Alkaloid and coumarins from the green fruits of Aegle marmelos

Phytochemistry 75 (2012) 108–113

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Alkaloid and coumarins from the green fruits of Aegle marmelos Suda Chakthong a,b,d,⇑, Paosiyah Weaaryee a,b, Pongsak Puangphet a, Wilawan Mahabusarakam a,b,d, Patimaporn Plodpai c,d, Supayang P. Voravuthikunchai c,d, Akkharawit Kanjana-Opas e a

Department of Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand Center for Innovation in Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand c Department of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand d Natural Products Research Center, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand e Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand b

a r t i c l e

i n f o

Article history: Received 1 September 2010 Received in revised form 17 November 2011 Available online 22 December 2011 Keywords: Aegle marmelos Rutaceae Marmesiline Marmelonine Smyrindiol

a b s t r a c t Five (1–5) and 15 known compounds (6–20) were isolated from the acetone extract of the green fruits of Aegle marmelos. The structure of compounds 1–5, marmesiline (1), 6-(4-acetoxy-3-methyl-2-butenyl)-7hydroxycoumarin (2), 6-(2-hydroxy-3-hydroxymethyl-3-butenyl)-7-hydroxycoumarin (3), marmelonine (4) and 8-hydroxysmyrindiol (5), were determined on the basis of spectroscopic analyses. Antifungal and antibacterial activities of selected compounds were also evaluated. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Aegle marmelos is a medicinal plant belonging to the family Rutaceae, commonly known as the ‘‘Bael tree’’ and known in Thai as ‘‘Matum’’. It is a deciduous large-sized tree found in India, Thailand, and various southeastern Asian countries. All parts of the trees have many medicinal properties: astringent, aphrodisiac, demulcent, haemostatic, antidiarrheal, antidysenteric, antipyretic, antiscourbutic, and as an antidote to snake venom (Kesari et al., 2006). Decoction of the leaves and ripe fruit are used in remedies for dysentery, diarrhoea, and diabetes mellitus (Kamalakkannan and Prince, 2003). The leaf extract has been found effective in the regeneration of damaged pancreas (b-cell) in diabetic rats (Das et al., 1996) and the extract also exhibits antidiabetic action in glucose-induced hyperglycaemic rats (Sachdewa et al., 2001). The decoction of the root and bark are used in the treatment of fever and show antimalarial activity as well (Arumugam et al., 2008), whereas the aqueous extract of the fruit exhibited hypoglycaemic effect in streptozotocin-induced diabetes in rats (Kamalakkannan and Prince, 2003). The seed extract exhibits significant activity against Vibrio cholerae, Staphylococus aureus and Escherichia coli (Acharyya et al., 2009). Essential oils isolated from the ⇑ Corresponding author at: Department of Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand. Tel.: +66 7428 8425; fax: +66 7455 8841. E-mail address: [email protected] (S. Chakthong). 0031-9422/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2011.11.018

leaves have shown promising antifungal activities (Rana et al., 1997). Previous chemical investigations reported alkaloids, coumarins and steroids from the fruit extracts (Saha and Chatterjee, 1957; Sharma et al., 1980, 1981; Sharma and Sharma, 1981). Our preliminary biological tests of crude acetone extract from the green fruits of A. marmelos have shown interesting antifungal activity against Rhizoctonia solani NPRC750, Rigidoporus microporus PR720 and Sclerotium oryzae NPRC760 with minimum inhibitory concentration (MIC) values ranging from 31.2 to 62.5 lg/ml. These results stimulated us to investigate the bioactive compounds from this plant. We describe herein the isolation and structural elucidation of five new compounds; an alkaloid (1), two coumarins (2 and 3) and two dihydrofuranocoumarins (4 and 5) along with 15 known compounds (6–20) from the green fruits of A. marmelos (Fig. 1). Furthermore, the isolated compounds were tested for antifungal and antibacterial activities. 2. Results and discussion Compound 1 was isolated as a white powder with a molecular formula of C22H25O4N, as determined from HREIMS analysis. The IR spectrum showed absorption bands indicating hydroxyl (3417 cm1), conjugated carbonyl (1661 cm1) and aromatic (1621, 1539, 1456 cm1) moieties. The UV spectrum of 1 had an absorption maxima at 217, 223 and 272 nm, suggesting the presence of a trans-cinnamide structure (Sharma et al., 1981). The 1H NMR spectroscopic data of 1 (Table 1) were closely comparable

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S. Chakthong et al. / Phytochemistry 75 (2012) 108–113

3' 4'

2'

5'

7'

1'

6''

O 8'

9' N

1

H

6'

2 1''

OH 1

5''

5'

OH 4'' O

1'''

3''

2''

2'''

3''' 4'''

4'

Me RO

Me

4'

3'

1' 2'

5

6

4a

4

HO 7 8 8a O O 2: R = C CH3

5'''

1''

HO

3 2

O

2''

Me

O 7

2''

8

8a O

HO 3'' 3' 6 5 4a 4 Me 3 1'' 2' 2 HO 8a O O 7 Me O 8 2'' OH 5

3 2

O

4

2'

3'

HO

HO Me HO Me

19: R = H 3'' O 3' 5 4 4a 6 HO 1'' 2'

5'

O

O

R

Me

3 2

O

3'

O

O

O

Me HO Me

O

8

O

O

O

O

rutaretin R3

O

O

R Me HO Me

14: R 1 = CH 2CH=CMe 2, R2 = OH, R 3 = H

O

15: R 1 = CHO, R 2 = OH, R 3 = H

16: R = H

18: R1 = OMe, R 2 = OH, R 3 = OMe

20: R = OH

Me

CO2 H

Me

4

2'

10: R = OCH 2CHC=CH2 11: R = OMe

4a

smyrindiol

12: R1 = OMe, R 2 = OMe, R 3 = H

6: R = OCH2 CH=CMe 2 O 8: R = OCH 2 CC=CH 2 Me 9: R = OH OH

5

OH

2

R

6

HO 7 8 8a O 3

R1 O

1'

O

Me Me

O

O

HO O

7

O

N H

O

Me Me

OH 17

13 Fig. 1. Compounds 1–20, smyrindiol and rutaretin.

with those of marmeline (17), which was previously isolated from the acetone extract of the same plant (Sharma et al., 1981) and also isolated in this study. Compound 1 differed from marmeline (17) in the side-chain: marmeline (17) has an oxyprenyl whereas that of 1 has a hydroxyloxyprenyl side-chain. The 1H NMR spectroscopic data of 1 displayed signals at dH 3.90 (1H, td, J = 9.5, 1.0 Hz, H-1000 a), dH 4.04 (1H, ddd, J = 9.5, 3.2, 1.0 Hz, H-1000 b); dC 71.2, dH 4.46 (1H, br d, J = 9.5 Hz, H-2000 ); dC 73.6, dH 5.00 (1H, s, H-4000 a), dH 5.13 (1H, s, H-4000 b); dC 112.8 and a singlet at dH 1.80 (3H); dC 18.9. In the HMBC spectrum, the methylene protons H2-1000 (d 3.90 and 4.04) correlated with C-2000 (d 73.6) and C-400 (d 158.2) and the methyl protons H3-5000 (d 1.80) correlated with C-2000 (d 73.6), C-3000 (d 143.3) and C-4000 (d 112.8) confirming the attachment

of the side-chain at C-400 of the benzene ring. Faizi et al. (2009) demonstrated that oxazoline ring derivatives were the parent compounds of cinnamide derivatives, thus the configuration at C-2 should be racemic. Cinnamide 1 was found, however to have a low specific rotation (½a25 D = +2.3 (c 0.5, CHCl3)). Therefore, compound 1 was identified as N-[2-hydroxy-2-[4-[(2-hydroxy-3methyl-3-butenyl)oxy]phenyl]ethyl]-3-phenyl-(2E)-2-propenamide and named as marmesiline. Compound 2 was isolated as a brownish powder and its molecular formula of C16H16O5, was obtained by HREIMS. The UV, IR, and 1 H NMR spectroscopic data (Table 2) of 2 were almost identical to those of isophellodenol C (19), which was previously isolated from the acetone extract of the roots of Heracleum candicans WALL

Table 1 1 H and 13C NMR spectroscopic data (500 MHz/CDCl3) for compound 1. 1 Position 1 2 10 2 , 60 30 , 50 40 70 80 90 100 0

dH (J in Hz) 3.43 (ddd, 13.8, 7.8, 5.2) 3.80 (ddd, 13.8, 5.2, 2.9) 4.86 (dd, 7.8, 2.9) – 7.49 (dd, 7.6, 1.9) 7.34–7.36 (m) 7.34–7.36 (m) 7.64 (d, 15.6) 6.37 (d, 15.6) – –

dC 47.7 73.4 134.5 127.9 128.9 129.9 141.8 119.9 167.1 134.5

Position

dH (J in Hz)

dC

200 , 600 300 , 500 400 1000

7.30 6.90 – 3.90 4.04 4.46 – 5.00 5.13 1.80 6.00

127.1 114.7 158.2 71.2

2000 3000 4000 5000 N–H

(d, 8.5) (d, 8.5) (td, 9.5, 1.0) (ddd, 9.5, 3.2, 1.0) (br d, 9.5) (s) (s) (s) (br t, 5.2)

73.6 143.3 112.8 18.9 –

110

S. Chakthong et al. / Phytochemistry 75 (2012) 108–113

(Nakamori et al., 2008), and also isolated in this study. The main difference between 2 and isophellodenol C (19) was the additional signal of an acetoxyl group in 2 shown as a singlet of an acetoxy methyl at dH 2.02; dC 20.9 and a carbonyl carbon at dC 171.0. Furthermore a singlet resonance of the methylene protons H2-40 (dH 4.46) of 2 was shifted more downfield than that of 19 (dH 3.98) suggesting that H2-40 of 2 was connected to an acetoxyl group instead of a hydroxyl group as in 19. In the HMBC spectrum, the methylene protons at d 4.46 (H2-40 ) correlated with the carbons at d 171.0 (C100 ), 132.7 (C-30 ), 125.6 (C-20 ) and 14.0 (C-50 ) and a methyl signal at d 2.02 (H3-200 ) correlated with the carbon at d 171.0 (C-100 ), confirming the attachment of an acetoxyl group at C-40 . In addition, the methylene protons at d 3.36 (H2-10 ) showed long-range correlations with C-5 (d 128.5), C-6 (d 124.5), C-7 (d 157.6) and C-30 (d 132.7) and the H-5 aromatic proton (d 7.12) with C-10 (d 27.9), indicating the location of 40 -acetoxy-30 -methyl-20 -butenyl side-chain at C-6 of the coumarin skeleton. The geometry of the 20 –30 double bond was in an E-orientation on the basis of NOESY cross-peaks of H-10 /H3-50 and H-20 /H2-40 . Thus structure 2 was assigned as 6-(4acetoxy-3-methyl-2-butenyl)-7-hydroxycoumarin. Compound 3 was obtained as a brownish powder and its molecular formula of C14H15O5 was determined from a HRAPCIMS analysis in the positive-ion mode. The IR, 1H and 13C NMR spectroscopic data (Table 2) of 3 were also closely related to those of isophellodenol C (19). The difference was shown in the side-chain of the coumarin skeleton; that of 3 was a 20 -hydroxy-30 -hydroxymethyl-30 -butenyl group, whereas that of isophellodenol C was a 40 -hydroxy-30 -methyl-20 -butenyl group. The 1H and 13C NMR spectroscopic data of the former side-chain were shown as two sets of doublet of doublets of the benzylic methylene protons at dH 2.81 (J = 14.3, 8.0 Hz, H-10 a) and 2.89 (J = 14.3, 4.3 Hz, H-10 b); dC 37.4, a doublet of doublets of an oxymethine proton at dH 4.43 (J = 8.0, 4.3 Hz, H-20 ); dC 73.4, two doublets of the hydroxymethylene protons at dH 4.10 and 4.07 (each 1H, d, J = 13.6 Hz, H2-50 ); dC 62.8 and two singlets of terminal olefinic methylene protons at dH 4.98, 4.99 (each 1H, H2-40 ); dC 111.3 including the carbon signal of C-30 at dC 149.5. The configuration at C-20 could not be defined due to the reactivity of OH-20 as an allylic hydroxyl group. The structure of the side-chain was supported by the HMBC correlations of H2-40 (d 4.98 and 4.99) and H2-50 (d 4.10 and 4.07) with C-20 (d 73.4) and of a methine proton H-20 (d 4.43) with C-40 (d 111.3), whereas

the HMBC correlations of the methylene protons H2-10 (d 2.81 and 2.89) with C-30 (d 149.5), C-5 (d 130.2) and C-7 (d 159.8) indicated the attachment of a butenyl side-chain at C-6 of the coumarin skeleton. Structure 3 was thus determined as 6-(2-hydroxy-3hydroxymethyl-3-butenyl)-7-hydroxycoumarin. Compound 4 was obtained as a yellow solid and its molecular formula of C14H12O5, was determined from HREIMS. The IR spectrum showed absorption bands due to a hydroxyl and a lactone carbonyl at 3416 and 1721 cm1, respectively. The 1H and 13C NMR spectroscopic data (Table 2) were closely related to those of smyrindiol (Fig. 1, Zou et al., 2005), indicating the same furanocoumarin skeleton. However, instead of two gem-dimethyl singlet signals as shown in smyrindiol, only one methyl singlet signal at dH 1.52 was evident in 4. Furthermore, the 1H NMR spectrum also showed signals of non-equivalent oxymethylene protons at d 3.27 and 3.67 (each 1H, d, J = 9.0 Hz, H2-300 ) which were linked to the carbon resonance at d 73.8 in the HMQC spectrum. The methyl protons at d 1.52 (H3-200 ) showed HMBC correlations with C-20 (d 90.7), C-100 (d 77.7) and a methylene carbon C-300 (d 73.8); in turn, the methylene protons at d 3.27 and 3.67 (H2-300 ) correlated with C-30 (d 80.8), C-20 (d 90.7) and a tertiary methyl carbon H3-200 (d 23.6), as well as the small coupling between H2-300 and the methyl protons (H3-200 ) in the COSY spectrum. This information suggested a dihydrofuran ring connected to a furanocoumarin parent skeleton with oxygens of the two furan rings in opposite directions and a methyl group connected to a dihydrofuran ring at C-100 . The relative configurations of compound 4 were examined following Asahara et al. (1984): the vicinal coupling constants of 2,3-cis- and 2,3-trans-dihydrobenzofuran rings are different in magnitude, J2,3-cis = 6.1 Hz but J2,3-trans = 3.2 Hz. The vicinal coupling constant (5.8 Hz) of two doublets at d 4.78 (H-20 ) and 5.70 (H-30 ) indicated their cis orientation. Moreover, the NOESY spectrum showed correlation between H-30 and H3-200 suggesting a cis-relationship between H-20 , H-30 and H3-200 . Thus, compound 4 was assigned as marmelonine. Compound 5 was isolated as a brownish powder. The HREIMS indicated a molecular formula of C14H14O6 and the IR spectrum had absorption bands corresponding to a hydroxyl (3393 cm1) and a lactone carbonyl (1707 cm1). The 1H and 13C NMR spectroscopic data of 5 (Table 2) were also similar to those of smyrindiol, except for the absence of a singlet signal for an aromatic proton H-8 at dH 6.83; dC 99.0 as in smyrindiol (Zou et al., 2005). The presence of an

Table 2 1 H and 13C NMR spectroscopic data (300 MHz/CDCl3) for compounds 2–5. Position

2

3

4

5

dH (J in Hz)

dC

dH (J in Hz)

dC

dH (J in Hz)

dC

dH (J in Hz)

dC

1 2 3 4 4a 5 6 7 8 8a 10

– – 6.17 7.56 – 7.12 – – 6.80 – 3.36

(d, 7.1)

– 161.6 112.9 143.7 112.0 128.5 124.5 157.6 103.2 154.1 27.9

– 162.5 111.5 144.3 111.6 130.2 123.6 159.8 103.1 154.4 37.4

– – 6.30 7.67 – 7.53 – – 6.87 – –

– 160.6 113.4 143.4 114.1 125.6 123.6 163.1 98.7 157.1 –

– – 6.20 (d, 9.5) 7.68 (d, 9.5) – 7.08 (s) – – – – –

– 161.7 111.9 145.1 114.1 114.8 128.4 150.5 129.3 144.3 –

20 30 40

5.58 (t, 7.1) – 4.46 (s)

125.6 132.7 69.7

73.4 149.5 111.3

4.78 (d, 5.8) 5.70 (d, 5.8) –

90.7 80.8 –

4.33 (d, 5.9) 5.36 (d, 5.9) –

91.0 72.3 –

50

1.72 (s)

14.0

– – 6.08 7.57 – 7.13 – – 6.67 – 2.81 2.89 4.43 – 4.98 4.99 4.10 4.07

–

–

–

–

00

– 2.02 (s)

171.0 20.9

1 200 300

(d, 9.5) (d, 9.5) (s)

(s)

(d, 9.4) (d, 9.4) (s)

(s) (dd, 14.3, 8.0) (dd, 14.3, 4.3) (dd, 8.0, 4.3) (s) (s) (d, 13.6) (d, 13.6)

62.8

(d, 9.6) (d, 9.6) (s)

(s)

– 1.52 (s) 3.27 (d, 9.0) 3.67 (d, 9.0)

77.7 23.6 73.8

– 1.54 (s) 1.56 (s)

72.4 27.5 25.7

111

S. Chakthong et al. / Phytochemistry 75 (2012) 108–113 Table 3 Antifungal activity (MIC/MFC, lg/ml) of 6, 8–12, and 15. No.

R. solani

R. microporus

S. oryzae

6 8 9 10 11 12 15 Propiconazole

62.5/250 7.8/7.8 62.5/250 62.5/250 62.5/125 31.2/125 15.6/31.2 1.9/1.9

62.5/250 15.6/15.6 125/250 62.5/250 125/250 62.5/125 31.2/62.5 1.9/1.9

31.2/250 7.8/7.8 62.5/250 62.5/250 62.5/250 62.5/250 31.2/250 1.9/1.9

Rhizoctonia solani NPRC750, Rigidoporus microporus PR720 and Sclerotium oryzae NPRC760.

aromatic quaternary carbon at d 129.3 (C-8) which was, however, in agreement with that of rutaretin (dC 128.0, Okuyama et al., 1989) supporting the substitution of a hydroxyl group at C-8 of the coumarin nucleus. Thus the presence of a hydroxyl group at C-8 was implied whose location was based on the HMBC correlations (4J) of H-4 (d 7.68) and H-5 (d 7.08) to the carbon at d 129.3 (C-8). A large vicinal coupling constant (5.9 Hz) of two doublets at d 4.33 (H-20 ) and 5.36 (H-30 ) supported the cis orientation. The absolute configurations of 5 were established by comparing its specific rotation with smyrindiol. Smyrindiol had 20 S,30 R configurations with a positive specific rotation value, +22.4 (c 0.25, CHCl3) (Zou et al., 2005) and +24.4 (c 2.8, CHCl3) (Jiménez et al., 2000). With a specific rotation value of +20.1 (c 1.0, MeOH), compound 5 was assigned to 20 S,30 R configurations and named as 8-hydroxysmyrindiol. In addition, 15 known compounds were also isolated: imperatorin (6) (Razdan et al., 1987), valencic acid (7) (Ito et al., 1988b), 8-[(300 -methyl-200 -oxo-300 -buten-100 -yl)oxy]-7H-furo[3,2-g]benzopyran-2-one (8) (De Mol et al., 1984), xanthotoxol (9) (Razdan et al., 1987), isogosferol (10) (Adebajo and Reisch, 2000), xanthotoxin (11) (Razdan et al., 1987), scoparone (12) (Razdan et al., 1987), (+)-decursinol (13) (Nemoto et al., 2003), demethylsuberosin (14) (Patre et al., 2009), 6-formylumbilliferone (15) (Ito et al., 1988a), (+)-marmesin (16) (Kim et al., 2006), N-2-hydroxy-2-[4-(30 ,30 -dimethyllallyloxy)phenyl]ethylcinnamide or marmeline (17) (Sharma et al., 1981), isofraxidin (18) (Banthorpe and Brown, 1989), isophellodenol C (19) (Nakamori et al., 2008) and xanthoarnol (20) (Ishii et al., 1973; Zou et al., 2005). Their structures were identified by comparison of the NMR spectroscopic data and the physical data with those previously reported in the literatures. Only isolated compounds in sufficient amounts were tested for antifungal activity against R. solani NPRC750, R. microporus PR720

and S. oryzae NPRC760 using a broth microdilution method (Table 3). Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values were evaluated for compounds 6, 8–12, and 15. These results demonstrated that compound 8 was most active against R. solani NPRC750 and S. oryzae NPRC760, with MIC and MFC of 7.8 lg/ml. Moreover, the isolated compounds (4– 12, 15, 17, 19, 20) were also evaluated for their antibacterial activities against both Gram-positive bacteria: Bacillus subtilis, S. aureus, Enterococcus faecalis TISTR459, Methicillin-Resistant S. aureus (MRSA) ATCC43300, Vancomycin-Resistant E. faecalis (VRE) ATCC51299 and Gram-negative bacteria: Salmonella typhi, Shigella sonei, and P. aeruginosa (Table 4). Only xanthoarnol (20) exhibited good activity against E. faecalis with the same MIC values of 18.75 lg/ml as vancomycin, whereas the rest of the compounds showed no antibacterial activities. 3. Concluding remarks In conclusion, five new compounds, one alkaloid (1) and four coumarin derivatives (2–5), were isolated from the green fruits of A. marmelos. Compound 4 was a coumarin with a unique structure of tetracyclic skeleton which could be derived from a dihydrofuranocoumarin precursor. In addition, xanthoarnol (20) exhibited antibacterial activity against E. faecalis with the same MIC values of 18.75 lg/ml as vancomycin, which can be considered as a potential antibacterial agent. 4. Experimental 4.1. General Melting points were recorded in °C on a digital Electrothermal 9100 Melting Point Apparatus, whereas ultraviolet spectra were measured in methanol solutions on a UV-160A spectrophotometer (SHIMADZU). The IR spectra were recorded neat on a Perkin–Elmer FTS FT-IR spectrometer. Optical rotations [a]D were measured in chloroform and methanol solution on a JASCO P-1020 digital polarimeter. The 1H and 13C NMR spectra were recorded on a FT-NMR Bruker Ultra Shield™ 300 and 500 MHz spectrometers, as well as a Unity Inova Varian 500 MHz spectrometer using tetramethylsilane (TMS) as internal standard. EI and HREI mass spectra were measured on ThermoFinnigan MAT 95 XL spectrometer. HRAPCIMS was recorded on a Bruker Daltonics microTOF spectrometer. Quick column chromatography (QCC) was carried out on silica gel 60

Table 4 Antibacterial activity (MIC, lg/ml) of 4–12, 15, 17, 19 and 20. No.

4 5 6 7 8 9 10 11 12 15 17 19 20 Vancomycin

Gram-positive bacteria

Gram-negative bacteria

B. subtilis

S. aureus

E. faecalis

MRSA

VRE

S. typhi

S. sonei

P. aeruginosa

>300 300 >300 >300 300 >300 >300 >300 >300 >300 >300 >300 >300 2.343

>300 300 300 300 >300 >300 >300 300 300 >300 >300 >300 >300 37.5

>300 >300 >300 >300 >300 >300 >300 >300 >300 >300 >300 >300 18.75 18.75

>300 300 >300 300 >300 300 300 300 300 >300 >300 >300 >300 2.343

>300 300 >300 300 >300 300 300 300 300 >300 >300 >300 >300 2.343

>300 300 >300 300 >300 >300 300 300 300 >300 >300 >300 >300 37.5

>300 300 >300 300 >300 >300 >300 300 300 300 >300 >300 >300 37.5

>300 300 300 300 300 150 >300 >300 300 300 >300 300 >300 2.343

Gram-positive bacteria: Bacillus subtilis, Staphylococcus aureus, Enterococcus faecalis TISTR459, Methicillin-Resistant S. aureus (MRSA) ATCC43300, Vancomycin-Resistant E. faecalis (VRE) ATCC51299. Gram-negative bacteria: Salmonella typhi, Shigella sonei, and P. aeruginosa.

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GF254 (Merck). Column chromatography (CC) was performed by using silica gel 100 (70–230 Mesh ASTM, Merck) or on Sephadex LH-20. For thin-layer chromatography (TLC), aluminium sheets of silica gel 60 F254 (20  20 cm, layer thickness 0.2 mm, Merck) were used for analytical purposes and the compounds were visualised under ultraviolet light. Solvents for extraction and chromatography were distilled at their boiling ranges prior to use except CHCl3 was analytical grade reagent. 4.2. Plant material The green fruits of A. marmelos Lour. were collected from Songkhla province in the Southern part of Thailand, in October, 2008. Identification was made by Mr. Ponlawat Pattarakulpisutti, Department of Biology, Facutly of Science, Prince of Songkla University. The specimen (Paosiyah 01) has been deposited in the Herbarium of Department of Biology, Facutly of Science, Prince of Songkla University, Songkhla, Thailand. 4.3. Extraction and Isolation Chopped-dried green fruits of A. marmelos (3.20 kg) were immersed in Me2CO (15 l) at room temperature for 5 days. After evaporation, a dark green gum of Me2CO extract (55.0 g) was subjected to QCC over silica gel and eluted with a gradient of n-hexane, CH2Cl2 and MeOH to furnish 15 fractions (A–O). Fraction F was further purified by CC over silica gel and eluted with CH2Cl2 to give 7 fractions (F1–F7). Subfraction F2, containing one major component, was recrystallized from CH2Cl2-hexane (50:50, v/v) to give 6 (0.83 g). Subfraction F6 was purified by CC over silica gel and eluted with MeOH-CH2Cl2 (2:98, v/v) to afford 10 fractions (F6A–F6J). Subfraction F6I yielded 7 (47.7 mg). Subfraction F6C was purified by CC over silica gel and eluted with MeOH-CH2Cl2 (1:99, v/v) to afford 5 fractions (F6C1–F6C5). Subfraction F6C2 was further purified on prep. TLC, eluted with MeOH-CH2Cl2 (2:98, v/v) to give 8 (9.3 mg). Subfraction F6E was separated by CC with Sephadex LH-20, eluted with MeOH to afford 3 subfractions (F6E1–F6E3). Subfraction F6E3 gave 9 (11.5 mg). Subfraction F6F was separated by CC with Sephadex LH-20, eluted with MeOH to afford 6 subfractions (F6F1–F6F6). Subfraction F6F4 was further purified on prep. TLC, eluted with MeOH-CH2Cl2 (2:98, v/v) to give 10 (3.6 mg). Fraction H was further purified by CC over silica gel and eluted with a gradient of CH2Cl2-MeOH to give 9 subfractions (H1–H9). Subfraction H2 afforded 11 (7.3 mg). Subfraction H6 was purified by CC over silica gel and eluted with a gradient of CH2Cl2MeOH to give 13 subfractions (H6A–H6M). Subfraction H6G gave 12 (48.0 mg). Subfraction H8 was purified by CC over silica gel and eluted with MeOH-CH2Cl2 (2:98, v/v) to give 9 subfractions (H8A–H8I). Subfraction H8E was further purified by CC over silica gel and eluted with MeOH-CH2Cl2 (1:99, v/v) to give 9 subfractions (H8E1–H8E9). Subfraction H8E3 was 15 (5.1 mg). Subfraction H8E6 was further purified on prep. TLC and eluted with MeOHCH2Cl2 (2:98, v/v) to give 13 (1.8 mg) and 14 (2.7 mg). Fraction I was further purified by CC over silica gel and eluted with a gradient of CH2Cl2-MeOH to give 7 subfractions (I1–I7). Subfraction I5 was purified by CC over silica gel and eluted with MeOH-CH2Cl2 (3:97, v/v) to give 10 subfractions (I5A–I5J). Subfraction I5G was further purified on prep. TLC and eluted with MeOH-CH2Cl2 (4:96, v/v) to give 1 (2.0 mg). Subfraction I6 was purified by CC over silica gel and eluted with a gradient of CH2Cl2-MeOH to give 8 subfractions (I6A–I6H). Subfraction I6D gave 16 (7.1 mg). Subfraction I6E was purified by CC over silica gel and eluted with MeOH-CH2Cl2 (5:95, v/v) to give 5 subfractions (I6E1–I6E5). Subfractions I6E3 was separated by CC with Sephadex LH-20, eluted with MeOH-CH2Cl2 (50:50, v/v) to afford 3 subfractions (I6E3AI6E3C). Subfractions I6E3C was further purified on prep. TLC and

eluted with MeOH-CH2Cl2 (3:97, v/v) to give 17 (2.8 mg) and 2 (1.9 mg). Fraction K was further purified by CC over silica gel and eluted with MeOH-CH2Cl2 (10:90, v/v) to give 10 subfractions (K1–K10). Subfraction K3 was purified by CC over silica gel and eluted with EtOAc-hexane (40:60, v/v) to give 11 subfractions (K3A–K3K). Subfractions K3J was further purified on prep. TLC and eluted with Me2CO-CH2Cl2 (10:90, v/v) to give 18 (3.2 mg) and 4 (3.9 mg). Subfraction K5 was purified by CC over silica gel and eluted with MeOH-CH2Cl2 (3:97, v/v) to give 8 subfractions (K5A–K5H). Subfraction K5D was further purified on prep. TLC and eluted with MeOH-CH2Cl2 (3:97, v/v) to give 5 (3.5 mg). Subfraction K5G was further purified on prep. TLC and eluted with MeOH-CH2Cl2 (5:95, v/v) to give 3 (2.5 mg). Subfraction K6 was purified by CC over silica gel and eluted with Me2CO-CH2Cl2 (15:85, v/v) to give 8 subfractions (K6A–K6H). Subfraction K6E was 20 (2.8 mg). Subfraction K6D was further purified on prep. TLC eluted with EtOAc-hexane (60:40, v/v) to give 19 (2.3 mg). 4.3.1. N-[2-hydroxy-2-[4-[(2-hydroxy-3-methyl-3butenyl)oxy]phenyl]ethyl]-3-phenyl-(2E)-2-propenamide or marmesiline (1) White powder, m.p. 163–164 °C, ½a25 D = + 2.3 (c 0.5, CHCl3); UV kmax (MeOH) (log e): 217 (3.59), 223 (3.58) and 272 (3.43) nm; IR (Neat) t (cm1): 3417 (O–H stretching), 1661 (C@O stretching), 1621, 1539, 1456 (aromatics). For 1H NMR (CDCl3, 500 MHz) and 13 C NMR (CDCl3, 125 MHz) spectroscopic data, see Table 1. EIMS m/z 367 [M]+ (0.3), 349 (36), 220 (57), 161 (100), 160 (62), 131 (100), 103 (38), 77 (16); HREIMS [M]+ m/z 367.1783 (calcd for C22H25O4N 367.1784). 4.3.2. 6-(4-Acetoxy-3-methyl-2-butenyl)-7-hydroxycoumarin (2) Brownish powder, m.p. 133–134 °C; UV kmax (MeOH) (log e): 205 (4.18), 297 (3.58), 330 (3.70) nm; IR (Neat) t (cm1): 3392 (O–H stretching), 1720 (C@O stretching), 1618, 1570, 1421 (aromatics). For 1H NMR (CDCl3, 300 MHz) and 13C NMR (CDCl3, 75 MHz) spectroscopic data, see Table 2. EIMS m/z 288 [M]+ (3), 229 (22), 228 (77), 213 (100); HREIMS [M]+ m/z 288.0999 (calcd for C16H16O5 288.0998). 4.3.3. 6-(2-Hydroxy-3-hydroxymethyl-3-butenyl)-7hydroxycoumarin (3) Brownish powder, m.p. 279–280 °C; ½a26 D = 9.4 (c 0.09, CHCl3); UV kmax (MeOH) (log e): 205 (4.42), 256 (3.49), 331 (3.93) nm; IR (Neat) t (cm1): 3432 (O–H stretching), 1726 (C@O stretching), 1621, 1557, 1488 (aromatics). For 1H NMR (CDCl3 + CD3OD (1 drop)), 300 MHz) and 13C NMR (CDCl3 + CD3OD (1 drop), 75 MHz) spectroscopic data, see Table 2. EIMS m/z 262 [M]+ (0.3), 244 (8), 176 (100), 175 (79), 147 (35); HRAPCIMS [M + H]+ m/z 263.0914 (calcd for C14H15O5 263.0920). 4.3.4. Marmelonine (4) Yellow solid, m.p. 195–196 °C, ½a26 D = + 1.7 (c 1.0, MeOH); UV kmax (MeOH) (log e): 205 (4.41), 257 (3.57) and 325 (3.95) nm; IR (Neat) t (cm1): 3416 (O–H stretching), 1721 (C@O stretching), 1625, 1575, 1491 (aromatics). For 1H NMR (CDCl3, 300 MHz) and 13 C NMR (CDCl3, 75 MHz) spectroscopic data, see Table 2. EIMS m/z 260 [M]+ (36), 215 (35), 186 (100), 158 (34), 131 (11), 77 (5); HREIMS [M]+ m/z 260.0685 (calcd for C14H12O5 260.0685). 4.3.5. (20 S,30 R)-8-hydroxysmyrindiol (5) Brownish powder, m.p. 179–180 °C, ½a26 D = +20.1 (c 1.0, MeOH); UV kmax (MeOH) (log e): 210 (4.39), 268 (3.72) and 326 (3.99) nm; IR (Neat) t (cm1): 3393 (O–H stretching), 1707 (C@O stretching), 1623, 1588, 1418 (aromatics). For 1H NMR (CDCl3 + CD3OD (1 drop), 300 MHz) and 13C NMR (CDCl3 + CD3OD (1 drop), 75 MHz) spectroscopic data, see Table 2. EIMS m/z 278 [M]+ (17), 203 (66),

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202 (100), 174 (17); HREIMS [M]+ m/z 278.0790 (calcd for C14H14O6 278.0790). 4.4. Antifungal susceptibility testing Broth microdilution method was performed according to the guidelines of Clinical and Laboratory Standards Institute document M38-A2 (Clinical and Laboratory Standards Institute, 2008). Compounds were dissolved in 10% DMSO in H2O (Merck, Darmstadt). Stock solutions of the compounds were diluted in RPMI 1640 medium (with L-glutamine, without bicarbonate), supplemented with 2% glucose, and buffered to pH 7.0 with 0.165 M 3-(N-morpholino)-propanesulfonic acid (Sigma–Aldrich, Steinheim). The compounds (100 ll) were diluted to final concentrations ranging from 1.9 to 250 lg/ml in 96-well microlitre plates. One hundred microlitre of each mycelial suspension containing approximately 104 CFU/ml were inoculated and incubated at 35 °C for 24 h. DMSO was used as negative control and propiconazole (Irvita plant protection, Curaçao) was used as the standard antifungal drug. The MIC was observed at least in duplicate as the lowest concentration that completely inhibited visible growth. To determine the MFC, 100 ll from each of the wells at or above the MIC were plated on potato dextrose agar (Difco, Detroit) and incubated at 25 °C for 72 h. The MFC was defined as the lowest concentration at which no colonies were detected on the agar plate. 4.5. Antibacterial assay The isolated compounds from the green fruits of A. marmelos were tested against both Gram-positive and Gram-negative bacteria: B. subtilis, S. aureus TISTR517, E. faecalis TISTR459, MethicillinResistant S. aureus (MRSA) ATCC43300, Vancomycin-Resistant E. faecalis (VRE) ATCC51299, S. typhi, S. sonei and P. aeruginosa. The microorganisms were obtained from the culture collections, Department of Industrial Biotechnology and Department of Pharmacognosy and Botany, PSU, except for the TISTR and ATCC strains, which were obtained from Microbial Research Center (MIRCEN), Bangkok, Thailand. The antimicrobial assay employed was the same as described in Boonsri et al. (2006). Vancomycin, which was used as a standard, showed antibacterial activity against E. faecalis TISTR459 at 18.75 lg/ml. Acknowledgments This work was supported by the Center for Innovation in Chemistry (PERCH-CIC), Office of the Higher Education Commission, Ministry of Education and the Graduate School, Prince of Songkla University. The authors also would like to thank Assoc. Prof. Chanita Ponglimanont and Dr. Thumnoon Mutarapat for helpful suggestion, Mr. T. Anantapong for the antibacterial test, the Scientific Equipment Center, Prince of Songkla University and Chulabhorn Research Institute for the MS analysis. References Acharyya, S., Patra, A., Bag, P.K., 2009. Evaluation of the antimicrobial activity of some medicinal plants against enteric bacterial with particular reference to multi-drug resistant Vibrio cholerae. Trop. J. Pharm. Res. 8, 231–237. Adebajo, A.C., Reisch, J., 2000. Minor furocoumarins of Murraya koenigii. Fitoterapia 71, 334–337.

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