignin glycosides from leaves of Clerodendrum formicarum Gürke (Lamiaceae) from Cameroon

1 Introduction

Clerodendrum L., a famous plant of the family Lamiaceae, is a large and diverse genus having 580 species found in Africa, Australia, America and Asia. Members of the genus are being used as medicines in Indian, Chinese, Thai, Korean and Japanese systems of medicine for the treatment of life-threatening diseases such as jaundice, cancer, typhoid and hypertension. Many species of the genus Clerodendrum are known for potent bioactivities. A decoction of C. phlomidis has been reported to have antidiabetic activity [1]. Central nervous system (CNS)-related activities were also observed in the same plant showing CNS depressant, tranquillizing and muscle relaxant in experimental mice and rats [2]. C. bungei shows antitumor activity in hepatic cells of mice [3]. C. inerme has been used as an antioxidant drug in various indigenous systems of medicines [4]. The alcoholic extracts of C. phlomidis exhibit antimalarial activity against Plasmodium falciparum [5]. Various species of Clerodendrum have also been tested for antimicrobial potential [6]. The major chemical components reported from the genus Clerodendrum are neolignans [7], flavonoids [8], flavonoid glycosides [9], chalcones [10], steroids [11], steroidal glycosides [12], iridoids [13], iridoid glucosides [14], terpenes [15] and macrocyclic alkaloids [16].

In continuation of our search for new natural products from higher plants, we have reported terpenoids [17], aromatic acids [18] and long-chained feruloyl esters [19] from C. formicarum. The present communication describes the isolation and characterization of two new lignin glycosides, named formiside (1) and seco-formiside (2), from the leaves of C. formicarum, collected from Obili-Yaounde (Cameroon).

2 Results and discussion

The crude ethanol soluble part of C.formicarum leaves was chromatographed using combinations of hexane-ethyl acetate and ethyl acetate-methanol as a mobile phase. Fractions eluted with 20% methanol in ethyl acetate were pooled and rechromatographed over Sephadex LH-20 using water–methanol (1:1) and finally purified by reversed phase high-performance liquid chromatography (HPLC). Water–methanol (2:3) was used as a mobile phase. Compounds 1 and 2 were obtained as viscous gums.

The IR spectrum of 1 displayed three prominent absorption bands at 3487, 1664 and 1590 cm⁻¹ attested for hydroxyl, ester carbonyl and aromatic carbon–carbon double bond functions in the molecule, respectively. Its molecular formula C₃₉H₄₈O₁₈ was determined on the basis of high-resolution electronspray impact mass spectroscopy (HR-ESIMS), which showed a pseudo molecular ion peak at m/z=805.2890 (calcd. 805.2919, [M+H]⁺). The parent skeleton of compound 1 was found to be similar with lariciresinol [20]. This was attested by the presence of diagnostic peaks in the ¹H and ¹³C NMR spectra.

The ¹H and ¹³C NMR spectra of 1 displayed signals of six aromatic methines at δ_H/δ_C=6.84 (d, J=1.2 Hz)/110.8, (CH-2); 6.72 (d, J=8.0 Hz)/116.1, (CH-5); 6.73 (d, J=8.0 Hz)/119.6, (CH-6); 6.63 (d, J=1.6 Hz)/113.0, (CH-2′); 6.67 (d, J=8.0 Hz)/116.0, (CH-5′) and 6.50 (dd, J=8.0, 1.6 Hz)/121.9, (CH-6′); three methylens at δ_H/δ_C=2.68 (dd J=13.4, 4.0 Hz), 2.50 (dd, J=13.4, 10.8 Hz)/33.8, (CH₂-7′); 3.63 (dd J=9.2, 8.0 Hz), 3.56 (dd, J=9.2, 6.5 Hz /69.2, (CH₂-9); 3.74 (dd J=8.5, 6.5 Hz), 3.56 (dd, J=8.5, 5.8 Hz /73.5, (CH₂-9); three methines δ_H/δ_C=4.82 (d, J=5.5 Hz)/85.5, (CH-7); 2.33 m/51.4, (CH-8); 2.44 m/43.7, (CH-8′); and six quaternary carbons at δ_C=136.2/C-1, 148.8/C-3, 146.7/C-4, 133.4/C-1′, 148.7/C-3′ and 154.7/C-4′. These data clearly indicated that lariciresinol is a part of molecule.

¹H and ¹³C NMR spectra also indicated the presence of two sugar units and a 3-methoxy, 4-hydroxy benzoyl moiety. The ¹H and ¹³C NMR spectra of 1 displayed signals for glucose at δ_H/δ_C=4.31 (d, J=7.2 Hz)/103.2, (CH-1″); 3.52 (t, J=7.2 Hz)/78.3, (CH-2″); 3.24 (t, J=7.2 Hz)/78.1, (CH-3″); 3.29 (t, J=7.2 Hz)/71.8, (CH-4″); 3.30 m/78.0, (CH-5″) and 3.86 (dd, J=11.4, 2.0 Hz), 3.66 (dd, J=11.4, 5.8 Hz /62.8, (CH₂-6″); signals for apiose at δ_H/δ_C=5.46 br s /109.6, (CH-1″′); 3.87 br. s/78.4, (CH-2″′); 4.31 (d, J=10.0 Hz), 3.61 (d, J=10.0 Hz)/75.5, (CH₂-4″′); 4.03 (d, J=10.5 Hz), 3.61 (d, J=10.5 Hz)/69.0, (CH₂-5″′); and a quaternary carbon at δ_C 79.2 (C-3″′). The ¹H and ¹³C NMR values of sugar units were assigned by the help of a 1D-total correlation spectroscopy experiment. These sugars were assigned as β-d-glucose and β-d-apiose by comparing their ¹³C NMR chemical shifts with reported values in the literature [21] and coupling constant values of anomeric protons. The ¹H and ¹³C NMR spectra of 1 also displayed peaks for a 3-methoxy, 4-hydroxy benzoyl moiety resonating at δ_H/δ_C=7.49 (d, J=1.2 Hz)/113.7, (CH-2″″); 6.77 (d, J=8.8 Hz)/110.4, (CH-5″″); 7.48 (dd, J=8.8, 1.2 Hz)/125.4, (CH-6″″) and four quaternary carbons at δ_C=123.0 (C-1″″), 153.4 (C-3″″), 148.9 (C-4″″) and 168.0 for carbonyl carbon of benzoyl ester. A complete picture of NMR spectral data is given in Table 1.

Position and connectivity of sugars and 3-methoxy, 4-hydroxy benzoyl moiety were determined with the aid of heteronuclear multiple-bond correlations (HMBCs). HMBC correlation between δ 3.52 and anomeric carbon of apiose C-1″′ (δ 109.6) clearly indicated the position of apiose at C-2″. Similarly, HMBC correlation of H-5″′ (δ 4.03/3.61) and H-6″″ (δ 7.48) to ester carbonyl carbon (δ 168.0) confirmed that 3-methoxy, 4-hydroxy benzoyl fragment substituted at C-5″″. Key HMBC and correlation spectroscopy (COSY) correlations in compound 1 are shown in Fig. 1. The stereochemistry in compound 1 was determined by nuclear Overhauser effect spectroscopy (NOESY) experiments and its circular dichroism (CD) data were compared with previously reported compounds in the literature [22]. In the NOESY spectrum, H-8 showed a correlation with H-8′, but there is no NOESY correlation between H-8 and H-7. This clearly indicates that H-8 and H-8′ are on the same side, whereas H-7 and H-8 are on the opposite sides of the five-membered ring. The CD spectrum of 1 showed a positive Cotton effect at around 280 nm [Δε=1.95] and a negative Cotton effect at around 250 nm [Δε=–9.36] [22].

Fig. 1:

Important HMBC and COSY interactions in 1.

The molecular formula C₃₉H₅₀O₁₈ of compound 2 was determined on the basis of HR-ESIMS, which showed a pseudo molecular ion peak at m/z=829.2824 (calcd. 829.2895 for [C₃₉H₅₀O₁₈+Na]⁺). Its IR spectrum exhibited absorption bands at 1591 (aromatic C=C), 1665 (conjugated ester carbonyl) and 3403 (OH) cm⁻¹.

The ¹H and ¹³C NMR spectra were found similar to compound 1, with only the difference in the chemical shift of C-7. In compound 1, this was methine resonated at δ_H/δ_C=4.82 (d, J=5.5 Hz)/85.5, whereas in compound 2, it was methylene resonated at δ_H/δ_C=2.51 dd (J=13.8, 6.6 Hz), 2.63 dd (J=13.8, 7.8 Hz)/35.7. This clearly indicated that compound 2 is a seco form of compound 1. Ether linkage between C-7 and C-9′ has disconnected which is further supported by HMBC correlation in compound 1 where HMBC correlation between H₂-9′ and C-7 was observed, whereas the same was absent in compound 2. Key COSY and HMBC correlations in compound 2 are shown in Fig. 2. Stereochemistry in compound 2 was determined by comparing its CD spectrum with previously reported compounds in the literature [22]. The CD spectrum of 2 showed two negative Cotton effects at around 286 nm [Δε=–5.80] and at 234 nm [Δε=–3.17], which are in accordance with values reported in the literature.

Fig. 2:

Important HMBC and COSY interactions in 2.

Both lignin glycosides are new additions to the list of natural products and named formiside (1) and seco-formiside (2).

3 Experimental section