Phytochemistry xxx (2014) xxx–xxx

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Biofilm blocking sesquiterpenes from Teucrium polium Wael A. Elmasri a, Mohamed-Elamir F. Hegazy b, Mina Aziz a, Ekrem Koksal a, Wail Amor c, Yehia Mechref a, Abdul N. Hamood c, David B. Cordes a, Paul W. Paré a,⇑ a

Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409-1061, USA Chemistry of Medicinal Plants/Center Excellence Science, National Research Centre, El-Tahrir St, Dokki, Giza 12311, Egypt c Department of Microbiology and Immunology, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA b

a r t i c l e

i n f o

Article history: Received 20 September 2013 Received in revised form 10 January 2014 Available online xxxx Keywords: Teucrium polium Lamiaceae Sesquiterpenes Biofilm blocking

a b s t r a c t The chemical composition and antibacterial activity of Teucrium polium L. (Lamiaceae) were assessed; sixteen compounds were isolated from a CH2Cl2/MeOH extract of the aerial parts of the plant including four 4b,5a-epoxy-7aH-germacr-10 sesquiterpenes 4b,5a-epoxy-7aH-germacr-10(14)-en-6b-ol-1-one, (14)-en,1b-hydroperoxyl,6b-ol, 4b,5b-epoxy-7aH-germacr-10(14)-en,1b-hydroperoxyl,6b-ol and 4a,5bepoxy-7aH-germacr-10(14)-en,1b-hydroperoxyl,6a-ol, together with seven known sesquiterpenes, one known iridoid glycoside, two known flavonoids, and one known phenylpropanoid glycoside. Structures were elucidated on the basis of spectroscopic (UV, 1H and 13C NMR) data, as well as two-dimensional NMR (1H–1H COSY, HMQC, NOESY and HMBC), and ESI-MS analysis. The relative stereochemistry of the ketone was established by X-ray crystallography, while its absolute configuration was attained by a modified Mosher’s method. Antibacterial activity of the crude extract, as well as with four of the isolated metabolites, was observed with Staphylococcus aureus anti-biofilm activity in the low lMol range. Diverse sesquiterpene-skeleton structure and corresponding comprehensive enzyme capacity is discussed. Ó 2014 Elsevier Ltd. All rights reserved.

Introduction Teucrium polium is a member of the Lamiaceae family with the genus including more than 300 species. Plant species in this genus are distributed ubiquitous around the world, with a concentration in the Mediterranean basin. Chemical investigations have shown that members of this genus are rich in monoterpenes, sesquiterpenes, sterols, saponins, iridoids, flavonoids, polyphenolic compounds, fatty acids, alkaloids and essential oils (Perez et al., 1993; Kamel and Sandra, 1994; Piozzi et al., 1998). This genus is also a rich source of diterpenes with a neoclerodane skeleton; more than 220 diterpenes have been described and many of these metabolites are of ecological interest as insect antifeedants and for their medicinal properties (Piozzi et al., 2005; Perez et al., 1993; Kamel and Sandra, 1994; Piozzi et al., 1998; Ulubelen et al., 2000). Several plants of this genus are used in folk medicine for treatment of fungal infections and abscesses (Batanouny, 2005). In addition, these plants are used for digestive disorders, inflammation, hypertension, fever, diabetes, rheumatism, parasitic diseases, ⇑ Corresponding author. Tel.: +1 (806) 742 3062; fax: +1 (806) 742 1289. E-mail address: [email protected] (P.W. Paré).

such as amoebiasis, and as tonics, stimulants and antiseptics (Moustapha et al., 2011). Previous studies have demonstrated the therapeutic efficacy of Teucrium species as antibacterial (Belmekki et al., 2013), antipyretic (Autore et al., 1984), anti-inflammatory (Menichini et al., 2009), antioxidant (Sharififar et al., 2009), hypoglycemic (Afifi et al., 2005; Esmaeili and Yazdanparast, 2004), antincancer (Nematollahi-Mahani et al., 2007), and anti-nociceptive agents (Abdollahi et al., 2003). T. polium contains phenylpropanoid glycosides, iridoid glycosides, flavonoids (De Marino et al., 2012), diterpenes (Fiorentino et al., 2010), and monoterpenes (Wassel and Ahmed, 1974), as well as sesquiterpenes (Cozzani et al., 2005). The plant is widely used in folk medicine for abdominal colic, headache, and kidney stones, as well as vermifuge, anti-inflammatory, and antipyretic (Aburjai et al., 2006). In Egypt, T. polium is used for wound healing, as well as an appetizer, expectorant, and hypoglycemic (Kamel, 1995). In addition, an extract showed activity against both yeast and carrageenin pyrexia in rats. The interest in this study was in T. polium’s marked antibacterial activity (Djaboua et al., 2013) against both Gram-positive and Gram-negative bacteria (Autore et al., 1984). Antibiotic resistance to pathogenic bacteria is a problematic and persistent issue in medicine. Biofilm formation is a physical strategy that bacteria employ to effectively block penetration and

http://dx.doi.org/10.1016/j.phytochem.2014.03.029 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Elmasri, W.A., et al. Biofilm blocking sesquiterpenes from Teucrium polium. Phytochemistry (2014), http://dx.doi.org/ 10.1016/j.phytochem.2014.03.029

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W.A. Elmasri et al. / Phytochemistry xxx (2014) xxx–xxx

toxicity of antibiotics. Blocking or retarding formation of biofilms improves the efficacy of antibiotics (Götz, 2002). Staphylococcus aureus is frequently the causal agent in biofilm-associated infections (Otto, 2008) and treatment is becoming increasing less effective due to antibiotic prevalent resistance (Begun et al., 2007; Darabpour and Hossein, 2010). The focus of this study is to mine for T. polium metabolites with antibiotic activity.

Results and discussion The CH2Cl2–MeOH extract of T. polium aerial plant material was partitioned with a gradient of n-hexane, dichloromethane, and methanol. Compounds from the eluted fractions were purified using a combination of Sephadex, and silica gel CC, as well as, by RP-HLPC. Four new compounds 1–4, along with 12 known 5–16 (Fig. 1) were isolated and characterized. Compound 1 was isolated as a colorless crystals with a specific rotation of ½a25 D = 8.3 (c 0.9, CHCl3). The mass spectrum of 1 exhibited a HRESI-MS molecular ion peak [2M+Na+] at m/z 527.3339 (calc. 527.3348), and [M+] at m/z 253.1794 (calc. 253.1798), suggesting a molecular formula C15H24O3. Its IR spectrum showed absorptions due to a hydroxyl group (3412 cm1), one terminal exocyclic double bond, and a carbonyl group (2954, 1669 and 928 cm1) (Barreroa et al., 1999). On the basis of a DEPT experiment, the fifteen 13C signals corresponded to one exocyclic double bond, three methyls, four methylenes, and four methines (two of which are oxygenated), as well as two quaternary carbon atoms one of which is a ketone. The 1H and 13C NMR spectra (Tables 1 and 2, respectively) established the following fragments: one tertiary methyl signal at dH 1.25, isopropyl methyl signals appearing at dH 0.88 and 0.99 (d, J = 6.8 Hz) respectively, and a hydroxyl-bearing methine at dH 3.43 (dd, J = 7.7), and an exocyclic methylene dH 5.88 and 6.22 (s). The degree of saturation indicated a monocyclic structure. Based on 1-D and 2-D NMR studies and previously reported metabolites isolated from Calamintha ashei (family Lamiaceae) (Menelaou et al., 2010), and genus Santolina (family Asteraceae) (Appendino et al., 2005; Barreroa et al., 1999), a germacrane ring system was proposed. Based on a literature report, an exocyclic double bond was positioned at C-10 (dC 151.46) and C-14 (dC 129.59) (Bedir et al., 1999); HMBC correlation between H2-14 (dH 5.78 and 6.02) and C-10 confirmed this positioning. In addition, HMBC correlations between H-14 and dC 206.34 and dC 33.8 established positions C-1 and C-9, respectively. The downfield chemical shift of C-1 allowed for the assigning of a ketone group. HMBC couplings were also observed between C-10 and C-1 with dH signals at 3.25 and 2.36 which were assigned to H2-2. A 1H–1H COSY correlation between H2-2 and dH resonances at 2.16 (m) and 1.39 (m) were assigned to H2-3. HMBC established

O

OOH

14

1

4 15

8

O

6

R2 11 R1

OR

1 1a 1b

R H R-MTPA S-MTPA

2 3 4

R1 α-Me α-Me β-Me

O R3

R2 β-H α-H α-H

Fig. 1. Identified germacrane metabolites.

R3 β-OH β-OH α-OH

carbon assignments at C-2 and C-3 (Fig. 2). The HMBC between H22, as well as H2-3 and dC signal at 59.6, allowed for the assignment of an oxygenated quaternary carbon at C-4 based on the chemical shift and DEPT. The HMBC between H2-3 and dC signals at dC 16.72 and 69.29 allowed for the assignment of C-15 and C-5, respectively. The up-field chemical shift of oxygenated C-4 and C-5 indicated the presence of an epoxide at these positions (Barreroa et al., 1999). Based on HMQC and COSY analysis dH signals at 2.55 and 3.43 were assigned to H-5 and H-6, respectively. The downfield carbon and proton shifts at position 6 indicated the presence of a hydroxyl group. The COSY correlation between H-6 and the dH signal at 0.85, which in turn correlated with the dH signal at 1.35 and 1.82, allowed for the assignment of H-7 and H-8, respectively. These assignments were confirmed by HMBC between H-6, C-7 and C-8. A COSY correlation between H-7 and dH signal at 1.67, which in turn correlated with 2 methyl signals at dH 0.88 and 0.99, allowed for the assignment of C-11, C-12 and C-13, respectively. Based on the relative up-field proton chemical shifts and clear doublet signals for H-12 and H-13 identified the presence of an isopropyl group. Moreover, HMBC between H-7 and C-11, as well as C-12, established a C-7 linkage of the isopropyl unit. COSY coupling of H2-8 to dH signals as 2.16 and 2.79 allowed for the assignment of C-9, which was supported by the HMBC between H-7, C-8 and C-9. A COSY correlation between H-9 (dH, 2.79 brd.) and H-14 (dH 5.88, d, 1.8), as well as a HMBC between H-9, C-10 and C-14, confirmed the connectivity of C-9 and C-10. The relative stereochemistry of the chiral centers in 1 was resolved by a combination of X-ray crystallography Fig. 4 and 2-D NOESY data, as well as analysis of the coupling constants and was supported by data from literature (Barreroa et al., 1999) (Fig. 3). The-cross peaks observed in the NOESY spectrum between H-6 and H-15, as well as between H-5 and H-11, implied that H-15, H-6, and H-7 were on the same molecular face of germacrane ring; thus H-5 was on the opposite side to H-15, H-6 and H-7 which was confirmed by X-ray crystallography of 1 (Fig. 4). The multiplicities of the carbon signals were deduced from DEPT experiments, whereas the assignment of all proton resonances and their connectivities to adjacent proton and carbon signals were established from 2-D HMBC and 2-D 1H–1H COSY experiments. A modified Mosher’s method was performed to determine the absolute configuration of the secondary alcohols at C-6 (Ohtani et al., 1991). Treatment of two aliquots of 11 with (S)- and (R)-MTPA chloride in dry pyridine gave the corresponding esters 1a and 1b, respectively, with a molecular ion peak at m/z 469.2201 consistent with derivatized products. The pattern of Dd(S  R) values (Fig. 5) allowed for the absolute configuration at C-6 to be R and the complete stereochemistry of 1 based on NMR, X-ray crystallography and Mosher data was assigned to 4S, 5S, 6R, and 7S as shown in Fig. 1 with the name 4b,5a-epoxy-7aH-germacr-10(14)-en-6b-ol-1-one. Compound 2 was isolated as a colorless syrup, with a specific rotation of ½a25 D = +12.5 (c 0.6, CHCl3). Its mass spectrum exhibited a HRESI-MS molecular ion peak [M]+ at m/z 271.1896 (calc. 271.1903) indicating a molecular formula of C15H26O4. The IR spectrum showed absorptions due to a hydroxyl group (3356 cm1), (C@O) and one terminal exocyclic double bond (2955, 2928, 2870. 2360, 1701, 1558, 1008, 906, 818 and 668 cm1). The structure of 2 is similar to 1, based on NMR spectroscopic data, except for the replacement of the ketone group in 1 with a methine-bearing hydroperoxide in 2 (Fig. 1). The hydroperoxide at C-1 followed from a downfield chemical shift for C-1 to dC 85.9 and a new proton signal at dH 4.44 corresponding to H-1. EIMS was consistent with the presence of a hydroperoxide. The HMBC between H2-14 (dH 5.11, 5.14) and dC signal at 85.9 confirmed the C-1 position (Fig. 2). The presence of a peroxide group was positive employing commercially available Baker Testrips (Thomas Scientific). The relative stereochemistry of the chiral centers for 2 was resolved by

Please cite this article in press as: Elmasri, W.A., et al. Biofilm blocking sesquiterpenes from Teucrium polium. Phytochemistry (2014), http://dx.doi.org/ 10.1016/j.phytochem.2014.03.029

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W.A. Elmasri et al. / Phytochemistry xxx (2014) xxx–xxx Table 1 H NMR spectroscopic data of 1–4 (d in ppm, J in Hz) (400 MHz, CDCl3). Signals were assigned on the basis of DEPT, 1H–1H COSY, HMQC, and HMBC experiments.

1

1⁄

Position 1 2

3.25 2.36 2.16 1.39 2.55 3.43 0.85 1.35 1.82 2.16 2.79 1.67 0.88 0.99 5.88 6.22 1.25

3 5 6 7 8 9 11 12 13 14 15 *

(H, m) (H, m) (H, m) (H, m) (H, d, 7.79) (H, dd, 7.79, 5.5) (H, m, 5.5) (H, m) (H, m) (H, m) (H, br d.) (H, m) (3H, d, 6.87) (3H, d, 6.87) (H, d, 1.8) (H, s) (3H, s)

2

3

4

4.44 (H, br d) 2.09 (2H, m)

4.43 (1H, t, 6.9) 2.02 (2H, m)

1.99 (H, m) 2.05 (H, m) 2.94 (H, d, 8.2) 3.60 (H, br d, 8.2) 1.12 (H, m) 1.67 (H, m) 1.91 (H, m) 2.56 (H, qd, 5.5) 2.1 (H, m) 1.64 (H, m) 0.92 (3H, d, 6.9) 0.99 (3H, d, 6.9) 5.15 (H, s) 5.19 (H, s) 1.29 (3H, s)

1.97 (2H, m)

4.18 (H, br d) 1.63 (H, m) 1.97 (H,m) 1.1 (H, m) 2.11 (H, m) 2.92 (H, d, 6.9) 3.62 (H, dd, 6.9) 1.14 (H, m) 1.92 (H, m) 2.16 (H, m) 2.41 (H, m) 2.31 (H, m) 1.7 (H, m) 0.90 (3H, d, 6.4) 0.98 (3H, d, 6.4) 5.17 (H, s) 5.44 (H, s) 1.18 (3H, s)

Solvent: methanol d3.

Table 2 C NMR spectroscopic data of 1–4 (d in ppm) (400 MHz, CDCl3). Signals were assigned on the basis of DEPT, 1H–1H COSY, HMQC, and HMBC experiments. 13

*

2.96 (H, d, 7.8) 3.59 (H, d, 7.8) 1.11 (H, m) 1.69 (H, m) 1.9 (H, m) 2.51 (qd, 5.5) 2.10 (H, m) 1.61 (H, m) 0.89 (3H, d, 6.4) 0.95 (3H, d, 6.4) 5.11 (H, s) 5.14 (H, s) 1.25 (3H, s)

Position

1⁄

2

3

4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

206.34 34.69 39.33 59.66 69.29 71.68 46.26 25.53 33.85 151.46 32.39 21.23 21.54 129.59 16.72

86.17 25.31 33.91 59.27 67.91 70.90 47.23 27.31 34.24 144.96 31.59 20.82 21.09 116.48 16.88

85.99 25.35 33.78 59.81 68.31 70.81 46.88 27.04 33.8 145.21 31.53 20.79 21.07 116.14 16.89

86.56 22.84 37.67 59.56 68.50 72.28 43.36 27.55 35.98 145.17 30.96 20.92 21.23 117.65 17.12

Solvent d3 MeOH.

O

OOH

O OR

1

O

2

OR

Fig. 2. Observed HMBC correlations for 1–2 as indicated by the arrows.

NOESY (Fig. 3) with cross-peaks observed between H-1, H-15, H-6 and H-7, indicating that these protons were on the same molecular face of the germacrane ring. Analysis of the vicinal couplings between H-5 and H-6 were found to be J5,6 (8.24 Hz) pointing to an anti-coplanar arrangement. Assuming the same stereochemistry for both 1 and 2, C-1 was assigned an S configuration and the structure of 2 was established as 4b,5a-epoxy-7aH-germacr-10(14)en,1b-hydroperoxyl,6b-ol, also a new natural product. Compound 3 was isolated as a colorless syrup with a specific rotation of ½a25 D = +74 (c 0.40, CHCl3). Its mass spectrum exhibited a HRESI-MS molecular ion peak [M]+ at m/z 271.1895 (calc.

271.1903) indicating a molecular formula C15H26O4. The IR spectrum showed absorptions due to a hydroxyl group (3386 cm1), (C@O) and one terminal exocyclic double bond (2957, 2870. 2707, 1670 and 925 cm1). The structure of 3 was similar to 2, based on NMR spectroscopic data, except for an inverted stereocenter at C-5, based on NOE data, in which a correlation was observed between C-5 and C-1, C-4, C-6, and C-7 which was not observed in 2 (Fig. 3). Structure 3 was established as 4b,5bepoxy-7aH-germacr-10(14)-en,1b-hydroperoxyl,6b-ol, also a new natural product. Compound 4 was isolated as a colorless syrup with a specific rotation of ½a25 D = +37.5 (c 0.11, CHCl3). The mass spectrum exhibited a HRESI-MS molecular ion peak [M]+ at m/z 271.1897 (calc. 271.1903) indicating a molecular formula C15H26O4. Its IR spectrum showed absorptions due to a hydroxyl group (3405 cm1), (C@O) and one terminal exocyclic double bond (2957, 2871, 2710, 1670, 1018, 910, and 736 cm1). The structure of 4 was similar to 3, based on NMR spectroscopic data except for the stereochemistry at C-4 and C-6 (Fig. 3). Based on NOE data, a correlation was observed between C-4 and C-6, these being in the opposite configuration to C-1, C-5, and C-7. The absence of NOE coupling between H-6 and H-7, as well as between H-1 and H3-15, confirmed these configuration assignments. So 4 was assigned the name 4a,5b-epoxy-7aH-germacr-10(14)-en,1b-hydroperoxyl,6a-ol, also a new natural product. In addition, 12 known compounds consisting of seven sesquiterpenes: 10a,1b;4b,5a-diepoxy-7aH-germacrm-6-ol (5) (Sanz and Alberto, 1991), teucladiol (6) (Bruno et al., 1993), 4b,6b-dihydroxy-1a,5b(H)-guai-9-ene (7) (Mahmoud, 1997), oplopanone (8) (Dastlik et al., 1989), oxyphyllenodiol A (9) (Muraoka et al., 2001), eudesm-3-ene-1,6-diol (10) (Mahmoud, 1997), rel1b,3a,6b-trihydroxyeudesm-4-ene (11) (Stavri et al., 2004), arteincultone (12) (Khafagy et al., 1983), and two flavonoids including 7,40 -O-dimethylscutellar-ein(5,6-dihydroxy-7,40 -dimethoxyflavone) (14) and salvigenin (15) (Okuda et al., 1975; Seshadri and Sharma, 1973) as well as, two glycosides: teucardoside (13) (Ruhdorfer and Rimpler, 1981) and poliumoside (16) (De Marino et al., 2012) were isolated and identified by direct comparison of their spectroscopic data with those reported in the literature. Biofilm inhibition was observed in the bacterial line S. aureus strain AH133-GFP via confocal laser scanning microscopy (CLSM) which showed that the depth of biofilm formed in DMSO containing-disk was greater than when treated with 3, 10, or 15 (Fig. 6). At the 0.18 and 0.67 lMol doses, 3 and 15, respectively were as

Please cite this article in press as: Elmasri, W.A., et al. Biofilm blocking sesquiterpenes from Teucrium polium. Phytochemistry (2014), http://dx.doi.org/ 10.1016/j.phytochem.2014.03.029

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W.A. Elmasri et al. / Phytochemistry xxx (2014) xxx–xxx

O

OOH

OOH

OOH

H

H

H

H

O

HO

H

H

O

1

H OH

H

2

O

H OH

H

3

H

H

O

HO

H

4

Fig. 3. NOESY correlations observed for metabolites 1–4.

germacrene A synthase, as well as germacrane cyclase that generates the guaiane and eudesmane skeletons, (Piet et al., 1995, 1996) are functional in the Egyptian ecotype of T. polium. Experimental General experimental procedures

Fig. 4. X-ray crystal structure of 1 with thermal ellipsoids drawn at the 50% probability level.

O

+0.01 +0.03 + 0.05

-0.01

+0.05

-0.11

-0.06

O -0.002

OR

R= S-MTPA and R-MTPA Fig. 5. Application of modified Mosher’s method for 1 to determine absolute configuration at C-6; Dd(S  R) values are in ppm.

Optical rotations were measured in CHCl3 on an Autopal IV automatic polarimeter (from Rudolph Research Analytical) equipped with a sodium lamp (kmax = 589 nm) and a 10 cm microcell. IR (KBr) spectra were recorded on a ThermoNicolet model IR 100 spectrophotometer. High-resolution ESI mass spectrometry (HRESI-MS) was carried out on a Micromass QTOF spectrometer and electrospray ionization mass spectrometry (ESI–MS) experiments were performed on an API 2000 triple-quadrupole mass spectrometer (Applied Biosystems, Foster City, CA). High-performance liquid chromatography (HPLC) separations in the isocratic mode were achieved on Agilent 1100 apparatus equipped with Rheodyne injector, refractive index, and with UV detectors, using an Agilent Prep-C18 column (21.2  250 mm, 10 lm). NMR spectra were obtained on a Varian (Palo Alto, CA) Unity Inova 500 NMR spectrometer (1H at 500 MHz and 13C at 125 MHz) equipped with V NMR 6.1C software and Sun hardware, d (ppm), J in Hz, spectra relative to CD3OH (dH = 3.31) and as internal standard. Chemical shifts are referenced to the residual solvent signal (CDCl3: dH 7.26, dC 77.0). The multiplicities of 13C NMR resonances were determined by DEPT experiments. One-bond heteronuclear 1H–13C connectivities were determined with the HMQC experiment. Two- and three-bond 1H–13C connectivities were determined by HMBC experiments. Nuclear Overhauser Effect (NOE) measurements were obtained from 2D NOESY experiments. Column chromatography (CC) was carried out using EMD silica gel 60 (70–230 mesh). Analytical TLC was performed on Merk silica gel 60 F254 sheets 0.25 mm thick. Plant material

effective in inhibiting biofilm formation as 0.02 lMol of a commercially used broad-spectrum inhibitor of biofilm formation for bacteria, gentamicin, which is potent antibiotic standard, while partial inhibition was observed with 0.79 lMol of 10. The rest of the compounds produced variable levels of inhibition (data not shown).

Air-dried aerial parts of T. polium were collected in June 2010, from North Sinai, Egypt. A voucher specimen SK-105 has been deposited in the Herbarium of St. Katherine protectorate, Egypt.

Concluding remarks

Extraction and isolation

Three distinct sesquiterpene skeletons have been reported from T. polium collected in Tunisia (Ghiglione et al., 1976), Saudi Arabia (Hassan et al., 1979), and Serbia (Kovacevic and Lakusic, 2001), eudesmol, guaiol, and germacrene, respectively. Plants collected from Egypt contain all three of these sesquiterpene skeletons. This establishes that the enzymes that convert the common precursor FPP (farnesyl diphosphate) to the germacrane skeleton,

Air-dried aerial plant tissue (2 kg) was crushed and extracted with CH2Cl2–MeOH (1:1) (4 L) at room temperature. After solvent removal, the residue (210 g) was subjected to silica gel column chromatography (CC) and eluted with n-hexanes, CH2Cl2 and MeOH in increasing order of polarity up to 100% CH2Cl2 and then to 15% MeOH in CH2Cl2 (a total solvent volume of 400 L to afford 398 1-L fractions.

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W.A. Elmasri et al. / Phytochemistry xxx (2014) xxx–xxx

Fractions (1–7) were combined (2.6 g) based on TLC similarities, concentrated in vacuo, re-dissolved in CH2Cl2 and defatted with MeOH. The lower layer was subjected to silica gel CC, eluted with n-hexanes with increasing amounts of EtOAc up to 1:1 n-hexanes:EtOAc to afford 29 fractions. Pooled fractions 2–9 (600 mg) were subjected to reversed phase HPLC (MeOH-H2O), 30:70) to obtain 9 (2.5 mg). Fractions (84–111) were pooled (1.1 g) based on TLC similarities, concentrated in vacuo and subjected to silica gel CC ; eluted with n-hexanes with increasing amounts of EtOAc up to 1:1 n-hexanes:EtOAc. Subfractions 8–10 (310 mg) were pooled, evaporated, then subjected to reversed phase HPLC to afford 12 (2 mg) and 8 (7 mg). Pooled fractions 112–221 (1.4 g) were subjected to series of silica columns eluting with gradients of n-hexane–EtOAc up to 100% EtOAc and EtOAc–acetone up to 100% acetone then gradient acetone–MeOH up to acetone–MeOH (50:50) to afford 122 fractions; subfractions 16–17 (30 mg) were pooled and subjected to reversed phase HPLC (MeOH–CH3CN / H2O(50/50), 20–80) to afford 6 (5 mg), and 7 (3 mg). Subfractions 26–31 (220 mg) were pooled and subjected to a reversed phase HPLC (MeOH–CH3CN/H2O (50/50), 25–75) to afford 5 (11 mg), and 1 (4.7 mg). Fractions (294–332) were re-combined (7 g) based on TLC similarities, concentrated in vacuo and subjected to silica gel CC eluting with a gradient of CH2Cl2–MeOH starting with (10:3) up to (7:3); fractions were monitored by TLC eluting with CH2Cl2–MeOH–H2O (7:3:1), to afford 50 fractions. Sub-fractions 21–28 (2 g) were pooled and subjected to Sephadex LH-20 gel eluted with an isocratic system of n-hexane–CH2Cl2–MeOH (7:4:0.5); 42 sub-fractions were obtained and sub-fractions 13– 18 (550 mg) were pooled and purified by RP HPLC eluting with an isocratic MeOH–H2O system(17:83) to afford 16 (24 mg) and 13 (30 mg). Pooled fractions 41–83 (12.6 g) were subjected to a series of silica gel CC procedures, eluting with gradients of n-hexane–EtOAc up to 100% EtOAc and EtOAc–acetone up to 100% acetone then gradient acetone–MeOH up to acetone–MeOH (50:50) to afford 40 fractions. Sub-fractions 26–30 (1.3 g) were pooled and subjected to silica gel CC eluting with a gradient system of n-hexane–EtOAc (7:1) up to 100% EtOAc to afford 45 fractions from which 14 (20 mg), and 15 (35 mg) were isolated. Sub-fractions (16–21) (1.8 g) were pooled and passed through a series of silica gel CC eluting with a gradient system of n-hexane–EtOAc (7:1) up to 100% EtOAc to afford 45 fractions, sub-fraction 14 was identified as 10 (8 mg). Sub-fractions 3–6 (1.1 g) were pooled and subjected to silica gel CC eluting with a gradient system of n-hexane– EtOAc (7:1) up to 100% EtOAc giving 85 fractions to afford 2 (6 mg), 3 (10 mg), 4 (6 mg) and 11 (2 mg). 4b,5a-Epoxy-7aH-germacr-10(14)-en-6b-ol-1-one (1) Colorless crystals (dichloromethane–methanol); ½a20 D = 8.33° 1 (CHCl3; c 0.9); mp 183–187° C; UVmax 223; IR (mKBr ) 3412 max cm (OH), 2954, 2840, 1669 (C@C), 1433, 1384, 1075, 1034, 928, 826, 605; EIMS (probe) 70 eV m/z (rel. int.): 527.3339 [2M+Na+], 253.1794 [M]+ (calc. 527.3348). For 1H and 13C NMR spectroscopic data, see Table 1. (R)-MTPA ester of 1 (1a) 1 H NMR (CDCl3, 400 MHz): 3.10(1H, m, H-2a), 2.40(1H, m, H-2b), 2.16⁄ (1H, m, H-3a), 1.46 (1H, m, H-3b), 2.62 (1H, d, 7.79, H-5), 5.023 (1H, dd, 7.79, H-6), 1.03 (1H, m, H-7), 1.43 (1H, m, H-8a), 1.89 (1H, m, H-8b), 2.13⁄ (1H, m, H-9a), 2.89 (1H, br d, H-9b), 0.90 (3H, d, 6.87, H-12), 0.97 (3H, d, 6.87, H-13), 5.81 (1H, d, Ha-14), 6.05 (1H, s, Hb-14), 1.344 (3H, s, H-15); 13C NMR (CDCl3, 400 MHz): 203.9 (C-1), 33.8 (C-2), 37.7 (C-3), 58 (C-4), 64.6 (C-5), 76.6 (C-6), 44.2 (C-7), 25 (C-8), 32.6 (C-9), 150 (C-10), 31.3 (C11), 20.7 (C-12), 21 (C-13), 127.6 (C-14), 16.6 (C-15), 55.4 (OCH3), 127.8, 128.3, 129.6, and 132.5 (benzene ring), 165.8

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(C@O); an astericks (⁄) indicates overlaping signals. HRESIMS: m/ z 469.2201 [M+] (calc. 469.2202). (S)-MTPA ester of 1 (1b) 1 H NMR (CDCl3, 400 MHz): 3.11 (1H, m, H-2a), 2.41 (1H, m, H-2b), 2.17⁄ (1H, m, H-3a), 2.73 (1H, d, 7.79, H-5), 5.023 (1H, dd, 7.79, H-6), 0.98⁄ (1H, m, H-7), 1.84 (1H, m, H-8b), 2.12⁄ (1H, m, H-9a), 2.84 (1H, br d, H-9b), 0.88⁄ (3H, d, 6.87, H-12), 0.92⁄ (3H, d, H-13), 5.79 (1H, d, Ha-14), 6.04 (1H, s, Hb-14), 1.346 (3H, s, H-15); 13C NMR (CDCl3, 400 MHz): 203.9 (C-1), 33.8 (C-2), 37.7 (C-3), 58.3 (C-4), 64.6 (C-5), 76.6 (C-6), 44.4 (C-7), 24.8 (C-8), 32.6 (C-9), 150.1 (C-10), 31.4 (C-11), 20.7 (C-12), 20.9 (C-13), 127.5 (C-14), 16.6 (C-15), 55.3 (OCH3), 127.5, 128.3, 129.6, and 132.1 (benzene ring), 165.8 (C@O); an astericks (⁄) indicates overlaping signals. HRESIMS: m/z 469.2196 [M+] (calc. 469.2202). 4b,5a-Epoxy-7aH-germacr-10(14)-en,1b-hydroperoxyl,6b-ol (2) Colorless syrup; ½a20 D = +74.1° (CHCl3; c 0.6); UVmax 211; IR KBr (mmax cm1) 3356 (br.) (OH), 2955, 2870, 2360, 1701 (C@C), 1460, 1385, 1258, 1144, 1064, 1008, 906, 818; EIMS (probe) 70 eV m/z (rel. int.): 271.1896 [M]+ (calc. 271.1903). For 1H and 13C NMR spectroscopic data, see Table 1. 4b,5b-Epoxy-7aH-germacr-10(14)-en,1b-hydroperoxyl,6b-ol (3) Colorless syrup; ½a20 D = +74.1° (CHCl3; c 0.4); UVmax 211; IR 1 (mKBr ) 3386(br.) (OH), 2957, 2870, 2707, 1670 (C@C), 1461, max cm 1386, 1265, 1147, 1065, 1013, 915, 736; EIMS (probe) 70 eV m/z (rel. int.): 271.1895 [M]+ (calc. 271.1903). For 1H and 13C NMR spectroscopic data, see Table 1. 4a,5b-Epoxy-7aH-germacr-10(14)-en,1b-hydroperoxyl,6a-ol (4) Colorless syrup; ½a20 D = +37.5° (CHCl3; c 0.11); UVmax 208; IR KBr (mmax cm1) 3405 (OH), 2957, 2871, 2710, 1644 (C@C), 1457, 1384, 1266, 1147, 1062, 1018, 910, 736; EIMS (probe) 70 eV m/z (rel. int.): 271.1897 [M]+ (calc. 271.1903). For 1H and 13C NMR spectroscopic data, see Table 1. X-ray crystallography analysis X-ray diffraction data for 1 were obtained at room temperature, on a Bruker Smart Apex II CCD diffractometer, using graphitemonochromated Mo Ka radiation (k = 0.71073 Å). Intensity data were collected using x-steps accumulating area detector images spanning at least a hemisphere of reciprocal space. Data were corrected for Lorentz polarization effects, and a multi-scan absorption correction was applied using SADABS (Sheldrick, 2008b). The structure was solved by direct methods and refined by full-matrix leastsquares against F2 using SHELXTL (Sheldrick, 2008a). The OH hydrogen was located from the difference Fourier map and refined isotropically, subject to a distance restraint. All other hydrogen atoms were assigned riding isotropic displacement parameters and constrained to idealized geometries. Due to weak anomalous scattering, the absolute structures could not be determined directly from the data. Crystal Data for 1 are summarized as follow: C15H24O3, colorless platelet, Mr = 252.34, crystal size 0.36  0.12  0.03 mm3, orthorhombic, space group P21212, a = 10.799(11) Å, b = 12.924(14) Å, c = 10.106(10) Å, V = 1411(3) Å3, Z = 4, T = 298(2) K, l = 0.081 mm–1, 4306 reflections collected, unique reflections (Rint) = 2587 (0.0240), R1(F2 > 2rF2) = 0. 0406, wR2 (all data) = 0.0932. CCDC assignment 958390 contains the supplementary crystallographic data for this paper. Peroxide colorimetric assay BAKER TESTRIPS for peroxides (Thomas Scientific) is a colorimetric assay for semi-quantitative determination of inorganic

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and organic compounds containing peroxide or a hydroperoxide group. The assay incorporates peroxidase enzyme which transfers oxygen from peroxide to an organic redox indicator converting it to a blue product (Kelly, 1996). The test strip was dipped briefly (ca. 1 s) into the test solution. After 5 s, a blue color indicates the presence of peroxide.

the inoculated plates and three different concentrations from each tested compound were spotted on the disk. The plates were incubated at 37 °C for 16–18 h and the zone of clearing around the disks was measured.

Preparation of the (R) and (S)-MTPA ester derivatives of 1

Overnight cultures of the tested AH133-GFP were pelleted, washed and re-suspended in fresh LB broth. The cultures were then serially diluted tenfold to obtain inoculums of 1  103 to 1  104 CFU. For each compound, a 6 mm cellulose disks (Becton Dickinson) were placed on the surface of freshly prepared LB agar plates. An aliquot (10 ll) of diluted culture was applied to each disk and plates were incubated at room temperature for 5 min. An aliquot (10 ll) of either DMSO or T. polium test compound (triplicates of each concentration) was added to each disk and plates were incubated at 37 °C for 24 h. Biofilms were quantified by counting CFUs per disk (Hammond et al., 2011). The cellulose disks were removed from the LB plates and placed in an individual 1.5 ml centrifuge tube containing 1 ml of phosphate buffered saline and vortexed to disrupt the biofilm and detach the bacteria. The bacterial suspension was then serially diluted tenfold and 10 ll aliquots of each dilution were spotted on LB agar plates. The number of bacteria per

An aliquot of 1 (1.5 mg of each) was dissolved in CH2Cl2 (2 mL) with dry pyridine (0.45 mL) under N2 (g). (S)- or (R)-a-methoxy-a(trifluoromethyl)phenylacetyl (MTPA) chloride (0.1 mL) was added into separate aliquots and the reaction was stirred overnight, quenched with a saturated NaHCO3 solution, and the CH2Cl2 layer was washed twice with distilled H2O. The reaction mixture was dried under reduced pressure to afford the (R) and (S)-MTPA esters. Biofilm inhibition assay S. aureus strain AH133-GFP, which carries the gene for the green fluorescent protein was grown overnight in Luria Bertani (LB) broth and diluted in LB to ca. 1  108 CFU/ml. The 100 ll culture was spread on an LB agar plate. Sterile cellulose disks were placed on

AH133-GFP biofilm regulation

Fig. 6. Biofilm inhibition of transgenic GFP S. aureus by 3 (50 lg) (A), 15 (200 lg) (B), and gentamicin (10 lg) (C) with solvent control (DMSO) comparison (D); representative 3D-generated confocal images exhibiting GFP expression are shown. (E) S. aureus biofilm inhibition by 3, 10, 15 and gentamicin (10 lg) as well as DMSO control is shown (n = 3, bars represent standard error).

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disk was calculated by using the following formula: CFU per disk = CFU counted  dilution factor  100. Acknowledgments This research was supported in part by the Robert Welch Foundation (D-1478), NSF equipment grant CHE-1048553 and NSF CRIF program. We are grateful to and Tianjiao Yang for providing massspectral technical support.

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