PHYTOTHERAPY RESEARCH Phytother. Res. 28: 784–787 (2014) Published online 22 August 2013 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.5053

SHORT COMMUNICATION Flavonoid glycosides from the stem bark of discoidea demonstrate antibacterial and free radical scavenging activities

Edmund Ekuadzi,1 Rita Dickson,1 Theophilus Fleischer,2 Kofi Annan,2 Dominik Pistorius,3 Lukas Oberer4 and Simon Gibbons5* 1Department of Pharmacognosy, Faculty of Pharmacy and Pharmaceutical Sciences, College of Health Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana 2Department of Herbal Medicine, Faculty of Pharmacy and Pharmaceutical Sciences, College of Health Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana 3Natural Product Unit, Novartis Institutes for Biomedical Research, Basel 4056, Switzerland 4Analytical Sciences, Novartis Institutes for BioMedical Research, Basel 4056, Switzerland 5Department of Biological and Pharmaceutical Chemistry, UCL School of Pharmacy, 29-39 Brunswick Square, London WC1N 1AX, UK

One new flavonoid glycoside, along with three known flavonoid glycosides were isolated from the stem bark of , which is traditionally used in the management of wounds and skin infections in Ghana. The new flavonoid glycoside was elucidated as hydroxygenkwanin-8-C-[α-rhamnopyranosyl-(1 → 6)]-β- glucopyranoside (1) on the basis of spectroscopic analysis. The isolated compounds demonstrated free-radical scavenging as well as some level of antibacterial activities. Microorganisms including Staphylococcus aureus are implicated in inhibiting or delaying wound healing. Therefore, any agent capable of reducing or eliminating the microbial load present in a wound as well as decreasing the levels of reactive oxygen species may facilitate the healing process. These findings therefore provide some support to the ethnopharmacological usage of the in the management of wounds. Copyright © 2013 John Wiley & Sons, Ltd.

Keywords: Margaritaria discoidea; flavonoid glycosides; margadiscoside; fagovatin; antibacterial activities; antioxidant activities.

INTRODUCTION MATERIALS AND METHODS

Margaritaria discoidea (Baill.) G.L. Webster (), General experimental procedures. NMR spectra were previously referred to as discoideus, is a tree that recorded on a Bruker AV-III-600 spectrometer equipped finds use in ethnomedicine as a treatment against wounds and with a 1.7 mm TXI cryoprobe. The mass spectra were infectious skin diseases (Abbiw, 1990; Irvine, 1961). Earlier performed by ESI in positive-ion mode using an LTQ phytochemical investigationshaveshownthepresenceof Orbitrap XL mass spectrometer (ThermoScientific). IR eight alkaloids (Fehler, 2000; Mensah et al., 1988; spectra were measured as a solid film with a Bruker Weenen et al., 1990) and betulinic acid (Calixto et al., 1998). Hyperion 2000 FTIR microscope connected to a Vertex In an earlier work, we reported the antibacterial, antioxidant 70 spectrometer. Flash chromatography was carried out and anti-inflammatory activities of the 70% ethanol extract on RediSep Rf normal phase disposable columns. Prep of the M. discoidea leaves and stem bark (Dickson et al., LC/MS was carried out on an Agilent Technologies 1200 2010). Here, we identify and characterize the compounds series Chromatograph fitted with an Agilent 1100 series that may be contributing to the biological activities LC/MSD quadrupole mass spectrometer. observed in our earlier study. The isolation, structure elucidation, antioxidant and antibacterial activities of one new flavone glycoside (1) along with three known Plant material. The stem bark of M. discoidea was flavonoid glycosides (2, 3 and 4) are reported herein for collected from Kente in the Amansie Central District the first time in this species. The structure of compound in June 2009. A voucher specimen (KNUST/HM1/2010/ 1 was established using NMR spectroscopy, IR, UV and S003) has been deposited at the Department of Pharma- MS data (Fig. 1). cognosy Herbarium. Extracts of Margaritaria discoidea are well known as wound healing remedies in Ghana. The presence of these compounds may contribute in part to the wound healing benefits derived from the plant when used traditionally. Extraction and isolation of compounds. The dried pow- dered stem bark (2.5 kg) was successively Soxhlet-extracted using petroleum ether (50.50 g), EtOAc (208.75 g) and 70% * Correspondence to: Professor Simon Gibbons, Department of Biological and Pharmaceutical Chemistry, UCL School of Pharmacy, 29–39 Brunswick EtOH (386.25 g). The resulting extracts were evaporated Square, London WC1N 1AX, UK. on a rotary evaporator under reduced pressure at a temper- Email: [email protected] ature of less than 40 °C. 20 g of the 70% EtOH extract was Received 26 March 2013 Revised 06 June 2013 Copyright © 2013 John Wiley & Sons, Ltd. Accepted 08 July 2013 ANTIBACTERIAL AND ANTIOXIDANT FLAVONOID GLYCOSIDES 785

polarities to yield fractions A (0.07 g), B (1.06 g), C (3.72 g), D (2.89 g) and E (3.66 g). Fraction D was subjected to prep-LC/MS using a SunFire C-18 Optimal Bed Density column (150 × 30 mm; 5 μm particle diameter; Waters) with a flow rate of 60 mL/min and a mobile phase of H2O and MeCN, each containing 0.1% HCOOH, with a linear gradient from 10% to 30% MeCN in 15 min to yield fractions D1 (58.20 mg), D2 (18.05 mg) and D3 (9.30 mg). Fraction D3 afforded pure compound 2 (9.30 mg; 0.0072%w/w). Further fractionation of D1 and D2 on a Luna Phenyl-Hexyl column (150 × 21 mm; 5 μm particle diameter; Phenomenex) with a flow rate of 24 mL/min and a mobile phase of H2O and MeCN, each containing 0.1% HCOOH, with a linear gradient from 15% to 40% MeCN in 15 min yielded compounds 3 (7.50 mg; 0.0058%w/w), 4 (7.88 mg; 0.0061%w/w) and 1 (1.42 mg; 0.0011% w/w), respectively.

Bioactivity of isolated compounds. Broth microdilution assay. Minimal inhibitory concentration values of the compounds were determined based on a broth micro-well dilution method (Eloff, 1998). The iocula of microorganisms Figure 1. Selected COSY (bold faced bonds) and HMBC (arrows) were prepared from 12-h broth cultures, and serial dilutions correlations of compound 1. were made to achieve a suspension of approximately 105 CFU/mL. Growth of the microorganisms was deter- mined by adding 20 μL of a 5% solution of tetrazolium salt and incubating for a further 30 minutes. Amoxycillin was included as the positive control. All experiments Table 1. Antibacterial activity of isolated compounds were carried out in triplicate. MIC (μg/mL)

Microorganisms 12 3 4 Free radical scavenging activity (DPPH method). The isolated compounds were compared to n-propyl gallate Gram-positive (in methanol), and the DPPH method (Blois, 1958) Bacillus subtilis NCTC 10073 500 500 >500 >500 was used. A 20 mg/L solution of DPPH in methanol Staphylococcus aureus 500 500 >500 >500 was prepared, and 3 mL of this solution was added to ATCC 25923 1 mL each of the test compounds at 1, 0.5, 0.1 and 0.05 mg/mL in methanol. After 3 min, the absorbance Gram-negative was measured at 517 nm using a T90+ UV/VIS Proteus vulgaris NCTC 4635 500 500 500 500 > > Spectrometer (PG Instruments Limited). 1 mL of methanol Pseudomonas aeruginosa 500 500 500 500 was added to 3.0 mL DPPH solution which served as a ATCC 27853 negative control. All experiments were carried out in All experiments were carried out in triplicates. MIC readings for all wells triplicate. Inhibition of radical scavenging was calculated were the same. 200 μg/mL of amoxicillin served as positive control. according to the following equation: MIC readings for all wells of the same compound were identical.

% DPPH scavenging activity ¼ ½ðÞA0 A1 =A0 x 100

Table 2. IC50 values (μg/mL) for free radical scavenging activity with A being the absorbance of the control, and A , the of the isolated compounds 0 1 absorbance in the presence of the test sample. Data were

Compounds IC50 DPPH (μg/mL) presented as % DPPH scavenging effect against concen- tration and the IC50 determined. 1 17.45 2 20.34 3 68.94 4 24.88 RESULTS AND DISCUSSION n-Propyl gallate 7.983

All experiments were carried out in triplicates. n-Propylgallate Compound 1 was obtained as a yellow amorphous solid. served as positive control. Its IR spectrum exhibited absorption bands for an α, β- unsaturated ketonic functional group (1652 and 1607 cm1) and hydroxyl groups (3381 cm1). The UV subjected to silica gel Flash Chromatography (≤4bar)using spectrum of 1 in methanol displayed two major a ternary gradient consisting of C6H14, EtOAc and MeOH absorption peaks at 245.4 (with a shoulder) and starting with nonpolar conditions and gradually of increasing 346.2 nm +indicative of a flavone natural product with

Copyright © 2013 John Wiley & Sons, Ltd. Phytother. Res. 28: 784–787 (2014) 786 E. EKUADZI ET AL.

Table 3. 1H and 13C NMR chemical shifts of compounds 1 and 2 (in DMSO-d6, 600 MHz)

12

Chemical shifts Chemical shifts Chemical shifts Chemical shifts Carbon no. (ppm) 1H NMR (ppm) 13C NMR (ppm) 1H NMR (ppm) 13C NMR

2 - 164.3; 164.1 - 165.5; 164.0 3 6.70 102.5 6.88 (s); 6.86 (s) 103.4 4 - 182.0; 181.7 - 182.5; 182.0 5 - 160.3; 159.5 - 159.5; 160.5 6 6.78; 6.80 91.0; 90.1 - 110.0 7 - 164.8; 163.6 - 165.3; 164.0 8 - 109.5; 109.4 6.85 (s); 6.84 (s) 91.0; 92.0 9 - 156.8; 156.6 - 156.8; 157.0 10 - 107.0 - 104.0; 104.5 C-1′ - 104.6; 104.1 - 121.0 C-2′ 7.42 112.8 7.98 (d, 7.3 Hz) 129.0 C-3′ - 146.1 6.95 (d, 8.8 Hz) 116.0 C-4′ - 156.8 - 161.7 C-5′ 6.83 115.8 6.95 (d, 8.8 Hz) 116.0 C-6′ 7.45 119.4 7.98 (d, 7.3 Hz) 129.0 8-C-Glc 6-C-Glc C- 1″ 4.58 (d, 9.9 Hz); 4.60 (d, 9.9 Hz) 72.9 4.58 (d, 9.9 Hz); 4.60 (d, 9.9 Hz) 73.0; 72.5 C- 2″ 3.97 (t, 8.8, 8. 8 Hz); 4.17 (t, 8.8, 8.8 Hz) 70.2 3.97 (t, 8.8, 8. 8 Hz); 4.17 (t, 8.8, 8.8 Hz) 70. 2 C- 3″ 3.20 (m); 3.22 (m) 70.7 3.20 (m); 3.22 (m) 79.5 C- 4″ 3.10 (m); 3.05 (m) 70.6 3.10 (m); 3.05 (m) 71.0 C- 5″ 3.17 (m); 3.15 (m) 78.9; 78.8 3.17 (m); 3.15 (m) 81.0 C- 6″ 3.88 (m); 3.30 (m) 66.6; 66.6 3.88 (m); 3.30 (m) 68.0 O-Rha-״O-Rha 6-״6 C – 1 4.51(d, 4.8 Hz) 100.6; 100.3 4.51(d, 4.8 Hz) 101.5 C – 2 3.73 (m) 71.9 3.73 (m) 70.0 C – 3 3.35 (m) 79.9 3.35 (m) 79.5 C – 4 3.14 (m) 70.5 3.14 (m) 70.8 C – 5 3.56 (d, 5.5 Hz) 70.2 3.56 (d, 5.5 Hz) 69.0 C – 6 1.12 (d, 6.2 Hz) 1.09 (d, 5.9 Hz) 17.9 1.12 (d, 6.2 Hz) 1.09 (d, 5.9 Hz) 18.2

7-OCH3 3.85 (s); 3.90 (s) 56.5; 56.3 3.88 (s); 3.90 (s) 57.0 5-OH 13.55 - 13.48 -

a3′,4′-ora3′,4′,5′-oxygenation pattern in the B-ring The HMBC spectrum of 1 showed 2 J and 3 J correla- (Mabry et al., 1970). The high-resolution MS signal at tions between the anomeric proton of Glc, H-1#x02BA; + m/z 609.18140 [M + H] , in combination with the ob- (δH 4.58) and C-8 (δC 104.6/104.0) and C-7 (δC164.8/ served isotopic intensities was indicative for a molecular 163.6), confirming C-8 as the position of the C-glycosidic formula of C28H32O15. linkage. Thus, the singlet at δH 6.78/6.80 was unambiguously The 1Hand13C NMR spectra of 11 showed duplica- assigned to H-6. The site of substitution of the additional tion of signals revealing two rotamers which slowly α-rhamnose moiety was confirmed to be the 6ʺ-OH of the interconverted via rotation about the C-glycosidic Glc moiety which was β-linked to the flavone aglycone. bond (Lewis et al., 2000). The 1HNMRspectrumof1 This was further confirmed by HMBC correlations showed signals at δH 6.83 (d), 7.42 (s) and 7.45 (d) between H-1‴ (δH 4.51) and C-6#x02BA; (66.59/66.57). assignable to H-5′,H-2′ and H-6′,respectively, On the basis of the above evidence, the flavonoid confirming that ring B was a 3′,4′-oxygenated phenyl glycoside was characterized as hydroxygenkwanin-8-C- moiety. A singlet at δH 3.85 indicated the presence of [α-rhamnopyranosyl-(1 → 6)]-β-glucopyranoside and trivially one methoxy group which was assignable to position called margadiscoside. 1 7. The H NMR spectrum also showed a signal at δH The known compounds, genkwanin-6-C-[α-rhamnopyranosyl- 6.70 that was characteristic for the H-3 resonance of (1 →6)]-β-glucopyranoside (2)(Qasimet al., 1987), kaempferol- a flavone. A broad singlet at δH 13.55 was due to the 3-O-α-rhamnopyranosyl-(1 → 2)-β-glucopyranoside-7-O- 5-OH group which formed a hydrogen bond with the α-rhamnopyranoside (3) (Mulinacci et al., 1995) and kaempferol- carbonyl group at C-4 of the flavone. The 1HNMR 3-O-α-rhamnopyranosyl-(1 →2)-[α-rhamnopyranosyl-(1 →6)]- spectrum also showed the presence of sugar moieties β-glucopyranoside-7-O-α-rhamnopyranoside (4)(Kiteet al., in 1 with two anomeric proton signals at δH 4.58 2007) are reported for the first time in this species. Also 13 (d, J =9.9 Hz) and δH 4.51 (d, J =4.8Hz).Thesignalat being reported for the first time are the full C NMR data δH 4.51 together with a methyl resonance at δH 1.15 of compound 2. (3H, d, 6.2 Hz) were both typical of a rhamnopyranosyl The antibacterial activity of compounds 1–4 is shown in residue (Yahara et al., 2000). Table 1. All of the test compounds exhibited weak

Copyright © 2013 John Wiley & Sons, Ltd. Phytother. Res. 28: 784–787 (2014) ANTIBACTERIAL AND ANTIOXIDANT FLAVONOID GLYCOSIDES 787 antibacterial activities with their minimum MIC values Hydroxygenkwanin-8-C-[α-rhamnopyranosyl (1 → 6)]- being 500 μg/mL. Even though the antibacterial activity is β-glucopyranoside (1) not high, the results could be said to be interesting as the various compounds isolated may be said to be contributing Yellow amorphous compound, UV: λmax (MeOH) 245.4 individually to the antibacterial effects observed. In the (with a shoulder) and 346.2 nm. IR: νmax 1652 and 1607 free radical scavenging assay using DPPH, the flavonoid (α, β-unsaturated ketonic function) and hydroxyls 3381 glycosides displayed predictably intense effects (Table 2). (O–H) cm1. 1H and 13C NMR data: Table 3. Flavonoids have been reported to exhibit free radical scavenging properties (Pietta, 2000). The wound healing process may be hampered by microbial infection and Genkwanin-6-C-[α-rhamnopyranosyl (1 → 6)]-β- oxygen free radicals leading to the formation of chronic glucopyranoside (2) non-healing ulcers (Houghton et al., 2005). For the isolated compounds to have shown antibacterial and Brown amorphous compound, UV: λmax (MeOH) 270.7 antioxidant activities, they may reduce the possibility of and 337.3 nm. IR: νmax 1652 and 1608 (α, β-unsaturated 1 1 this happening. Thus, the antibacterial and antioxidant ketonic function) and hydroxyls 3393 (O–H) cm. H properties of the isolated compounds, however mild, and 13C NMR data: Table 3. may be contributing in part to the wound healing effects of the plant. The presence of betulinic acid and the Acknowledgement alkaloids, securinine and phyllantidine, which have been isolated from the ethyl acetate fraction of the plant, was Novartis is hereby gratefully acknowledged for sponsoring Edmund reported in literature to possess antibacterial, antioxi- Ekuadzi to participate in the Novartis Diversity and Inclusion Next dant and anti-inflammatory effects (Nguemfo et al., Generation Scientist Programme in their Natural Product Unit in 2009; Fulda et al., 1999; Schuhly et al., 1999; Wachter Basel, Switzerland. et al., 1999, Nick et al., 1995; Mensah et al., 1990 & 1988). These may also be contributing to the wound Conflict of Interest healing effects of the plant as observed in folk medicine in Ghana. The authors have declared that there is no conflict of interest.

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