Antibacterial Activities of Pyrenylated Coumarins from the Roots of Prangos hulusii

The dichloromethane extract of the roots of Prangos hulusii, a recently described endemic species from Turkey, has yielded nine known and one new prenylated coumarins. The structures were elucidated by spectroscopic methods and direct comparison with the reference compounds where available. The root extract and its prenylated coumarins exhibit antimicrobial activity against nine standard and six clinically isolated strains at a concentration between 5 and 125 µg/mL. In particular, the new coumarin, 4′-senecioiloxyosthol (1), displayed 5 µg/mL MIC (Minimum Inhibitory Concentration) value against Bacillus subtilis ATCC 9372, murraol (4) and auraptenol (5) showed 63 µg/mL MIC value against Klebsiella pneumoniae ATCC 4352 and Bacillus subtilis ATCC 9372, and isoimperatorin (9) exhibited 16 µg/mL MIC value.


Results and Discussion
4′-Senecioiloxyosthol (1) was obtained as a colorless gum. The HRESIMS spectrum of 1 suggests a molecular formula of C20H22O5 with 10 degrees of unsaturation based on the [M + H] + molecular peak at m/z 343.1544 (calcd m/z 343.1545). The 1 H-NMR spectrum (Table 1) of 1 was very similar to that of osthol [13,16], with the exception of a missing vinylic methyl group signal of the prenyl side chain of osthol. Instead of two vinylic methyl signals, the 1 H-NMR spectrum of 1 displayed a vinylic methyl group signal at δH 1.72 (3H) and a methylene singlet at δH 4.87 (2H), indicating that the second vinylic methyl group of osthol side chain was replaced with an acyloxy bearing methylene group. Furthermore, the typical vinylic narrow quintet proton signal observed at δH 5.72 (J = 1.3 Hz) along with the two vinylic methyl group doublets at δH 2.19 and 1.90 (each 3H, J = 1.3 Hz) suggest the presence of a senecioil group as the acyl group. The 2D-ROESY spectrum of 1 exhibited interactions between C-5′ methyl group protons and H-2′ proton of the prenylated side chain of osthol ( Figure 2) as well as displayed interactions between H-6 and the methoxy group protons at C-7, and H-2″ proton and H-4″ methyl protons of the senecioiloxy acyl group, which clearly confirms the presence of a senecioiloxy acyl group at the C-4′ methyl group of osthol in 1. Furthermore, 13 C-NMR (Table 1), 2D COSY, UV and IR spectroscopic data (see experimental section and supplemental data) of 1 corroborated the structure as 4′-senecioiloxyosthol. Previously, 4′-angeloiloxy derivative of osthol (2)  (1) [13,16], with the exception of a missing vinylic methyl group signal of the prenyl side chain of osthol. Instead of two vinylic methyl signals, the 1 H-NMR spectrum of 1 displayed a vinylic methyl group signal at δ H 1.72 (3H) and a methylene singlet at δ H 4.87 (2H), indicating that the second vinylic methyl group of osthol side chain was replaced with an acyloxy bearing methylene group. Furthermore, the typical vinylic narrow quintet proton signal observed at δ H 5.72 (J = 1.3 Hz) along with the two vinylic methyl group doublets at δ H 2.19 and 1.90 (each 3H, J = 1.3 Hz) suggest the presence of a senecioil group as the acyl group. The 2D-ROESY spectrum of 1 exhibited interactions between C-5 methyl group protons and H-2 proton of the prenylated side chain of osthol ( Figure 2) as well as displayed interactions between H-6 and the methoxy group protons at C-7, and H-2 proton and H-4 methyl protons of the senecioiloxy acyl group, which clearly confirms the presence of a senecioiloxy acyl group at the C-4 methyl group of osthol in 1. Furthermore, 13 C-NMR (Table 1), 2D COSY, UV and IR spectroscopic data (see experimental section and supplemental data) of 1 corroborated the structure as 4 -senecioiloxyosthol. Previously, 4 -angeloiloxy derivative of osthol (2) (i.e., macrocarpin) was reported from Lomatium macrocarpum (Hook. & Arn.) C. & R., another Apiaceaen plant [21]. The 1 H-NMR spectroscopic data reported for macrocarpin (2) were similar to that of 1 with the exception of the presence of angeloiloxy acyl group signals [i.e., δ H 6.04 (1H, br t), 1.97 (3H) and 1.88 (3H)] instead of a senecioiloxy acyl group signals.  [21]. The 1 H-NMR spectroscopic data reported for macrocarpin (2) were similar to that of 1 with the exception of the presence of angeloiloxy acyl group signals [i.e., δH 6.04 (1H, br t), 1.97 (3H) and 1.88 (3H)] instead of a senecioiloxy acyl group signals.  The antimicrobial activity of extracts and isolated coumarins of Prangos hulusii was evaluated against Gram-positive and Gram-negative nine reference standards and six clinically isolated microorganism strains. The results of minimum inhibition concentration (MIC, in μg/mL) values are summarized in Table 2. The best antimicrobial activity was observed against Escherichia coli with the dichlormethane (DCM) extract (i.e., MIC at 156 μg/mL), followed by the petroleum ether (PE) and methanol (MeOH) extracts (i.e., each MIC at 313 μg/mL). All three extracts showed good activity The antimicrobial activity of extracts and isolated coumarins of Prangos hulusii was evaluated against Gram-positive and Gram-negative nine reference standards and six clinically isolated microorganism strains. The results of minimum inhibition concentration (MIC, in µg/mL) values are summarized in Table 2. The best antimicrobial activity was observed against Escherichia coli with the dichlormethane (DCM) extract (i.e., MIC at 156 µg/mL), followed by the petroleum ether (PE) and methanol (MeOH) extracts (i.e., each MIC at 313 µg/mL). All three extracts showed good activity against Enterococcus faecalis (MIC at 313 µg/mL). Similar activities were detected with the DCM extract against Proteus mirabilis, with the PE extract against Staphylococcus aureus and with the MeOH extract against Klebsiella pneumoniae ATCC 4352. No activity was observed with all of the tested extracts against clinical isolates K. pneumoniae, Acinetobacter baumannii, and E. coli, and only a weak activity was detected against other reference and clinical isolate bacteria.

Plant Material
The roots of Prangos hulusii were collected from Ödemiş by Hulusi Kütük,İzmir on March 2012, in Turkey. The plant was identified by Professor Emine Akalin Uruşak and a voucher specimen was deposited in the Herbarium of Istanbul University, Faculty of Pharmacy (ISTE 99676).

Determination of Antibacterial Activity
The MIC values of extracts and isolated compounds were determined using microbroth dilution method in 96-well microtitre plates. The bacterial cultures were prepared from overnight cultures on Tryptic Soy Agar (TSA) at 37 • C for 24 h by diluting in Mueller Hinton Broth (MHB) from approx. 10 8 CFU/mL to 2 × 10 6 CFU/mL. Then, 50 µL Mueller Hinton Broth (MHB) was added to the wells starting from the first well and continuing up to the twelfth. The extracts and isolated compounds were prepared 1/10 (v/v) in DMSO and 50 µg/mL of these were added to the first wells. Two-fold serial dilutions were made, achieving a final concentration ranging from 5000 to 10 µg/mL. The positive controls for Ciprofloxacin (CPR), Tetracycline (TTR), Cefotaxime (CEF), and Oxacillin (OXA) were determined with the final concentrations from 64 to 0.1 µg/mL. In addition, an extra row of DMSO was used as a vehicle control to determine its possible inhibitory activity. Finally, 25 µL of bacterial suspensions and % 0.001 resazurin solution were added to each well.
After incubating the bacteria at 37 • C for 24 h, the microtitre plates were examined visually for microbial growth which appeared as pink, colored by resazurin dye. In each row, the well containing the least concentration that showed no visible growth was considered the MIC. The bacterial samples were inoculated on TSA plates and incubated at 37 • C for 24 h.

Conclusions
Investigation of the dichloromethane extract of the roots of P. hulusii yielded several pyrenylated coumarins and furanocoumarins with antimicrobial activities. Prangos species frequently used for the treatment of burns and wounds in traditional folk medicine [3][4][5][6], perhaps the presence of pyrenylated coumarins with antimicrobial activity may play an important role for the aforementioned folkloric use of Prangos species.