Antibacterial activity of ethanolic extract and compounds from fruits of Tectona grandis (Verbenaceae)

Melting points of the isolated compounds were determined using an Electrothermal IA9000 Series digital melting point apparatus (Bibby Scientific, Great Britain). MS detection was carried out using a Waters Micromass ESI-Q-TOF II instrument with ESI ionization in the positive mode. EIMS spectra were recorded on a Finnigan MAT 95 spectrometer (70 eV) with perfluorokerosine as reference substance for HR-ESI-TOF-MS (Japan). IR spectra were recorded on a Shimadzu FTIR-8400S spectrophotometer (Japan). UV spectra were recorded on a Shimadzu UV-160A spectrometer (Japan) in absolute ethanol and alkaline ethanol. The NMR spectra were measured on Bruker 500 MHz NMR Avance II spectrometer equipped with cryoprobe, with TMS as an internal reference. Chemical shifts were recorded in δ (ppm) and the coupling constants (J) are in hertz (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.

Dried fruits of Tectona grandis (2.5 Kg) were extracted with ethanol (10 L) for 72 h at room temperature to yield a crude extract (55 g) after evaporation under reduced pressure. This extract (50 g) was subjected to silica gel column chromatography eluted with gradients of n-hexane-EtOAc and EtOAc-MeOH. Ninety fractions of 300 mL each were collected using mixtures of n-hexane-EtOAc 85:15, 70:30, 30:70 and combined on the basis of their TLC profiles into four main fractions coded A-D (A: 1–19; B: 20–46; C: 47–68; D: 69–90). Fraction A (20 g) contained mostly fatty material and was not further investigated. Fraction B (6.5 g) was separated by a column chromatography over silica gel using a gradient of n-hexane-EtOAc (100:0, 95:5, 90:10, 85:15, 80:20, 75:25 and 70:30) to afford five sub-fractions (FrB1-FrB5). Following their TLC profiles, only FrB3 was retained for further purification over silica gel column chromatography with n-hexane-EtOAc to afford 2β-hydroxyursolic acid (3) (10 mg). Fraction C (10 g) was subjected to column chromatography over silica gel eluted with n-hexane-EtOAc (90:10, 85:15, 80:20, 75:25 and 70:30). The collected fractions which contained the major compound 6-methyl-1,4-dihydroxyanthraquinone (2) were combined and applied on a Sephadex LH-20 column (n-hexane-dichloromethane-methanol, 7:4:0.5) to give 8 mg while the remaining complex material was kept aside for further investigation. Similary, repeated column chromatography of fraction D (2 g) yielded tectograndone (1) (8 mg).

Tectograndone (1): Red powder in acetone, ¹³C NMR (CDCl₃- DMSO-d ₆ , 125 MHz) δ (ppm) : 187.5 (C-1’), 185.5 (C-6), 183.8 (C-11), 188.2 (C-4’), 157.9 (C-8’), 157.2 (C-5’), 155.8 (C-5), 150.7 (C-12a), 149.0 (C-12), 145.7 (C-8), 138.4 (C-9), 135.6 (C-2’), 132.4 (C-10), 132.3 (C-3), 131.5 (C-6’), 130.2 (C-10a), 128.9 (C-7), 128.1 (C-7’), 126.6 (C-3’), 125.8 (C-6a), 117.0 (C-4a), 115.7 (C-4), 113.4 (C-11a), 111.6 (C-4a’), 79.8 (C-2).

6-methyl-1,4-dihydroxyanthraquinone (2) : Red powder in acetone, ¹³C NMR (DMSO-d ₆ , 125 MHz) δ (ppm) : 186.6 (C-9), 186.3 (C-10), 156.6 (C-1), 156.6 (C-4), 146.0 (C-6), 135.6 (C-7), 132.6 (C-5a), 130.4 (C-8a), 129.2 (C-2), 129.1 (C-3), 126.6 (C-5), 126.6 (C-8), 112.6 (C-4a), 112.4 (C-9a).

2β-hydroxyursolic acid (3) :White powder in MeOH, ¹³C NMR (DMSO-d ₆ , 125 MHz) δ (ppm) : 178.1 (C-28); 138.2 (C-13); 124.4 (C-12); 79.0 (C-3); 64.5 (C-2); 52.3 (C-5); 47.5 (C-18); 46.8 (C-1); 46.7 (C-17); 41.6 (C-9); 40.1 (C-14); 39.9 (C-4); 38.4 (C-8); 38.3 (C-20); 37.8 (C-19); 37.6 (C-10); 36.2 (C-22); 32.5 (C-7); 30.1 (C-21); 28.7 (C-23); 27.3 (C-15); 23.7 (C-16); 23.2 (C-27, C-11); 22.8 (C-30); 20.9 (C-6); 17.5 (C-24); 16.8 (C-26, C-29); 16.4 (C-25).

The qualitative analysis of the ethanol extract of the teak fruit was also conducted by using the method described by Harbone (1973) [15] with slight modifications.

The inoculum was prepared as described by Tereshuck et al. [16] from 24 h old cultures by picking numerous colonies and suspending them in sterile saline (NaCl, 0.9 %) solution. Absorbance was read at 530 nm and adjusted with the saline solution to match to that of a 0.5 McFarland standard solution, corresponding to about 1.5 × 10⁸ Colony Forming Units (CFU).

The antibacterial activity was investigated by determining the minimum inhibitory concentrations (MICs) and the minimum bactericidal concentrations (MBCs). MICs were determined by a broth micro-dilution method with slight modification of the method described by Newton et al. (2012) [17].

Stock solutions of the extract and compounds were prepared in the Mueller Hinton Broth (MHB) (Titan Biotech Ltd Rajasthan India) in 5 % (v⁄v) dimethylsulfoxide (DMSO) solution (Fisher chemicals, Strasbourg, France) for a final concentration of 4096 μg/mL and 1024 μg/mL respectively for extract and compounds.

Into each well of 96-microplate (Nunclon, Roskilde, Denmark) 100 μL of MHB and 100 μL of the test substance solution were introduced. Twofold were serial dilutions was made to obtain a concentration range of 8–1024 μg/mL for crude extract and 8–256 μg/mL compounds. Bacterial inoculums (400 μL) prepared above was added to MHB (15 mL) for a final concentration of 4 × 10⁶ CFU/mL which was used for this test. One hundred microliters of this inoculum was introduced to each well containing 100 μL of MHB and extract mixture to a final volume of 200 μL. The final concentration of DMSO in the well was less than 1 % (preliminary analyses show that 1 % (v/v) DMSO does not inhibit the growth of the test organisms). A sterility check (5 % DMSO, media, inoculum and water soluble antibiotic) was included in the experiment. The plates were covered with a sterile lid, and incubated at 35 °C for 24 h under shaking using a plate shaker (Flow Laboratory Germany) at 300 rpm. After this incubation, the MICs were assessed by adding 40 μL of 2 % solution of p-iodonitrotétrazolium (INT) (Sigma-Aldrich, South Africa) in each well. Viable bacteria cause the appearance of pink coloration in the presence of this solution [18]. The concentration that did not show the appearance of pink solution was considered as the inhibition concentration and the smallest one was noted as the MIC. For the well that did not present color changes, 50 μL aliquots of solution of the corresponding well which did not receive INT were put into the well of a new plate containing 150 μL of freshly prepared MHB and re-incubated at 35 °C for 48 h on the shaker. After this re-incubation, 40 μL of INT were introduced in each well and all the concentrations that did not present color change were considered as the bactericidal concentration and the smallest one was noted as MBC. The assay was repeated thrice. Ciprofloxacine at the concentration range of 0.039-5 μg/mL served as positive control.

In this study, the phytochemical composition of the fruits extract of T. grandis was evaluated and we investigated the antibacterial activity of extract together with isolated compounds investigated. Qualitatively, the crude extract contains anthraquinones, polyphenols, sterols, triterpenes and tannins but not flavonoïds, xanthocyanates and saponins (Table 1).

Moreover, the results shown in Table 2 demonstrate that the ethanol extract of teak fruit displayed good activities against Escherichia coli (ATCC 8739), Pseudomonas aeruginosa (PA 01), Klebsiella pneumonia (ATCC 11296) and Escherichia aerogenes (ATCC 13048) with the MIC values ranging from 64 to 256 μg/mL. The isolated compounds(1–3) showed potent antibacterial activity toward these microorganisms. This supports the use of this plant in traditional medicine in the treatment of skin diseases. Compounds 1 and 2, with the same basic quinoidal skeletal units, were the most active against E. coli (32 μg/mL) and E. aerogene (16 μg/mL) respectively. In addition, compound 2 also presented a considerable antibacterial activity against P. aeruginosa (128 μg/mL).