A novel antimicrobial flavonoid from the stem bark of Commiphora pedunculata (Kotschy & Peyr.) Engl.

A new flavonoid, 2-(3,5-dihydroxy-4-methoxy-phenyl)-3,5-dihydroxy-8,8-dimethyl-2,3-dihydro-8H-pyrano[3,2]chromen-4-one, together with previously reported epicatechin was isolated from the ethyl acetate soluble fraction of the methanol extract of the stem bark of Commiphora pedunculata. The structures of these compounds were elucidated based on extensive analysis of their spectral data, including 1 and 2D NMR. The compounds were active against 9 out of 12 tested microorganisms including a resistant strain; vancomycin-resistant entrococci (VRE), Escherichia coli, Staphylococcus aureus and Candida albicans. The zones of inhibition ranged between 22 and 34 mm against the microorganisms. The minimum inhibitory concentration was as low as 6.25 μg/mL against Shigella dysentriae, Bacillus cereus and S. aureus while the minimum bactericidal concentration was as low as 50 μg/mL against Pseudomonas aeruginosa, VRE and C. albicans. This is the first report of the isolation of the compound.


5'
A new flavonoid, 2-(3,5-dihydroxy-4-methoxy-phenyl)-3,5-dihydroxy-8,8-dimethyl-2,3-dihydro-8H-pyrano [3,2]chromen-4-one, together with previously reported epicatechin was isolated from the ethyl acetate soluble fraction of the methanol extract of the stem bark of Commiphora pedunculata. The structures of these compounds were elucidated based on extensive analysis of their spectral data, including 1 and 2D NMR. The compounds were active against 9 out of 12 tested microorganisms including a resistant strain; vancomycin-resistant entrococci (VRE), Escherichia coli, Staphylococcus aureus and Candida albicans. The zones of inhibition ranged between 22 and 34 mm against the microorganisms. The minimum inhibitory concentration was as low as 6.25 mg/mL against Shigella dysentriae, Bacillus cereus and S. aureus while the minimum bactericidal concentration was as low as 50 mg/mL against Pseudomonas aeruginosa, VRE and C. albicans. This is the first report of the isolation of the compound.

Introduction
Commiphora pedunculata (Kotschy & Peyr.) Engl. belongs to Burseraceae, a family composed of both trees and shrubs of tropical and sub-tropical geographical distribution (Watson & Dallwitz 1992). Over 200 species of Commiphora are recorded to occur in Africa, Arabia, the Indian Ocean Islands and India (Weeks et al. 2005). The Burseraceae family members (also known as the frankinscence or Myrhh family) are characterised by the non-allergic resin produced in virtually all plant tissues and the distinctive smooth, yet flaking aromatic bark (Judd et al. 2008). The resins are of substantial economic, medicinal and cultural value (Langenheim 2003). Traditionally, the stem and root are chewed for treating coughs and the decoction of the leaves and stem bark is used for the treatment of dysentery and diarrhoea (Baba Mai Wada, personal communication). Recently, the antimicrobial activity of the hexane, ethyl acetate and methanol extracts of the stem bark of C. pedunculata was reported , also two flavonoids with antimicrobial activity have been isolated from the ethyl acetate soluble fraction of the methanol extract of the plant . Other species of Commiphora are known to produce flavonoids and other phenolic compounds (Hanuš et al. 2005). In this paper, we report the isolation and characterisation of a new flavonoid: 2-(3,5-dihydroxy-4-methoxyphenyl)-3,5-dihydroxy-8,8-dimethyl-2,3-dihydro-8H-pyrano[3,2]chromen-4-one together with a known flavonoid (epicatechin) from the ethyl acetate soluble fraction of the methanol extract of the stem bark.

Results and discussion
Compound 1 was isolated as pale yellow crystals. It gave a positive result in the test for flavonoids (Harborne & Mabry 1975). Compound 1 was assigned the molecular formula C 21 H 20 O 8 based on HR-ESI-MS m/z at 401.3787 cald. 400.4896. The existence of a l max at 265 nm (band II) and a shoulder peak at 320 nm (band I) in the UV spectrum of compound 1 is an indication that it might be a dihydroflavonol (Bohm 1999). The IR spectrum of compound 1 showed a very broad band at 3350 cm 21 , characteristic of OZH stretching for phenolic hydroxyl groups, the band at 1680 cm 21 is characteristic of carbonyl stretching while the bands observed in the fingerprint region are due to various CZH stretching and bending modes (Wade 2006). The proton NMR spectrum of compound 1 revealed signals for a pair of ortho-coupled unsaturated protons at d H 6.61 (1H, d, J ¼ 10.0 Hz, H-4 00 ) and 5.61 (1H, d, J ¼ 10.0 Hz, H-3 00 ), these signals are typical of the dimethyl substituted pyran ring of 2H-1-benzopyran skeleton (Ellis 1977;Waffo et al. 2000). In addition, two aromatic signals at d H 6.55 (2H, s H-2 0 , 6 0 ) and 5.92 (1H, s, H-8) were revealed by the proton NMR, they were assigned to a tetra-substituted ring B and penta-substituted ring A of a flavonoid. Further observed in the proton NMR of compound 1 are two oxygenated methine proton signals at d H 4.54 (1H, d, J ¼ 11.4 Hz, H-3) and d H 4.90 (1H, d, J ¼ 11.4 Hz, H-2), a methoxy proton signal at d H 3.83 (3H, s, HZOCH 3 ) and a methyl signal at d H 1.43 (6H, s, H-1 000 , 2 000 ).
Compound 2 was isolated as a brown solid. The five aromatic proton signals of compound 2 were assigned to an AB type ring A [d H 5.96 (2H, d, J ¼ 2.1 Hz, H-6), 5.94 (2H, d, J ¼ 2.0 Hz, H-8)] and an ABX type ring B [d H 6.9 (2H, d, J ¼ 1 Hz, H-2 0 ), 6.7 (2H, d, J ¼ 8.1 Hz, H-5 0 ), 6.8 (2H, dd, J ¼ 8.4 and 1.6 Hz, H-6 0 )] (Kazuo et al. 1999). Ring A therefore is a 1,3,4,5-tetrasubstituted benzene ring while ring B is a 1,3,4-tri-substituted benzene ring. The signals between 6.7 and 6.9 ppm are characteristic of the ABX spin pattern in the ring B of epicatechin (Antonelli et al. 2007). Also the two methylene protons at d H : 2.8 ppm (dd, J ¼ 16.76 and 4.5 Hz, ax.) and 2.7 ppm (dd, J ¼ 16.8 and 2.6 Hz, eq.) with their small J values are typical of position 4 of epicatechin. The close assemblage of signals about 156 ppm in the 13 C spectrum of compound 2 is characteristic of positions 5, 7 and 9 of the ring A of epicatechin. A comparison of the 13 C NMR data of compound 2 with data reported in the literature for epicatechin showed very close agreement, for example, 29.3 ppm (C-4), 79.9 ppm (C-2), 67.5 ppm (C-3), 115.9 ppm (C-5 0 ) and 119.4 ppm (C-6 0 ) (Markham & Ternai 1976). Co-TLC of 2 with standard sample of epicatechin showed the two to have the same R f value. The structure of 2 was therefore elucidated as epicatechin (Figure 1).
The result of the antimicrobial activity of the compounds is shown in Table 1. Twelve microorganisms were screened, nine bacteria (including vancomycin resistant enterococci (VRE)) and three fungi. Compound 2 was active against 8 out of the 12 tested microorganisms while compound 1 was active against 6. The highest inhibition zone was observed with 2 against Shigella dysentriae (34 mm), followed by Bacillus cereus (32 mm) and Staphylococcus aureus (27 mm). The highest inhibition zone observed for compound 1 was against S. aureus (29 mm), Escherichia coli (27 mm) and Candida albicans (25 mm). VRE, S. aureus, E. coli, C. albicans and C. krusei showed sensitivity to both compounds, with inhibition zones of 24, 29, 27, 25 and 22 mm for compound 1 and 24, 27, 25, 25 and 24 mm for compound 2 against the microorganisms, respectively. The two compounds were inactive against Streptococcus pyogenes, Proteus mirabilis and Candida tropicalis.
The zones of inhibition observed with the compounds were slightly lower than those of the standard drugs used (Ciprofloxacin and Fluconazole). The minimum inhibitory concentration (MIC) value was lowest against S. dysentriae, S. aureus and B. cereus (6.25 mg/mL) with compound 1. Other MIC values were also considerably low at 12.5, 25.0 and 50 mg/mL for both compounds. The low MIC values suggest that the compound possesses good antimicrobial activity, considering that compounds with MIC values less than 100 mg/mL are regarded as having strong antimicrobial activity (Tang et al. 2003). Although the antimicrobial activity of epictatechin has been previously investigated (Betts et al. 2011), this will be the first report of its efficacy against VRE, also this is the first report of the antimicrobial activity of the isolated new flavonoid. The antimicrobial activity observed with the isolated compounds might be due to the hydroxyl groups present on their structures.

Plant sample
The plant material was collected from the bushes around Basawa in Zaria, Kaduna State, Nigeria in the month of October 2011. It was authenticated by Mallam Galla at the herbarium unit of the Biological Sciences Department, Ahmadu Bello University Zaria-Nigeria, where a sample of it was deposited and assigned the specimen voucher number 219. The stem bark was separated from the stem, air-dried for 21 days and crushed to a coarse powder.

Extraction and isolation
The dried powder (500 g) was extracted with methanol (2.5 L) in a Soxhlet apparatus for 48 h at 658C. After evaporation of the solvent, the concentrated methanol extract was separated into water-soluble and water-insoluble portions. The water-soluble portion (1 L) was further partitioned with n-hexane (2.5 L) and ethyl acetate (5 L) to give 2.0 g of the n-hexane fraction and 14.7 g of the ethyl acetate fraction after removal of the solvents. A portion of the ethyl acetate fraction (7.5 g) was chromatographed on silica gel column eluting with hexane 100%, hexane -ethyl acetate mixtures (80:20, 60:40 and 30:70), ethyl acetate 100% and methanol 100% as solvent systems to give 25 fractions. The 25 fractions were pooled together based on similarity in their TLC profile to give four sub-fractions. Repeated Sephadex LH-20 gel filtration chromatography (eluting with MeOH) of sub-fraction 2 led to the isolation of compounds 1 and 2 (6.8 and 7.4 mg, respectively).

Antimicrobial assay
The isolated compound was tested for antimicrobial activity against clinical isolates of VRE, S. dysentriae, S. aureus, S. pyogenes, S. pneumonia, E. coli, B. cereus, P. mirabilis, Pseudomonus aeruginosa, C. albicans, C. tropicalis and Candida krusei, as described previously (Volleková et al. 2001;Bonev et al. 2008;Londonkar et al. 2013). Ciprofloxacin and fluconazole were used as positive standards. The agar well diffusion and broth dilution methods were used. The standardised inocula of the isolates were uniformly streaked onto freshly prepared Mueller Hinton agar plates. Using a sterile cork borer (6 mm in diameter), appropriately labelled wells were punched into each agar plate. Then 0.2 mL of the appropriate compound concentration was added into each well and then allowed to diffuse into the agar. The plates were incubated at 378C for 24 h for bacteria, while the incubation period was 48 h at 258C for fungi. The antimicrobial activities were expressed as diameter of inhibition zones produced by the compounds. The experiment was done in duplicates. The MICs of the compounds were determined using the broth dilution method. Various concentrations of the compound (50 -3.125 mg/mL) were prepared by twofold serial dilution in test tubes containing Mueller Hinton broth. The organisms (0.2 mL) were inoculated into each tube containing the compound. The tubes were incubated at 378C for 24 h for bacteria and 48 h at 258C for fungi. The lowest concentration in the series showing no visible growth of the test organisms was considered to be the MIC. The contents of the MIC tubes in the serial dilution were sub-cultured onto appropriately labelled Mueller Hinton agar plates and incubated at 378C for 24 h, then they were observed for colony growth. The lowest concentration of the sub-culture with no growth was considered as the minimum bactericidal concentration (MBC).

Conclusions
This study is a continuation of our investigations into the chemistry of C. pedunculata from Nigeria. Interestingly it led to the isolation of a new flavonoid with antimicrobial activity, indicating that the ethnomedicinal claims ascribed to the plant have scientific backing.

Supplementary material
Supplementary material relating to this paper is available online, alongside Table S1 and Figures S1 -S8.