Ericoside, a new antibacterial biflavonoid from Erica mannii (Ericaceae)

Fitoterapia 109 (2016) 206–211

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Ericoside, a new antibacterial biflavonoid from Erica mannii (Ericaceae) Gabin Thierry M. Bitchagno a, Simplice B. Tankeo b, Apollinaire Tsopmo c, James D. Simo Mpetga a, Alembert T. Tchinda d, Serge Alain T. Fobofou a,e, Antoine Honoré L. Nkuete a, Ludger A. Wessjohann e, Victor Kuete b, Pierre Tane a,⁎ a

Department of Chemistry, University of Dschang, P.O. Box 67, Dschang, Cameroon Department of Biochemistry, University of Dschang, P.O. Box 67, Dschang, Cameroon Food Science and Nutrition, Department of Chemistry, Carleton University, Ottawa, ON, Canada d Institute of Medical Research and Medicinal Plants Studies (IMPM), P.O. Box 6163, Yaounde, Cameroon e Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Weinberg 3, 06120 Halle (Saale), Germany b c

a r t i c l e

i n f o

Article history: Received 25 October 2015 Received in revised form 13 December 2015 Accepted 15 December 2015 Available online 21 January 2016 Keywords: Ericaceae Erica mannii Biflavonoid Ericoside Antibacterial activity

a b s t r a c t A new dihydroflavonol–flavonol biflavonoid derivative, named ericoside was isolated from the ethanol extract of the whole plant of Erica mannii along with the known flavonoid, taxifolin 3-O-α-L-rhamnopyranoside; and two readily available sterols (sitosterol, sitosterol 3-O-β-D-glucopyranoside). The isolation was performed using chromatographic methods and the structure of purified molecules were elucidated using spectroscopic techniques (e.g. MS, NMR) and by comparison with literature data. The crude ethanol extract, ericoside, and taxifolin 3-O-α-L-rhamnopyranoside were tested against ten Gram-negative bacteria including multidrug resistant clinical isolates using a broth microdilution method. The crude ethanol extract showed no noteworthy activity. Of the purified compounds, ericoside displayed moderate activity against the resistant Escherichia coli AG100 with a MIC of 64 μg/mL. © 2016 Elsevier B.V. All rights reserved.

1. Introduction

2. Materials and methods

Erica mannii is one of the 700 species of the genus Erica (Ericaceae family), many of which are distributed throughout Europe, the Middle East and Africa. Traditionally uses such as hypotensive, antiinflammatory, urinary antiseptic, diuretic [1], and wounds healing [2] have been reported for members of this genus. Erica species are chemotaxonomically characterized based on their flavonoids [3] although, they are also known to possess 1,9-diarylnonanoids [4], phenylpropanoids [5], triterpenoids [6], and tannins [7,8]. In the course of our ongoing research on new bioactive compounds from Cameroonian medicinal plants, we have undertaken the isolation of the chemical constituents of E. mannii. We herein report, the isolation, the characterization and antibacterial activity of a new dihydroflavonol– flavonol biflavonoid derivative. Moreover, this is the first report of a biflavonoid from the genus Erica.

2.1. General experimental procedures

⁎ Corresponding author. E-mail address: [email protected] (P. Tane).

http://dx.doi.org/10.1016/j.fitote.2015.12.022 0367-326X/© 2016 Elsevier B.V. All rights reserved.

High resolution ESI mass spectra and the corresponding higher collision dissociation (HCD) measurements (normalized collision energy 50%) were obtained on an Orbitrap Elite™ mass spectrometer (ThermoFisher Scientific, Bremen, Germany) equipped with an HESI ion source (spray voltage 4 kV; capillary temperature 275 °C, source heater temperature 40 °C; FTMS resolution 30.000). Nitrogen was used as the sheath gas. Sample solutions were introduced continuously via a 500 μL Hamilton syringe pump at 5 μL/min. Data were evaluated by Xcalibur™ software 2.7 SP1. LC-ESI/MS was performed using a Waters 2795 separations module coupled to a 2998 photodiode array detector and Waters Micromass Quatro Ultima triple quadrupole mass spectrometer. The column was Phenomenex Kinetix C18 (100 × 4.60 mm, 2.6 μm, 100 Å (Torrance, California)) and the mobile phase consisted of acetonitrile–water (ACN–H2O) with formic acid (FA); [0.1%, (v/v)]. The solvent gradient was linear programmed from 5 to 100% ACN over 15 min at a flow rate of 1.0 mL min−1. Positive ESI conditions included: capillary voltage 3.50 kV, cone voltage 20 V, source temperature 80 °C, desolvation temperature 180 °C, cone gas flow (N2) 90 L/h, desolvation

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gas flow (N2) 540 L/h, and multiplier voltage of 650 V. Compounds were injected at a concentration of 100 μg/mL. Data were collected from 100 to 1500 Da and processed using MassLynx 4.0 (Waters, Milford, MA). UV spectra were obtained online in the range 200–400 nm using the 2998 photodiode array detector. IR spectra were obtained on a Shimadzu FTIR-8400S spectrophotometer (Japan). NMR experiments (1H, 13C, HSQC, HMBC, COZY) were recorded using a Bruker Avance 400 Spectrometer (Milton, On) operating at 400.13 for 1H and 100 MHz for 13C and comprising a 5 mm auto-tuning broadband probe with a Z-gradient. Samples were dissolved in methanol-d4 (CDN Isotopes, Point Claire, Quebec). Experiments were performed at room temperature and the spectra were calibrated based on the residual non-deuterated solvent (δH 3.31 and δC 49.1). Chemical shifts were recorded in δ (ppm) and the coupling constants (J) are in Hz. Silica gel 60 F254 (70–230; Merck; Darmstadt, Germany) was used for column chromatography. Precoated silica gel Kieselgel 60 F254 plates (0.25 mm thick) were used for TLC, and spots detected by spraying with 50% H2SO4 followed by heating at 100 °C.

2.2. Plant material The whole plant of E. mannii was collected in the Bamboutos Mountain, West Region of Cameroon in December 2010. The identification was done at the Cameroon National Herbarium (Yaoundé) by comparison with the voucher specimen kept under the number 60598 HNC.

2.3. Extraction and purification Dried materials of E.mannii (1.5 Kg) were ground and extracted with ethanol (10 L) for 72 h at room temperature to yield a crude extract (150 g) after evaporation under reduced pressure. 140 g of this extract were suspended in water and successively extracted with n-Hexane, EtOAc and n-butanol to afford the corresponding extracts weighing 20 g, 40 g and 60 g respectively. The n-Hexane and EtOAc extracts were combined on the basis of their TLC profiles. Fifty five grams of the resulting extract were subjected to silica gel column chromatography eluted with gradients of n-hexane-EtOAc and EtOAc-MeOH as mobile phases. 85 fractions of 300 mL each were collected and combined on the basis of their TLC profiles (using mixtures of n-hexane-EtOAc 85:15, 70:30, 30:70) into four main fractions coded A–D (A: 1–12; B: 13–38; C: 39–73; D: 74–85). Fraction A (10 g) contained mostly lipids and was not investigated further. Fraction B (8 g) was separated by column chromatography over silica gel using a gradient of n-HexaneEtOAc (100:0, 95:5, 90:10, 85:15, 80:20, 75:25 and 70:30) to afford sitosterol (10 mg) and a mixture of non-resolved triterpenes. Fraction C (10 g) was separated by column chromatography over silica gel using a gradient of CH2Cl2–MeOH (100:0, 95:5, 90:10, 85:15, 80:20, 75:25 and 70:30) to afford sitosterol 3-O-β-D-glucopyranoside (10 mg) and taxifolin 3-O-α-L-rhamnopyranoside (2, 23 mg). Fraction D (20 g) was subjected to silica gel column chromatography using CH2Cl2–MeOH as solvent and gave seven sub-fractions (D1–D7). D3 was purified over silica gel followed by Sephadex LH-20 column chromatography with an isocratic CH2Cl2–MeOH (70:30) solvent system to give ericoside (1, 25 mg).

2.4. Ericoside (1) Yellow amorphous powder, λmax (log ε): 230 (0.64), 291 (0.64), and 340 (3.72); IR (KBr) νmax: 3548, 3236, 1652, 1595, 1500, 1475, 1452, 1 13 C NMR 1174, and 825 cm−1; [α]24 D — 96.8, (c. 0.64, MeOH); H and − see Table 1; (−) HRESIMS: [M + OH] at m/z 865.2166 (calcd. for C42H41O20 865.2197); and (+) ESIMS: [M + Na-2 H]+ at m/z 869.

207

Table 1 1 H and 13C NMR data for compound 1 (400 and 100 MHz, in CD3OD), δ in ppm, J in Hz. Position

2 3 4 5 5a 6 7 8 8a 9 10/14 11/13 12 15 16 17 18 19 20 2′ 3′ 4′ 5′ 5a′ 6′ 7′ 8′ 8a′ 9′ 10′/14′ 11′/13′ 12′ 15′ 16′ 17′ 18′ 19′ 20′

1 δC

δH

HMBC (H→C)

83.6 78.5 196.5 165.3 102.0 97.2 168.5 96.1 163.9 128.4 129.8 116.3 159.1 102.4 72.0 72.2 73.4 70.8 18.1 159.2 136.0 179.4 163.0 105.7 99.6 165.7 94.6 158.3 122.4 131.7 116.2 161.4 103.6 72.3 72.4 74.0 72.1 17.8

5.12, d (10.5) 4.59, d (10.5) – – – 5.89, d (2.0) – 5.86, d (2.0) – – 7.33, d (8.5) 6.81, d (8.5) – 3.99, d (1.0) 3.50, dd (1.0; 3.1) 3.64, dd (3.1; 9.5) 3.32, m 4.25, m 1.19, d (6.2) – – – – – 6.17, d (2.0) – 6.35, d (2.0) – – 7.74, d (8.5) 6.92, d (8.5) – 5.37, d (1.5) 4.21, dd (1.5; 3.2) 3.70, m 3.30, m 3.28, m 0.90, d (5.6)

3, 4, 8a, 10/14 2, 4, 9, 5a – – – 4, 8, 7, 5 – 6, 7, 8a – – 2, 10/14, 12, 11/13 9, 11/13, 12 – 3, 19 17 16 17 17, 20 19 – – – – – 4′, 5′, 5a′, 7′, 8′ – 4′, 7′, 8a′, 5a′, 6′ – – 2′, 10′/14′, 11′/13′, 12′ 9′, 10′/14′, 11′/13′, 12′ – 3′, 18′ 17′ 18′ 19′ 17′, 20′ 18′

2.5. Taxifolin 3-O-α-L-rhamnopyranoside (2) Yellow amorphous powder; IR (KBr) 3571, 3309, 1645, 1600, 1515, 1475, 1178, and 821 cm− 1; 1H NMR (400 MHz, CD3OD): δ (ppm) 11.85 (1 H, s, OH-5); 6.95 (1 H, d, J = 8.0 Hz, H-6′); 6.82 (1 H, d, J = 8.0 Hz, H-5′); 6.81 (1 H, brs, H-2′); 5.91 (1 H, d, J = 1.5 Hz, H-6); 5.89 (1 H, d, J = 1.5 Hz, H-8); 5.07 (1 H, d, J = 11.0 Hz, H-2); 4.57 (1 H, d, J = 11.0 Hz, H-3); 4.25 (1 H, dd, J = 6.0; 9.5 Hz, H-5″); 3.99 (1 H, d, J = 1.0 Hz, H-1″); 3.50 (1 H, brd, J = 3.0 Hz, H-2″); 3.30 (1 H, dd, J = 3.0; 9.5 Hz, H-3″); 3.65 (1 H, m, H-4″); 1.19 (1 H, d, J = 6.5 Hz, H-6″); 13 C NMR (100 MHz, CD3OD): δ (ppm) 196.5 (C-4); 169.1 (C-7); 166.0 (C-5); 164.6 (C-8a); 147.9 (C-4′); 147.1 (C-3′); 129.6 (C-1′); 121.1 (C5′); 116.7 (C-2′); 116.0 (C-6′); 103.0 (C-5a); 102.6 (C-1″); 97.9 (C-6); 96.7 (C-8); 84.4 (C-2); 79.1 (C-3); 72.3 (C-2″); 72.6 (C-3″); 74.3 (C-4″); 71.0 (C-5″); 18.2 (C-6″); and (+)-ESIMS m/z (rel. Int): 901 [2 M + H]+ (100). 2.6. Antibacterial assays 2.6.1. Bacterial strains, culture media and chemicals Ten Gram-negative multi-drug resistant bacterial strains from laboratory UMR-MD1 of the University of Mediterranean in Marseille (France) and from the American Type Culture Collection (ATCC) were used. These bacteria included five strains of Escherichia coli composed of two reference strains (ATCC8739 and ATCC10536) and three clinical isolates (AG100, AG102 and AG100ATet), one strain of Enterobacter aerogenes (ECCI69), two strains of Klebsiella pneumoniae (KP55 and

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Fig. 1. Structures of ericoside (1) and taxifolin 3-O-α-L-rhamnopyranoside (2).

ATCC11296) and two strains of Providencia stuartii (ATCC29916 and NAE16). Their bacterial features have been reported previously [9,10, 11]. Two culture media, the Mueller Hinton Agar (MHA) for bacterial activation, and Mueller Hinton Broth (MHB) for the determination of the antibacterial parameters (minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC)) were used. Dimethylsulfoxide (DMSO) was used to dissolve the crude extract and compounds; p-iodonitrotetrazolium (INT) was used to detect the bacterial growth. These substances were purchased from SigmaAldrich (St. Quentin Fallavier, France). 2.6.2. Susceptibility assay The bacterial susceptibility was determined using the microplate dilution method as described by Newton et al. (2002) [12]. The crude extract and isolated compounds were dissolved in DMSO (2.5% W/V) in sterile MHB to obtain a stock concentration of 4096 μg/mL (crude extract), 1024 μg/mL (compounds 1 and 2) and 512 μg/mL (ciprofloxacin). Ciprofloxacin served as the positive control. 100 μL of each solution was added in the first columns of a 96-well microplate initially containing 100 μL of MHB. Serial dilutions of each sample were made yielding new concentrations in the range of 2048 to 16 μg/mL (crude extract), 256 to 8 μg/mL (compounds 1 and 2), and 256 to 4 μg/mL (ciprofloxacin). Then, one hundred microliter (100 μL) of bacterial inoculum (1.5 × 108 CFU/mL obtained from a McFarland turbidity standard n° 0.5 and a final concentration of 106 CFU/mL) prepared in MHB was added to each well containing the samples (100 μL) and mixed thoroughly to give final concentrations ranging from 1024 to 8 μg/mL for

the crude plant extract, 128 to 4 μg/mL for compounds 1 and 2, and 128 to 2 μg/mL for ciprofloxacin. The negative control consisted of wells containing MHB, DMSO (2.5%) and inoculum. DMSO at that concentration did not show inhibitory effects on the growth of the tested bacteria. Microplates were covered, sealed with parafilm and incubated at 37 °C for 18 h. Bacterial growth was detected by adding 40 μL of INT (0.2 mg/mL) to all wells by incubation at 37 °C for 30 min [13], during which viable bacteria reduced the yellow dye to a pink color. The MIC was noted as the lowest concentration of sample that prevented this change of color and exhibited complete inhibition of bacterial growth. The MBC was determined by adding 50 μL of the content of the wells (which did not received INT and which corresponded to those which had not shown any bacterial growth during MIC assay) to 150 μL of MHB contained in new microplates. After 48 h of incubation at 37 °C, the MBC was defined as the lowest concentration of sample that prevented any change of color after adding 40 μL of INT as described above. All tests were made in triplicate. 3. Results and discussion 3.1. Isolation and determination of structures Four compounds including a new biflavonoid (Fig. 1) were isolated from the ethanol extract of the whole plant of E. mannii through silica gel and Sephadex LH-20 column chromatography. Their structures were determined by interpretation of their spectroscopic data and by comparison with values previously reported in the literature for similar or related molecules.

Fig. 2. MS2 fragmentation behavior of ericoside (1) under negative ion electrospray conditions.

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209

Fig. 3. Selected HMBC correlations of ericoside (1).

Ericoside (1) was obtained as a yellow powder in CH2Cl2, [α]24 D — 96.8, (c. 0.64, MeOH). The molecular formula C42H40O19 was deduced from the negative ion FT-HRESI-MS which revealed an ion peak [M + OH]− at m/z 865.2166 (calcd. for C42H40O19 865.2197). In positive mode, ESI-MS analysis showed an ion peak [M + Na-2 H]+ at m/z 869. Collision induced dissociation (MS2) of the fragment ion at m/z 865 afforded two predominant fragment ions at m/z 433 [M-415]− and 431 [M-417]− (Fig. 2) corresponding to the loss of dihydroflavonol ([M-415]−) and flavonol ([M-417]−) moieties, respectively. The infrared spectrum exhibited a broad band absorption centered at 3236 cm− 1 attributed to one or more hydroxyl groups. The 1H NMR spectrum of 1 revealed the presence of two p-disubstituted benzene rings characterized by signals at δH 7.74 (d, 2 H, J = 8.5 Hz), 7.33 (d, 2 H, J = 8.5 Hz), 6.92 (d, 2 H, J = 8.5 Hz), and 6.81 (d, 2 H, J = 8.5 Hz) assigned to aromatic methines H-10′/H-14′, H-10/H-14, H-11′/H-13′, and H-11/H-13 respectively. In addition, the 1H spectrum also displayed resonances of four meta coupled doublets at δH 6.35 (d, 1 H, J = 2.0 Hz, H-8′); 6.17 (d, 1 H, J = 2.0 Hz, H-6′); 5.89 (d, 1 H, J = 2.0 Hz, H-6) and

5.86 (d, 1 H, J = 2.0 Hz, H-8), together with two aliphatic doublets of a dihydroflavonol moiety at δH 5.12 (d, 1 H, J = 10.5 Hz, H-2) and 4.59 (d, 1 H, J = 10.5 Hz, H-3) [14,15]. A β-axial position was attributed to H-2 with a trans-diaxial relationship to H-3 as suggested by the value of their coupling constant (J = 10.5 Hz). The 13C NMR/APT spectrum displayed resonances of forty-two carbons (Table 1) including two carbonyl groups at δC 196.5 (C-4) and 179.4 (C-4′) characteristics of a dihydroflavonol and a flavonol moieties respectively [15,16]. Two sugar moieties were identified as L-rhamnose by comparison of their 13 C NMR data [δC 102.4 (C-15), 72.0 (C-16), 72.2 (C-17), 73.4 (C-18), 70.8 (C-19), 18.1 (C-20) and 103.6 (C-15′), 72.3 (C-16′), 72.4 (C-17′), 74.0 (C-18′), 72.1 (C-19′), 17.8 (C-20′)] with those reported in the literature [16,17,18]. An α-orientation was attributed to the anomeric protons of each sugar moieties at δH 5.37 (d, 1 H, J = 1.5 Hz, H-15′) and 3.99 (d, 1 H, J = 1.0 Hz, H-15) on the basis of their coupling constants [16,17,18]. The linkage of the rhamnose units at the C-3 position of each flavonoid unit is common in the genus Erica [3,16] and was established by the HMBC spectrum which shows interactions

Fig. 4. Important NOESY correlations of ericoside (1).

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Table 2 Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBC) of crude extract and isolated compounds 1 and 2 from E. mannii against bacterial strains. Bacterial strains

Substances (μg/mL)

Reference antibiotic

Crude extract

1

2

Ciprofloxacin

MIC

MBC

MIC/ MBC

MIC

MBC

MIC/ MBC

MIC

MBC

MIC/ MBC

MIC

MBC

MIC/ MBC

Escherichia coli ATCC8739 ATCC10536 AG100Atet AG100 AG102

– 1024 – 1024 –

nt – nt – nt

nd N1 nd N1 nd

N128 128 N128 64 N128

nt – nt – nt

nd N1 nd N2 nd

– N128 – N128 –

nt – nt – nt

nd N1 nd N1 nd

b1 1 64 16 4

4 4 128 64 16

N4 4 2 4 4

Klebsiella pneumoneae ATCC11296 KP55

– 512

nt –

nd N2

128 N128

– nt

N1 nd

N128 N128

– –

N1 N1

b1 4

128 16

N128 4

Providencia stuartii ATCC29916 NAE16

1024 1024

– –

N1 N1

N128 N128

nt nt

nd nd

N128 –

– nt

N1 nd

32 128

256 256

8 2

Enterobacter cloacae ECCI69

–

nt

nd

N128

nt

nd

–

nt

nd

256

N256

N1

nt: not tested; nd: not determined; –: MIC or MBC greater than 1024 μg/mL for crude extract and 128 μg/mL for isolates compounds.

from H-15 to the carbon at δC 78.5 (C-3) and H-15′ to the carbon at δC 136.0 (C-3′) (Fig. 3). Further important HMBC correlations are depicted in Fig. 3. The HR-MS and MS/MS fragmentations of ericoside (1, Fig. 2) in addition to the down-field resonance of C-12/C-12′ (δC 159.1/161.4) signals suggest that the two flavonoid units are linked via the C-12 and C-12′ through an ether bridge. These carbons resonate at δC 157– 158 when bearing hydroxyl substituents [19,20,21]. The relative configuration of the dihydroflavonol moiety was elucidated by NOESY experiment along with the analysis of the vicinal 1H–1H coupling constants. Interaction revealed by the NOESY spectrum from H-3 to H-15 indicates that these protons are located in the same side of the molecule. Important NOESY correlations are depicted in Fig. 4. Compound 1 was therefore characterized as a new compound to which the trivially name ericoside (1) is given. The known compounds taxifolin 3-O-α-L-rhamnopyranoside (2), sitosterol, and sitosterol 3-O-β-D-glucopyranoside were identified by comparison of their spectroscopic data with those reported in the literature [22,23].

membranes [27]. Polyphenols can have tanning effects on proteins and bacteria resulting in unspecific toxic effects however, responses of bacteria to the tested samples varied from one microorganism to another indicating that unspecific tanning effects were not dominant.

4. Conclusions This study presents the characterization of a new antibacterial biflavonoid from the whole plant of E. mannii. To the best of our knowledge, this is the first report on the antibacterial activity of the crude extract and compounds from this plant species. The overall results of this study can be considered promising in view of the development of new phytodrugs to fight against MDR bacterial infections of public health importance since the tested microorganisms are highly resistant to antibiotics. However, future work is needed to clarify if the activity is really based on hitting a specific bacterial target or if general and unspecific tanning and protein denaturing properties of these compounds are responsible for antibiotic effects in vitro.

3.2. Antibacterial activity The crude ethanol extract, ericoside (1), and taxifolin 3-O-α-Lrhamnopyranoside (2) were tested for their activity against ten Gramnegative bacterial strains and the results are summarized in Table 2. The ethanol extract (crude) displayed moderate activity against K. pneumoniae KP55 (MIC 512 μg/mL) and very weak to no activity on other bacteria (MIC ≥ 1024 μg/mL). Compound 1 had a significantly greater activity against E. coli AG100 (MIC 64 μg/mL) and weak activity against E. coli ATCC10536 (MIC 128 μg/mL) and K. pneumoniae ATCC11296 (MIC 128 μg/mL), but it did not show any activity against other tested bacteria. Compound 2 showed weak activities against all tested bacteria (MIC N 128 μg/mL). The antimicrobial activity of the crude extract and pure compounds was appreciated according to established cutoff points [24]. Previous studies have reported that the number and position of hydroxyl groups in phenolic compounds such as flavonoids can significantly influence their antimicrobial activity [14,25,26]. In addition, the antibacterial profile of the tested samples was established using Gram-negative strains which presented a remarkable resistance mechanism than that of Gram-positive bacterial strains. The moderate or no activity found with compound 1 may then be due to both of these factors. It is also possible that compound 1 poorly interacts with membranes as it has been suggested that polar and large molecular weight compounds were not easily penetrate bacterial cell

Authors' contributions GTMB and SBT were field investigators and drafted the manuscript. AHLK collected the plant material. JDSM contributed to the interpretation of NMR data and correction of the manuscript. VK provided the bacterial strains and chemicals for antibacterial assays. AT, SAFT, ATT, and LAW participated in spectroscopic and physical data analysis and with VK revised and finalized the manuscript. PT designed and supervised the work.

Competing interests The authors declare that they have no competing interests.

Acknowledgments We are grateful to the University of Dschang for financing some consumables used in this work. We thank Dr. Jürgen Schmidt (IPB, Halle) for HR-MS measurements. SAFT is grateful to the German Academic Exchange Service (DAAD) for the financial support.

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