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New depside from Citrus reticulata Blanco a
bc
Uraiwan Phetkul , Souwalak Phongpaichit , Ramida d
Watanapokasin & Wilawan Mahabusarakam
ac
a
Department of Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand b
Department of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand c
Natural Products Center of Excellence, Faculty of Science, Prince of Songkla,University, Hat Yai, Songkhla 90112, Thailand d
Department of Biochemistry, Faculty of Medicine, Srinkharinwirot University, Bangkok 10110, Thailand Published online: 18 Mar 2014.
To cite this article: Uraiwan Phetkul, Souwalak Phongpaichit, Ramida Watanapokasin & Wilawan Mahabusarakam (2014) New depside from Citrus reticulata Blanco, Natural Product Research: Formerly Natural Product Letters, 28:13, 945-951, DOI: 10.1080/14786419.2014.896010 To link to this article: http://dx.doi.org/10.1080/14786419.2014.896010
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Natural Product Research, 2014 Vol. 28, No. 13, 945–951, http://dx.doi.org/10.1080/14786419.2014.896010
New depside from Citrus reticulata Blanco
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Uraiwan Phetkula, Souwalak Phongpaichitbc, Ramida Watanapokasind and Wilawan Mahabusarakamac* a Department of Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand; bDepartment of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand; cNatural Products Center of Excellence, Faculty of Science, Prince of Songkla, University, Hat Yai, Songkhla 90112, Thailand; dDepartment of Biochemistry, Faculty of Medicine, Srinkharinwirot University, Bangkok 10110, Thailand
(Received 20 December 2013; final version received 14 February 2014) A new depside, named depcitrus A (1), and 31 known compounds were isolated from the peels, leaves and branch barks of Citrus reticulata Blanco. Methylation of the high polarity fractions from the branch barks and peels gave one new methylated compound named depcitrus B (14) and five known compounds. Their structures were established based on spectroscopic evidence. The antioxidant, antimicrobial and cytotoxic activities of some pure compounds were evaluated. Keywords: Rutaceae; Citrus reticulata; depside; resorcylic derivatives; antibacterial activity; cytotoxicity activity
1. Introduction Citrus species have been used to treat many symptoms of diseases in humans such as coughs, skin inflammation, muscle pains, stomach upsets and ringworm infections (Li et al. 2009). They have been reported to be a rich source of bioactive compounds (Abeysinghe et al. 2007). In Thailand, various Citrus reticulata species are grown. In order to understand the chemical constituents and to search for useful bioactive metabolites, we have previously investigated the extract from the wood of C. reticulata, a tree that grows in the southern part of Thailand (Phetkul et al. 2013). In this work, we further investigated the extracts from leaves, peels and branch bark, and evaluated their antioxidant, antimicrobial and cytotoxic activities. 2. Results and discussion Extraction and purification of the CH2Cl2 extract of branch barks, CH2Cl2 and Me2CO extracts of the peels and the leaves resulted in the isolation of 13 compounds (1– 13) from the branch barks, 12 compounds (18 –29) from the peel and 11 compounds (10, 18– 20, 32 –38) from the leaves (Figure 1). The high polar fractions were methylated and further purified to give four (14– 17) and two (30, 31) methylated compounds, respectively. The structures of these compounds were elucidated by analyses of spectroscopic data as well as comparison of their spectroscopic data with the previously published data. The compounds were identified as depcitrus A 1, atranorin 2, 5-hydroxynoracronycine 3, citflavanone 4, 8-hydroxy-6-methoxy-pentylisocoumarin 5, citracridone-I 6, citrusinol 7, citrusinine-I 8, citramine 9, scopoletin 10, limonin 11, citracridone-III 12, 4-hydroxybenzoic
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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Figure 1. Structure of compounds from C. reticulata Blanco (1– 38), 4-hydroxy-2-methoxy-6-[(8Z)pentadec-8-en-1-yl]phenyl acetate, 4-hydroxy-2-methoxy-6-pentadecylphenyl acetate and 1-O-methyl-6acetoxy-5-(pentadec-10Z-enyl)resorcinol.
acid 13, depcitrus B 14, sphaerophorol dimethyl ether 15, 6,8-dimethoxypentylisocoumarin 16, citracridone-II 17, 5-demethylnobiletin 18, tangeretin 19, nobletin 20, tetramethyl-O-isocutellarein 21, natsudaidai 22, 3,4-dihydroxy benzoic acid 23, 8,30 -b-glucosyloxy-20 -hydroxy-30 -methylbutyl7-methoxy-coumarin 24, 5-hydroxy-7,40 -dimethoxyflavone 25, hesperidin 26, eriocitrin 27, narirutin 28, rutin 29, naringenin trimethyl ether 30, 2,3-dihydro-5-hydroxy-40 ,7-dimethoxyflavanone 31, butulinic acid 32, gardenin B 33, tymusin 34, 7-geranyloxy coumarin 35, 4hydroxybenzaldehyde 36, crenulatin 37 and 5-(2-enyl-3-methylbut)oxy-7-hydroxycoumarin 38. Depcitrus A (1) is a new depside whereas depcitrus B (14) is a new resorcylic derivative. Compounds 2 – 10, 12, 13, 15 –17, 23, 24, 25, 30, 31, 34, 35, 36, 37 and 38 were reported for the first time from C. reticulata Blanco. Compounds 11, 18– 22, 26– 29 and 33 were previously isolated. Depcitrus A (1) was obtained as an amorphous powder, m.p. 125– 1278C. Its UV spectrum showed absorption peaks at 265, 286, 311 and 400 nm. The IR spectrum showed the absorption band of CvO stretching at 1726 cm21. A molecular ion in the FAB-MS at m/z 501.2848 corresponded to a molecular formula of C29H40O7. The 13C NMR spectrum showed the resonances of four methine aromatic carbons: d 100.1 (C-3), 113.4 (C-5), 103.2 (C-30 ), 115.5 (C-50 ), three quaternary aromatic carbons: d 104.4 (C-1), 139.0 (C-6), 119.5 (C-60 ), five oxy aromatic
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carbons 166.6 (C-2), 169.1 (C-4), 158.0 (C-40 ), 151.6 (C-20 ), 145.4 (C-10 ), two methoxyl carbons: d 55.5 (4-OMe), 56.4 (20 -OMe), a carbonyl ester carbon: d 164.9 (C-14) and a carbonyl ketone: d 207.2 (C-8). The 1H NMR spectrum showed doublet resonances of meta-aromatic protons H-3 and H-5 at d 6.45 and d 6.28, and meta-aromatic protons H-30 and H-50 at d 6.52 and d 6.58, which indicated the presence of two 1,2,3,5-tetrasubstituted benzene rings. The COSY correlations of H-9 (2.37, t) to H-10 (1.42, m), H-10 to H-11 (1.20, m), H-11 to H-12 (1.10, m) and H-12 to H-13 (0.86, t), together with the HMBC correlations of H-9 (d 2.37) and H-7 (d 4.06, s) to the carbonyl carbon C-8 (d 207.2), indicated the presence of a 2-oxoheptyl side chain. Proton H-7 and a hydrogen bonded hydroxyl group resonating at d 11.27 showed a HMBC correlation to C-1 (d 104.4), which indicated the hydroxyl group (2-OH) and 2-oxoheptyl side chain ortho to the carbonyl ester. A methoxyl group at d 3.82 was 4-OMe due to H-3, H-5 and 4-OMe showing HMBC correlations to C-4. A heptyl side chain was deduced from the COSY correlations of H-70 (d 2.75, t) to H-80 (1.53, m), H-80 to H-90 (1.42, m), H-90 to H-100 (1.35, m), H-100 to H-110 (1.20, m), H-110 to H-120 (1.10, m) and H-120 to H-130 (0.82, t), and it was attached at C-60 because H-70 showed HMBC correlation to C-50 (d 115.5) and C-10 (d 145.4). The remaining methoxyl group (d 3.84, 20 -OMe) was assigned at C-20 according to the HMBC correlations of the methoxyl group and H-30 (d 6.52) to C-20 (d 151.6). The evidence that H-70 was further correlated to the oxy carbon at 145.4, while H-30 and H-50 were correlated to the oxy carbon at 145.4 and 158.0, together with the interpretation of the molecular formula of C29H40O7 ([M þ 1]þm/z 501.2848), implied the presence of a hydroxyl group and an ester bond formed between two aromatic rings. Two possible structures could be proposed with the ester bond attached at either the C-10 or C-40 and the hydroxyl group attached at either the C-40 or C-10 , respectively. A comparison of the 13C chemical shifts published for alkenylresorcinols; 4-hydroxy-2-methoxy-6-[(8Z)-pentadec-8-en-1-yl]phenyl acetate, 4-hydroxy-2-methoxy-6-pentadecylphenyl acetate and 1-O-methyl-6-acetoxy-5-(pentadec-10Z-enyl)resorcinol demonstrated that the carbons with an ester bond ortho to the alkyl and alkoxy groups consistently resonated at least 10 ppm further up field compared with the hydroxyl group meta to the alkyl and alkoxyl groups. Accordingly, the ester bond was assigned to attach at the C-10 (145.4) and the hydroxyl group was at C-40 (158.0) (Bao et al. 2010; Al-Mekhlafi et al. 2012). Therefore 2-hydroxy-4-methoxy-6-(2-oxoheptyl)- 20 -methoxy-40 -hydroxy-6-(hetyl)phenyl ester was assigned for 1. Depsitrus B (14) was obtained as a yellow viscous liquid. The UV spectrum showed maximum absorptions at 266.5 nm. The IR spectrum showed the absorption band of CvO stretching at 1725 cm21. A molecular ion in the FAB-MS at m/z 294.1472 corresponded to a molecular formula of C16H22O5. The 1H NMR spectrum showed resonances of meta-aromatic protons at d 6.45 (H-3), 6.28 (H-5), a hydrogen bonded hydroxy proton at d 11.27 (2-OH), a methoxyl group at d 3.84 (4-OMe), a methyl ester group at d 3.83 (1-CO2Me) and a 2-oxoheptyl group. The COSY and HMBC experiments indicated that the proton resonances at d 4.06 (H-7), 2.37 (H-9), 1.42 (H-10), 1.20 (H-11), 1.10 (H-12), 0.86 (H-13) and the carbonyl carbon resonance at d 207.2 (C-8) corresponded to a 2-oxoheptyl side chain as for depcitrus A (1). The NOSEY correlations of H-7 to H-5, and 4-OMe to H-5 and H-3 allowed an assignment to a 2-oxoheptyl group ortho to H-5, while a 4-OMe was ortho to H-5 and H-3. The hydroxyl proton was assigned to a 2-OH according to HMBC correlation of the 2-OH to C-3 (d 97.0). The chemical shift value of 2-OH (d 11.27) revealed that it formed a hydrogen bond to the adjacent group. Consequently, the remaining methyl ester group was placed at C-1 (d 107.0). Compound 14 was then assigned as methyl-2-hydroxy-4-methoxy-6-(2-oxoheptyl)-benzoate. Compounds 2– 4, 6 –8, 21, 35 and 38 were tested for antioxidative activity using the DPPH assays. Compounds 2 – 4, 6– 8, 21, 35 and 38 showed very weak activity with per cent scavenging of DPPH radical in the range of 0.65 – 23.27%. Compounds 5, 9, 10, 14– 17, 19, 22 –25, 30 –36 were not tested for antioxidative activity due to insufficient amounts.
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Compounds 2, 3, 6 –9, 11, 13, 18– 21, 35 and 38 were evaluated for cytotoxicity against cell lines A431, SKBR-3, T47D and AU565. Compounds 3, 6, 7, 8, 11 and 35 affected the growth of cell lines with IC50 values of less than 100 mg/mL. Compounds 2, 9, 13, 18 –21 and 38 showed no cytotoxicity against the tested cancer cell lines up to the final concentration of 100 mg/mL. Compounds 4, 5, 14 – 17, 22 – 34, 36 and 37 were not tested due to insufficient amounts. Compounds 2 –5, 7 –8, 10, 12, 19 –20 and the extracts of the peel, leaves and branch bark were tested for their antibacterial activity on Staphylococcus aureus ATCC25923, methicillinresistant S. aureus (MRSA) SK1 and Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC27853 and Cryptococcus neoforman ATCC90113, antifungal activity on Candida albicans NCPF3153, C. neoforman and Microsporum gypseum. The extracts and pure compounds 2, 3, 5, 7, 8, 10, 12, 19 and 20 had no effect on these microorganisms up to a dose of 200 mg/mL. Only 4 inhibited the growth of S. aureus ATCC25923, MRSA SK1with MIC values of 64, 64 mg/mL, respectively. Compounds 11, 13, 20, 23, 26 – 29 and 37 have previously been reported to have antioxidative activity. Compounds 3, 7, 8, 10 – 12, 18 –21 and 26 have been previously reported for their cytotoxicity on other cell lines. Compounds 2 and 11 have been previously reported for their antibacterial activity, therefore, we did not retest. 3. Experimental 3.1. General Melting points were recorded with a Fisher-Johns melting point apparatus and are uncorrected (Fisher-Johns Scientific Co, Waltham, MA, USA). Ultraviolet spectra were measured with a UV-160A spectrophotometer (Shimadzu, Kyoto, Japan). Principle bands (lmax) were recorded in wavelengths (nm) and log 1 in methanol solutions. Infrared spectra were obtained on a FTS165 FT-IR spectrophotometer and were recorded in wave numbers (cm21) (Perkin-Elmer, Shelton, CA, USA). 1H and 13C NMR spectra were recorded on an FT-NMR Bruker Ultra ShieldTM 300 MHz spectrometer (Bruker, Rheinstetten, Germany). The FAB-MS and HR-FABMS were obtained using a MAT 95 XL mass spectrometer (Thermo Finnigan, Bremen, Germany). Preparative thin-layer chromatography (PTLC) was carried out on 20 cm £ 20 cm glass plates with a 0.2 mm, thick layer of silica gel 60 GF254 (Merck, Darmstadt, Germany). Quick column chromatography (QCC) was performed using silica gel 60 H (Merck, Darmstadt, Germany) and column chromatography (CC) was performed using silica gel 100 (Merck, Darmstadt, Germany) or sephadex LH-20 gel (Pharmacia, Uppsala, Sweden) or silica gel RP-18 (40 – 63 mm, Merck, Darmstadt, Germany). Precoated plastic plates (20 £ 20 cm) with a 0.2 mm thick layer of silica gel 60 GF254, (Merck, Darmstadt, Germany) were used for TLC analysis. 3.2. Plant material The branch barks, leaves and peels of C. reticulata Blanco were collected from Sadao district, Songkhla province in the southern part of Thailand, in April 2011. Identification of the plants was made by J. Wai, Department of Biology, Faculty of Science, Prince of Songkla University. A voucher specimen U.Phetkul 1 (PSU) has been deposited in the Herbarium of the Department of Biology, Faculty of Science, Prince of Songkla University, Thailand. 3.3. Extraction and isolation Dried branch barks of C. reticulata Blanco (1.4 kg) were chopped and immersed in CH2Cl2 (7.5 L) at room temperature. After removal of the solvent by evaporation, a dark-brown gum (18.13 g) was obtained. This gum was subjected to QCC (a gradient of 10% acetone in hexane to
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80% MeOH in acetone) to give 14 fractions (A– N). Fraction A gave a colourless solid 2 (7.00 mg). Fraction H (714.2 mg) was purified by CC (20 – 50% acetone in hexane) to afford an orange solid 3 (17.0 mg), a yellow solid 4 (2.3 mg) and an amorphous powder 5 (2.1 mg). Fraction I (527.2 mg) was subjected to CC (20% acetone in hexane) to give an orange solid 6 (3.7 mg). Fraction J (966.3 mg) was purified by CC (20 – 100% acetone in hexane) to afford yellow solids 7 (11.7 mg), 8 (2.3 mg) and 9 (0.8 mg). Fraction L (890.2 mg) was subjected to separation on a Sephadex LH-20 column (20% CH2Cl2 in MeOH) to give fractions LA – LF. Fraction LC was further purified by CC (30% acetone in hexane) to give a white solid 10 (2.0 mg). Fraction M (651.0 mg) was subjected to separation on a Sephadex LH-20 column (20% CH2Cl2 in MeOH) to give fractions MA –MI. Fraction MC was further purified by CC (30 – 100% acetone in hexane) to give amorphous powder 1 (2.1 mg). Fraction N (2.9 g) was subjected to separation on a Sephadex LH-20 column (20% CH2Cl2 in MeOH) to give fractions NA –NI. Purification of fraction NB by CC (2% MeOH in CH2Cl2) provided a white solid 11 (18.0 mg), a yellow solid 12 (2.3 mg) and a white solid 13 (2.1 mg). Methylation of fraction LE (58.95 mg) with MeI (2.0 mL) and K2CO3 (10.0 mg) in Me2CO (1.0 mL) for 8 h and purified by CC (20% acetone in hexane) gave nine fractions (LEA-LEI). Fraction LEG (15.713 mg) was purified by PTLC (20% acetone in hexane) to give an amorphous powder 14 (0.71 mg). Fractions ND to NI were combined (NDI, 559.13 mg) and methylated with MeI (4.0 mL) and K2CO3 (20.0 mg) in Me2CO (2.0 mL). Separation of the reaction mixture on a Sephadex LH-20 column (20% CH2Cl2 in MeOH) gave three fractions (NDIA – NDIC). Fraction NDIC was purified by PTLC (20% acetone in hexane) to give yellow viscous liquids 15 (0.71 mg) and 16 (0.81 mg), white solids 11 (1.20 mg) and 16 (1.10 mg) and a yellow solid 17 (0.91 mg). Chopped-dried peel from fruits (893. 9 g) of C. reticulata was successively immersed in CH2Cl2 (1.5 L) and Me2CO (1.5 L) at room temperature (3 days £ 2 times). After removal of the solvent by evaporation, a dark-brown viscous CH2Cl2 extract (12.0124 g) and Me2CO extract (15.287 g) were obtained, respectively. The CH2Cl2 extract was dissolved in hexane to give a soluble (5.1007 g) and insoluble (6.9087 g) fraction. The CH2Cl2 insoluble fraction (6.9087 g) was subjected to a QCC (a gradient of 10% CH2Cl2 in hexane to 95% MeOH in CH2Cl2) to give 12 fractions (A– L). Fraction G (2.7178 mg) was purified by CC (1% MeOH in CH2Cl2) to afford yellow solids 18 (2.1 mg), 19 (2.3 mg) and 20 (4.1 mg). Fraction I (86.00 mg) was subjected on CC (20 –100% acetone in hexane) to give a yellow solid 21 (2.1 mg). Fraction J (70.1 mg) was purified by CC (20 – 100% acetone in hexane) to give fractions JA –JK. Fraction JF was purified by CC (30% acetone in hexane) to give a yellow solidd 22 (1.0 mg). The Me2CO extract was subjected to CC (a gradient of 30% CH2Cl2 in hexane to 95% MeOH in CH2Cl2) to give 16 fractions (A– P). Fraction H (873.2 mg) was purified by CC (30% CH2Cl2 in hexane) to afford yellow solids 18 (1.8 mg), 19 (3.0 mg) and 20 (5.1 mg). Fraction J (1.339 g) was subjected to CC (20 –100% acetone in hexane) to give a yellow solid 21 (2.5 mg). Fraction K (879.2 mg) was purified by CC (20% acetone in hexane) to afford a yellow solid 23 (1.2 mg). Fraction L (1.455 mg) was subjected to a reverse phase column (50% H2O in MeOH) to give a colourless solid 24 (2.0 mg). Fraction N (1.989 g) was purified on a reverse phase column (50% H2O in MeOH) to afford yellow solids 25 (2.1 mg), 26 (1.3 mg) and 27 (2.0 mg). Chromatography of fraction O (2.756 g) on a reverse phase column (50% H2O in MeOH) gave six fraction (OA –OF). Yellow solids 28 and 29 (2.0 mg) were obtained from fraction OC and OD, respectively. Methylation of fraction OF (238.9 mg) with MeI (4.0 mL) and K2CO3 (20.0 mg) in Me2CO (4.0 mL) for 8 h and purified by PTLC (20% acetone in hexane) gave yellow solids 30 (0.9 mg) and 31 (1.3 mg). Choppeddried leaves (1.4 kg) from C. reticulata were successively immersed in CH2Cl2 (14.0 L) and Me2CO (14.0 L) at room temperature (3 days £ 2 times). After removal of the solvent by evaporation, a dark-brown viscous CH2Cl2 extract (13.788 g) and Me2CO extract (17.1245 g) were obtained, respectively. The CH2Cl2 extract was dissolved in hexane to give soluble (5.18 g) and insoluble (8.599 g) fractions. The Me2CO extract was further fractionated in hexane to give a
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soluble (1.89 g) and an insoluble (15.235 g) fraction. The CH2Cl2 insoluble fractions (8.599 g) were separated by QCC (a gradient of 10% acetone in hexane to 50% MeOH in acetone) to give 12 fractions (A – L). Fraction F (876.3 mg) was purified by CC (20 – 50% acetone in hexane) to afford a white solid 32 (1.8 mg). Fraction I (541.2 mg) was purified by CC (20 –50% acetone in hexane) to afford yellow solids 18 (1.9 mg) and 19 (2.1 mg). Fraction J (556.8 mg) was purified on a Sephadex LH-20 column (20% MeOH in CH2Cl2) to give fractions JA –JE. Fraction JC was further purified by CC (20% acetone in hexane) affording yellow solids 20 (2.4 mg) and 33 (1.1 mg) and 34 (1.0 mg). Fraction K (987.3 mg) was purified on a Sephadex LH-20 column and eluted with CH2Cl2 – MeOH (1:4) to give fractions KA –KF. Fraction KD was further purified by CC (20 –50% acetone in hexane) to afford a yellow solid 35 (3.8 mg). The Me2CO insoluble fraction (15.235 g) was separated by QCC (a gradient of 5% acetone in hexane to 50% MeOH in acetone) to give 16 fractions (A – P). Fraction I (978.8 mg) was purified by CC (40 –100% CH2Cl2 in hexane) to afford yellow solids 18 (1.8 mg), 19 (2.1) and 20 (3.2). Fraction L (970.2 mg) was purified by CC (80 – 95% CH2Cl2 in hexane) to give fractions LA – LK. Fraction LG was further purified by CC (80 – 95% CH2Cl2 in hexane) to afford a colourless solid 36 (1.3 mg). Fraction N (244.4 mg) was purified on a Sephadex LH-20 column (20% MeOH in CH2Cl2) to give fractions NA – ND. Fraction NC was further purified by CC (1 –3% MeOH in CH2Cl2) to provide 10 (0.7 mg). Fraction O (2.0776 g) was purified on a Sephadex LH-20 column (20% MeOH in CH2Cl2) to give fractions OA –OE. Fraction OD was further purified by CC (40 –95% acetone in hexane) to give a yellow solid 37 (1.5 mg). Fraction P (2.3726 mg) was purified on a Sephadex LH-20 column (20% MeOH in CH2Cl2) to give fractions PA –PG. Fraction PE was further purified by CC (50% acetone in hexane) to afford a yellow solid 38 (2.3 mg). Depcitrus A (1): amorphous powder, m.p. 125– 1278C; HR-FAB-MS at [M þ 1]þ, m/z 501.2848 for C29H40O7 (calcd 501. 2870). UV lmax (CHCl3) (log 1): 265.0 (3.52), 286 (3.25), 311(3.88) and 400 (1.09). IR (neat) n (cm21) 1651 (CvO stretching). 1H NMR (300 MHz, CDCl3) d 11.27 (s, 2-OH), 6.58 (d, 2.1, H-50 ), 6.45 (d, 3.0, H-3), 6.28 (d, 3.0, H-5), 6.52 (d, 2.1, H-30 ), 4.06 (s, H-7), 3.84 (s, 20 -OMe), 3.82 (s, 4-OMe), 2.37 (t, H-9), 1.42 (m, H-10), 1.20 (m, H-11), 1.10 (m, H-12), 0.86 (t, H-14), 2.75 (t, 7.5, H-70 ), 1.53 (m, H-80 ), 1.42 (m, H-90 ), 1.35 (m, H-100 ), 1.20 (m, H-110 ), 1.10 (m, H-120 ), 0.82 (t, H-130 ), and 13C NMR (75 MHz, CDCl3) d 207.2 (C-8), 166.6 (C-2), 169.1 (C-4), 164.9 (C-14), 158.0 (C-40 ), 151.6 (C-20 ), 145.4 (C-10 ), 139.0 (C-6), 119.5 (C-60 ), 115.5 (C-50 ), 113.4 (C-5), 104.4 (C-1), 103.2 (C-30 ), 100.1 (C-3), 56.4 (20 -OMe), 42.5 (C-9), 33.9 (C-70 ), 31.7 (C-11), 31.3, (C-80 ), 30.7(C-100 ), 29.7 (C-12, C-110 ), 23.3 (C-10, C-90 ), 22.4 (C-120 ), 13.9 (C-13), 13.8 (C-130 ). Depcitrus B (14): HR-FAB-MS m/z: HR-EI-MS at m/z 294.1472 for C16H22O5 (calcd for 294.1462). UV lmax (MeOH) (log 1): 266.5 (3.61). IR (neat) n (cm21): 1765 (CvO stretching). 1 H NMR (500 MHz, CDCl3) d 11.27 (2 – OH), d 6.45 (d, 2.0, H-3), 6.28 (d, 2.0,H-5), 3.84 (s, 4-OMe), 3.83 (s, 1-CO2Me) 4.06 (s, H-7), 2.37 (t, 7.5, H-9), 1.42 (H-10), 1.20 (H-11), 1.10 (H-12), 0.86 (t, H-14), and 13C NMR (125 MHz, CDCl3) d 207.2(C-8), 172.0 (1-CO2Me), 167.0 (C-2), 164.0 (C-4), 140.0 (C-6), 110.0 (C-5), 107.0 (C-1), 97.0 (C-3), 55.4 (4-OMe), 51.9 (CO2Me), 48.9 (C-7), 41.9 (C-9), 30.1 (C-10, C-11), 23.4 (C-12). 13 * C Chemical shifts were obtained from HMQC, HMBC and DEPT 135. 4. Conclusions In conclusion, a phytochemical investigation of the extracts from the peel, leaves and branch bark of C. reticulata Blanco species, found eight polymethoxy flavonoids, six acridone alkaloids, four flavonoids, four flavonoid glycosides, four coumarins, two isocoumarins, two depsides, two resorcylic derivatives, one coumarin glucosides, one limonoid, one triterpenoid and three benzoic derivatives. This is the first report for the isolation of resorcylic derivatives
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and depside from Rutaceae. The chemical composition and their antioxidative, cytotoxic and antimicrobial results may contribute to the establishment of future use of this plant. Further investigation on their bioactivity may be worth in future studies for better exploiting the function of these substances. Supplementary material Supplementary material relating to this article is available online, alongside Table S1 and Figures S1 –S12.
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Acknowledgements We are grateful to the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program [grant number PHD/0129/2552] and PSU Ph.D. Scholarship for a scholarship to U. Phetkul. The Graduate School, Prince of Songkla University is gratefully thanked for partial financial support. Thanks to Dr Brian Hodgson for assistance with the English
References Abeysinghe DC, Li X, Sun Ch, Zhang W, Zhou Ch, Chen K. 2007. Bioactive compounds and antioxidant capacities in different edible tissues of citrus fruit of four species. Food Chem. 104:1338–1344. doi:org/10.1016/j.foodchem. 2007.01.047 Al-Mekhlafi NA, Shaari K, Abas F, Kneer R, Jeyaraj EJ, Stanslas J, Yamamoto N, Honda T, Lajis HN. 2012. Alkenylresorcinols and cytotoxic activity of the constituents isolated from Labisia pumila. Phytochemistry. 80:42–49. doi:org/10.1016/j.phytochem.2012.04.008 Bao L, Wang M, Zhao F, Zhao Y, Liu H. 2010. Two new resorcinol derivatives with strong cytotoxicity from the roots of Ardisia brevicaulis Diels. Chem Biodiv. 7:2901–2907. Li S, Pan MH, Lo CY, Tan D, Wang Y, Shahidi F, Ho CT. 2009. Chemistry and health effects of polymethoxyflavones and hydroxylated polymethoxyflavones. J Funct Food. 1:2–12. doi: 10.1021/jf102730b Phetkul U, Wanlaso N, Mahabusarakam W, Phongpaichit S, Carroll AR. 2013. New acridone from the wood of Citrus reticulata. Nat Prod Res. 27(20):1922–1926. doi: 10.1080/14786419.2013.793687
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
New depside from Citrus reticulata Blanco a
bc
Uraiwan Phetkul , Souwalak Phongpaichit , Ramida d
Watanapokasin & Wilawan Mahabusarakam
ac
a
Department of Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand b
Department of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand c
Natural Products Center of Excellence, Faculty of Science, Prince of Songkla,University, Hat Yai, Songkhla 90112, Thailand d
Department of Biochemistry, Faculty of Medicine, Srinkharinwirot University, Bangkok 10110, Thailand Published online: 18 Mar 2014.
To cite this article: Uraiwan Phetkul, Souwalak Phongpaichit, Ramida Watanapokasin & Wilawan Mahabusarakam (2014) New depside from Citrus reticulata Blanco, Natural Product Research: Formerly Natural Product Letters, 28:13, 945-951, DOI: 10.1080/14786419.2014.896010 To link to this article: http://dx.doi.org/10.1080/14786419.2014.896010
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Natural Product Research, 2014 Vol. 28, No. 13, 945–951, http://dx.doi.org/10.1080/14786419.2014.896010
New depside from Citrus reticulata Blanco
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Uraiwan Phetkula, Souwalak Phongpaichitbc, Ramida Watanapokasind and Wilawan Mahabusarakamac* a Department of Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand; bDepartment of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand; cNatural Products Center of Excellence, Faculty of Science, Prince of Songkla, University, Hat Yai, Songkhla 90112, Thailand; dDepartment of Biochemistry, Faculty of Medicine, Srinkharinwirot University, Bangkok 10110, Thailand
(Received 20 December 2013; final version received 14 February 2014) A new depside, named depcitrus A (1), and 31 known compounds were isolated from the peels, leaves and branch barks of Citrus reticulata Blanco. Methylation of the high polarity fractions from the branch barks and peels gave one new methylated compound named depcitrus B (14) and five known compounds. Their structures were established based on spectroscopic evidence. The antioxidant, antimicrobial and cytotoxic activities of some pure compounds were evaluated. Keywords: Rutaceae; Citrus reticulata; depside; resorcylic derivatives; antibacterial activity; cytotoxicity activity
1. Introduction Citrus species have been used to treat many symptoms of diseases in humans such as coughs, skin inflammation, muscle pains, stomach upsets and ringworm infections (Li et al. 2009). They have been reported to be a rich source of bioactive compounds (Abeysinghe et al. 2007). In Thailand, various Citrus reticulata species are grown. In order to understand the chemical constituents and to search for useful bioactive metabolites, we have previously investigated the extract from the wood of C. reticulata, a tree that grows in the southern part of Thailand (Phetkul et al. 2013). In this work, we further investigated the extracts from leaves, peels and branch bark, and evaluated their antioxidant, antimicrobial and cytotoxic activities. 2. Results and discussion Extraction and purification of the CH2Cl2 extract of branch barks, CH2Cl2 and Me2CO extracts of the peels and the leaves resulted in the isolation of 13 compounds (1– 13) from the branch barks, 12 compounds (18 –29) from the peel and 11 compounds (10, 18– 20, 32 –38) from the leaves (Figure 1). The high polar fractions were methylated and further purified to give four (14– 17) and two (30, 31) methylated compounds, respectively. The structures of these compounds were elucidated by analyses of spectroscopic data as well as comparison of their spectroscopic data with the previously published data. The compounds were identified as depcitrus A 1, atranorin 2, 5-hydroxynoracronycine 3, citflavanone 4, 8-hydroxy-6-methoxy-pentylisocoumarin 5, citracridone-I 6, citrusinol 7, citrusinine-I 8, citramine 9, scopoletin 10, limonin 11, citracridone-III 12, 4-hydroxybenzoic
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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Figure 1. Structure of compounds from C. reticulata Blanco (1– 38), 4-hydroxy-2-methoxy-6-[(8Z)pentadec-8-en-1-yl]phenyl acetate, 4-hydroxy-2-methoxy-6-pentadecylphenyl acetate and 1-O-methyl-6acetoxy-5-(pentadec-10Z-enyl)resorcinol.
acid 13, depcitrus B 14, sphaerophorol dimethyl ether 15, 6,8-dimethoxypentylisocoumarin 16, citracridone-II 17, 5-demethylnobiletin 18, tangeretin 19, nobletin 20, tetramethyl-O-isocutellarein 21, natsudaidai 22, 3,4-dihydroxy benzoic acid 23, 8,30 -b-glucosyloxy-20 -hydroxy-30 -methylbutyl7-methoxy-coumarin 24, 5-hydroxy-7,40 -dimethoxyflavone 25, hesperidin 26, eriocitrin 27, narirutin 28, rutin 29, naringenin trimethyl ether 30, 2,3-dihydro-5-hydroxy-40 ,7-dimethoxyflavanone 31, butulinic acid 32, gardenin B 33, tymusin 34, 7-geranyloxy coumarin 35, 4hydroxybenzaldehyde 36, crenulatin 37 and 5-(2-enyl-3-methylbut)oxy-7-hydroxycoumarin 38. Depcitrus A (1) is a new depside whereas depcitrus B (14) is a new resorcylic derivative. Compounds 2 – 10, 12, 13, 15 –17, 23, 24, 25, 30, 31, 34, 35, 36, 37 and 38 were reported for the first time from C. reticulata Blanco. Compounds 11, 18– 22, 26– 29 and 33 were previously isolated. Depcitrus A (1) was obtained as an amorphous powder, m.p. 125– 1278C. Its UV spectrum showed absorption peaks at 265, 286, 311 and 400 nm. The IR spectrum showed the absorption band of CvO stretching at 1726 cm21. A molecular ion in the FAB-MS at m/z 501.2848 corresponded to a molecular formula of C29H40O7. The 13C NMR spectrum showed the resonances of four methine aromatic carbons: d 100.1 (C-3), 113.4 (C-5), 103.2 (C-30 ), 115.5 (C-50 ), three quaternary aromatic carbons: d 104.4 (C-1), 139.0 (C-6), 119.5 (C-60 ), five oxy aromatic
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carbons 166.6 (C-2), 169.1 (C-4), 158.0 (C-40 ), 151.6 (C-20 ), 145.4 (C-10 ), two methoxyl carbons: d 55.5 (4-OMe), 56.4 (20 -OMe), a carbonyl ester carbon: d 164.9 (C-14) and a carbonyl ketone: d 207.2 (C-8). The 1H NMR spectrum showed doublet resonances of meta-aromatic protons H-3 and H-5 at d 6.45 and d 6.28, and meta-aromatic protons H-30 and H-50 at d 6.52 and d 6.58, which indicated the presence of two 1,2,3,5-tetrasubstituted benzene rings. The COSY correlations of H-9 (2.37, t) to H-10 (1.42, m), H-10 to H-11 (1.20, m), H-11 to H-12 (1.10, m) and H-12 to H-13 (0.86, t), together with the HMBC correlations of H-9 (d 2.37) and H-7 (d 4.06, s) to the carbonyl carbon C-8 (d 207.2), indicated the presence of a 2-oxoheptyl side chain. Proton H-7 and a hydrogen bonded hydroxyl group resonating at d 11.27 showed a HMBC correlation to C-1 (d 104.4), which indicated the hydroxyl group (2-OH) and 2-oxoheptyl side chain ortho to the carbonyl ester. A methoxyl group at d 3.82 was 4-OMe due to H-3, H-5 and 4-OMe showing HMBC correlations to C-4. A heptyl side chain was deduced from the COSY correlations of H-70 (d 2.75, t) to H-80 (1.53, m), H-80 to H-90 (1.42, m), H-90 to H-100 (1.35, m), H-100 to H-110 (1.20, m), H-110 to H-120 (1.10, m) and H-120 to H-130 (0.82, t), and it was attached at C-60 because H-70 showed HMBC correlation to C-50 (d 115.5) and C-10 (d 145.4). The remaining methoxyl group (d 3.84, 20 -OMe) was assigned at C-20 according to the HMBC correlations of the methoxyl group and H-30 (d 6.52) to C-20 (d 151.6). The evidence that H-70 was further correlated to the oxy carbon at 145.4, while H-30 and H-50 were correlated to the oxy carbon at 145.4 and 158.0, together with the interpretation of the molecular formula of C29H40O7 ([M þ 1]þm/z 501.2848), implied the presence of a hydroxyl group and an ester bond formed between two aromatic rings. Two possible structures could be proposed with the ester bond attached at either the C-10 or C-40 and the hydroxyl group attached at either the C-40 or C-10 , respectively. A comparison of the 13C chemical shifts published for alkenylresorcinols; 4-hydroxy-2-methoxy-6-[(8Z)-pentadec-8-en-1-yl]phenyl acetate, 4-hydroxy-2-methoxy-6-pentadecylphenyl acetate and 1-O-methyl-6-acetoxy-5-(pentadec-10Z-enyl)resorcinol demonstrated that the carbons with an ester bond ortho to the alkyl and alkoxy groups consistently resonated at least 10 ppm further up field compared with the hydroxyl group meta to the alkyl and alkoxyl groups. Accordingly, the ester bond was assigned to attach at the C-10 (145.4) and the hydroxyl group was at C-40 (158.0) (Bao et al. 2010; Al-Mekhlafi et al. 2012). Therefore 2-hydroxy-4-methoxy-6-(2-oxoheptyl)- 20 -methoxy-40 -hydroxy-6-(hetyl)phenyl ester was assigned for 1. Depsitrus B (14) was obtained as a yellow viscous liquid. The UV spectrum showed maximum absorptions at 266.5 nm. The IR spectrum showed the absorption band of CvO stretching at 1725 cm21. A molecular ion in the FAB-MS at m/z 294.1472 corresponded to a molecular formula of C16H22O5. The 1H NMR spectrum showed resonances of meta-aromatic protons at d 6.45 (H-3), 6.28 (H-5), a hydrogen bonded hydroxy proton at d 11.27 (2-OH), a methoxyl group at d 3.84 (4-OMe), a methyl ester group at d 3.83 (1-CO2Me) and a 2-oxoheptyl group. The COSY and HMBC experiments indicated that the proton resonances at d 4.06 (H-7), 2.37 (H-9), 1.42 (H-10), 1.20 (H-11), 1.10 (H-12), 0.86 (H-13) and the carbonyl carbon resonance at d 207.2 (C-8) corresponded to a 2-oxoheptyl side chain as for depcitrus A (1). The NOSEY correlations of H-7 to H-5, and 4-OMe to H-5 and H-3 allowed an assignment to a 2-oxoheptyl group ortho to H-5, while a 4-OMe was ortho to H-5 and H-3. The hydroxyl proton was assigned to a 2-OH according to HMBC correlation of the 2-OH to C-3 (d 97.0). The chemical shift value of 2-OH (d 11.27) revealed that it formed a hydrogen bond to the adjacent group. Consequently, the remaining methyl ester group was placed at C-1 (d 107.0). Compound 14 was then assigned as methyl-2-hydroxy-4-methoxy-6-(2-oxoheptyl)-benzoate. Compounds 2– 4, 6 –8, 21, 35 and 38 were tested for antioxidative activity using the DPPH assays. Compounds 2 – 4, 6– 8, 21, 35 and 38 showed very weak activity with per cent scavenging of DPPH radical in the range of 0.65 – 23.27%. Compounds 5, 9, 10, 14– 17, 19, 22 –25, 30 –36 were not tested for antioxidative activity due to insufficient amounts.
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Compounds 2, 3, 6 –9, 11, 13, 18– 21, 35 and 38 were evaluated for cytotoxicity against cell lines A431, SKBR-3, T47D and AU565. Compounds 3, 6, 7, 8, 11 and 35 affected the growth of cell lines with IC50 values of less than 100 mg/mL. Compounds 2, 9, 13, 18 –21 and 38 showed no cytotoxicity against the tested cancer cell lines up to the final concentration of 100 mg/mL. Compounds 4, 5, 14 – 17, 22 – 34, 36 and 37 were not tested due to insufficient amounts. Compounds 2 –5, 7 –8, 10, 12, 19 –20 and the extracts of the peel, leaves and branch bark were tested for their antibacterial activity on Staphylococcus aureus ATCC25923, methicillinresistant S. aureus (MRSA) SK1 and Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC27853 and Cryptococcus neoforman ATCC90113, antifungal activity on Candida albicans NCPF3153, C. neoforman and Microsporum gypseum. The extracts and pure compounds 2, 3, 5, 7, 8, 10, 12, 19 and 20 had no effect on these microorganisms up to a dose of 200 mg/mL. Only 4 inhibited the growth of S. aureus ATCC25923, MRSA SK1with MIC values of 64, 64 mg/mL, respectively. Compounds 11, 13, 20, 23, 26 – 29 and 37 have previously been reported to have antioxidative activity. Compounds 3, 7, 8, 10 – 12, 18 –21 and 26 have been previously reported for their cytotoxicity on other cell lines. Compounds 2 and 11 have been previously reported for their antibacterial activity, therefore, we did not retest. 3. Experimental 3.1. General Melting points were recorded with a Fisher-Johns melting point apparatus and are uncorrected (Fisher-Johns Scientific Co, Waltham, MA, USA). Ultraviolet spectra were measured with a UV-160A spectrophotometer (Shimadzu, Kyoto, Japan). Principle bands (lmax) were recorded in wavelengths (nm) and log 1 in methanol solutions. Infrared spectra were obtained on a FTS165 FT-IR spectrophotometer and were recorded in wave numbers (cm21) (Perkin-Elmer, Shelton, CA, USA). 1H and 13C NMR spectra were recorded on an FT-NMR Bruker Ultra ShieldTM 300 MHz spectrometer (Bruker, Rheinstetten, Germany). The FAB-MS and HR-FABMS were obtained using a MAT 95 XL mass spectrometer (Thermo Finnigan, Bremen, Germany). Preparative thin-layer chromatography (PTLC) was carried out on 20 cm £ 20 cm glass plates with a 0.2 mm, thick layer of silica gel 60 GF254 (Merck, Darmstadt, Germany). Quick column chromatography (QCC) was performed using silica gel 60 H (Merck, Darmstadt, Germany) and column chromatography (CC) was performed using silica gel 100 (Merck, Darmstadt, Germany) or sephadex LH-20 gel (Pharmacia, Uppsala, Sweden) or silica gel RP-18 (40 – 63 mm, Merck, Darmstadt, Germany). Precoated plastic plates (20 £ 20 cm) with a 0.2 mm thick layer of silica gel 60 GF254, (Merck, Darmstadt, Germany) were used for TLC analysis. 3.2. Plant material The branch barks, leaves and peels of C. reticulata Blanco were collected from Sadao district, Songkhla province in the southern part of Thailand, in April 2011. Identification of the plants was made by J. Wai, Department of Biology, Faculty of Science, Prince of Songkla University. A voucher specimen U.Phetkul 1 (PSU) has been deposited in the Herbarium of the Department of Biology, Faculty of Science, Prince of Songkla University, Thailand. 3.3. Extraction and isolation Dried branch barks of C. reticulata Blanco (1.4 kg) were chopped and immersed in CH2Cl2 (7.5 L) at room temperature. After removal of the solvent by evaporation, a dark-brown gum (18.13 g) was obtained. This gum was subjected to QCC (a gradient of 10% acetone in hexane to
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80% MeOH in acetone) to give 14 fractions (A– N). Fraction A gave a colourless solid 2 (7.00 mg). Fraction H (714.2 mg) was purified by CC (20 – 50% acetone in hexane) to afford an orange solid 3 (17.0 mg), a yellow solid 4 (2.3 mg) and an amorphous powder 5 (2.1 mg). Fraction I (527.2 mg) was subjected to CC (20% acetone in hexane) to give an orange solid 6 (3.7 mg). Fraction J (966.3 mg) was purified by CC (20 – 100% acetone in hexane) to afford yellow solids 7 (11.7 mg), 8 (2.3 mg) and 9 (0.8 mg). Fraction L (890.2 mg) was subjected to separation on a Sephadex LH-20 column (20% CH2Cl2 in MeOH) to give fractions LA – LF. Fraction LC was further purified by CC (30% acetone in hexane) to give a white solid 10 (2.0 mg). Fraction M (651.0 mg) was subjected to separation on a Sephadex LH-20 column (20% CH2Cl2 in MeOH) to give fractions MA –MI. Fraction MC was further purified by CC (30 – 100% acetone in hexane) to give amorphous powder 1 (2.1 mg). Fraction N (2.9 g) was subjected to separation on a Sephadex LH-20 column (20% CH2Cl2 in MeOH) to give fractions NA –NI. Purification of fraction NB by CC (2% MeOH in CH2Cl2) provided a white solid 11 (18.0 mg), a yellow solid 12 (2.3 mg) and a white solid 13 (2.1 mg). Methylation of fraction LE (58.95 mg) with MeI (2.0 mL) and K2CO3 (10.0 mg) in Me2CO (1.0 mL) for 8 h and purified by CC (20% acetone in hexane) gave nine fractions (LEA-LEI). Fraction LEG (15.713 mg) was purified by PTLC (20% acetone in hexane) to give an amorphous powder 14 (0.71 mg). Fractions ND to NI were combined (NDI, 559.13 mg) and methylated with MeI (4.0 mL) and K2CO3 (20.0 mg) in Me2CO (2.0 mL). Separation of the reaction mixture on a Sephadex LH-20 column (20% CH2Cl2 in MeOH) gave three fractions (NDIA – NDIC). Fraction NDIC was purified by PTLC (20% acetone in hexane) to give yellow viscous liquids 15 (0.71 mg) and 16 (0.81 mg), white solids 11 (1.20 mg) and 16 (1.10 mg) and a yellow solid 17 (0.91 mg). Chopped-dried peel from fruits (893. 9 g) of C. reticulata was successively immersed in CH2Cl2 (1.5 L) and Me2CO (1.5 L) at room temperature (3 days £ 2 times). After removal of the solvent by evaporation, a dark-brown viscous CH2Cl2 extract (12.0124 g) and Me2CO extract (15.287 g) were obtained, respectively. The CH2Cl2 extract was dissolved in hexane to give a soluble (5.1007 g) and insoluble (6.9087 g) fraction. The CH2Cl2 insoluble fraction (6.9087 g) was subjected to a QCC (a gradient of 10% CH2Cl2 in hexane to 95% MeOH in CH2Cl2) to give 12 fractions (A– L). Fraction G (2.7178 mg) was purified by CC (1% MeOH in CH2Cl2) to afford yellow solids 18 (2.1 mg), 19 (2.3 mg) and 20 (4.1 mg). Fraction I (86.00 mg) was subjected on CC (20 –100% acetone in hexane) to give a yellow solid 21 (2.1 mg). Fraction J (70.1 mg) was purified by CC (20 – 100% acetone in hexane) to give fractions JA –JK. Fraction JF was purified by CC (30% acetone in hexane) to give a yellow solidd 22 (1.0 mg). The Me2CO extract was subjected to CC (a gradient of 30% CH2Cl2 in hexane to 95% MeOH in CH2Cl2) to give 16 fractions (A– P). Fraction H (873.2 mg) was purified by CC (30% CH2Cl2 in hexane) to afford yellow solids 18 (1.8 mg), 19 (3.0 mg) and 20 (5.1 mg). Fraction J (1.339 g) was subjected to CC (20 –100% acetone in hexane) to give a yellow solid 21 (2.5 mg). Fraction K (879.2 mg) was purified by CC (20% acetone in hexane) to afford a yellow solid 23 (1.2 mg). Fraction L (1.455 mg) was subjected to a reverse phase column (50% H2O in MeOH) to give a colourless solid 24 (2.0 mg). Fraction N (1.989 g) was purified on a reverse phase column (50% H2O in MeOH) to afford yellow solids 25 (2.1 mg), 26 (1.3 mg) and 27 (2.0 mg). Chromatography of fraction O (2.756 g) on a reverse phase column (50% H2O in MeOH) gave six fraction (OA –OF). Yellow solids 28 and 29 (2.0 mg) were obtained from fraction OC and OD, respectively. Methylation of fraction OF (238.9 mg) with MeI (4.0 mL) and K2CO3 (20.0 mg) in Me2CO (4.0 mL) for 8 h and purified by PTLC (20% acetone in hexane) gave yellow solids 30 (0.9 mg) and 31 (1.3 mg). Choppeddried leaves (1.4 kg) from C. reticulata were successively immersed in CH2Cl2 (14.0 L) and Me2CO (14.0 L) at room temperature (3 days £ 2 times). After removal of the solvent by evaporation, a dark-brown viscous CH2Cl2 extract (13.788 g) and Me2CO extract (17.1245 g) were obtained, respectively. The CH2Cl2 extract was dissolved in hexane to give soluble (5.18 g) and insoluble (8.599 g) fractions. The Me2CO extract was further fractionated in hexane to give a
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soluble (1.89 g) and an insoluble (15.235 g) fraction. The CH2Cl2 insoluble fractions (8.599 g) were separated by QCC (a gradient of 10% acetone in hexane to 50% MeOH in acetone) to give 12 fractions (A – L). Fraction F (876.3 mg) was purified by CC (20 – 50% acetone in hexane) to afford a white solid 32 (1.8 mg). Fraction I (541.2 mg) was purified by CC (20 –50% acetone in hexane) to afford yellow solids 18 (1.9 mg) and 19 (2.1 mg). Fraction J (556.8 mg) was purified on a Sephadex LH-20 column (20% MeOH in CH2Cl2) to give fractions JA –JE. Fraction JC was further purified by CC (20% acetone in hexane) affording yellow solids 20 (2.4 mg) and 33 (1.1 mg) and 34 (1.0 mg). Fraction K (987.3 mg) was purified on a Sephadex LH-20 column and eluted with CH2Cl2 – MeOH (1:4) to give fractions KA –KF. Fraction KD was further purified by CC (20 –50% acetone in hexane) to afford a yellow solid 35 (3.8 mg). The Me2CO insoluble fraction (15.235 g) was separated by QCC (a gradient of 5% acetone in hexane to 50% MeOH in acetone) to give 16 fractions (A – P). Fraction I (978.8 mg) was purified by CC (40 –100% CH2Cl2 in hexane) to afford yellow solids 18 (1.8 mg), 19 (2.1) and 20 (3.2). Fraction L (970.2 mg) was purified by CC (80 – 95% CH2Cl2 in hexane) to give fractions LA – LK. Fraction LG was further purified by CC (80 – 95% CH2Cl2 in hexane) to afford a colourless solid 36 (1.3 mg). Fraction N (244.4 mg) was purified on a Sephadex LH-20 column (20% MeOH in CH2Cl2) to give fractions NA – ND. Fraction NC was further purified by CC (1 –3% MeOH in CH2Cl2) to provide 10 (0.7 mg). Fraction O (2.0776 g) was purified on a Sephadex LH-20 column (20% MeOH in CH2Cl2) to give fractions OA –OE. Fraction OD was further purified by CC (40 –95% acetone in hexane) to give a yellow solid 37 (1.5 mg). Fraction P (2.3726 mg) was purified on a Sephadex LH-20 column (20% MeOH in CH2Cl2) to give fractions PA –PG. Fraction PE was further purified by CC (50% acetone in hexane) to afford a yellow solid 38 (2.3 mg). Depcitrus A (1): amorphous powder, m.p. 125– 1278C; HR-FAB-MS at [M þ 1]þ, m/z 501.2848 for C29H40O7 (calcd 501. 2870). UV lmax (CHCl3) (log 1): 265.0 (3.52), 286 (3.25), 311(3.88) and 400 (1.09). IR (neat) n (cm21) 1651 (CvO stretching). 1H NMR (300 MHz, CDCl3) d 11.27 (s, 2-OH), 6.58 (d, 2.1, H-50 ), 6.45 (d, 3.0, H-3), 6.28 (d, 3.0, H-5), 6.52 (d, 2.1, H-30 ), 4.06 (s, H-7), 3.84 (s, 20 -OMe), 3.82 (s, 4-OMe), 2.37 (t, H-9), 1.42 (m, H-10), 1.20 (m, H-11), 1.10 (m, H-12), 0.86 (t, H-14), 2.75 (t, 7.5, H-70 ), 1.53 (m, H-80 ), 1.42 (m, H-90 ), 1.35 (m, H-100 ), 1.20 (m, H-110 ), 1.10 (m, H-120 ), 0.82 (t, H-130 ), and 13C NMR (75 MHz, CDCl3) d 207.2 (C-8), 166.6 (C-2), 169.1 (C-4), 164.9 (C-14), 158.0 (C-40 ), 151.6 (C-20 ), 145.4 (C-10 ), 139.0 (C-6), 119.5 (C-60 ), 115.5 (C-50 ), 113.4 (C-5), 104.4 (C-1), 103.2 (C-30 ), 100.1 (C-3), 56.4 (20 -OMe), 42.5 (C-9), 33.9 (C-70 ), 31.7 (C-11), 31.3, (C-80 ), 30.7(C-100 ), 29.7 (C-12, C-110 ), 23.3 (C-10, C-90 ), 22.4 (C-120 ), 13.9 (C-13), 13.8 (C-130 ). Depcitrus B (14): HR-FAB-MS m/z: HR-EI-MS at m/z 294.1472 for C16H22O5 (calcd for 294.1462). UV lmax (MeOH) (log 1): 266.5 (3.61). IR (neat) n (cm21): 1765 (CvO stretching). 1 H NMR (500 MHz, CDCl3) d 11.27 (2 – OH), d 6.45 (d, 2.0, H-3), 6.28 (d, 2.0,H-5), 3.84 (s, 4-OMe), 3.83 (s, 1-CO2Me) 4.06 (s, H-7), 2.37 (t, 7.5, H-9), 1.42 (H-10), 1.20 (H-11), 1.10 (H-12), 0.86 (t, H-14), and 13C NMR (125 MHz, CDCl3) d 207.2(C-8), 172.0 (1-CO2Me), 167.0 (C-2), 164.0 (C-4), 140.0 (C-6), 110.0 (C-5), 107.0 (C-1), 97.0 (C-3), 55.4 (4-OMe), 51.9 (CO2Me), 48.9 (C-7), 41.9 (C-9), 30.1 (C-10, C-11), 23.4 (C-12). 13 * C Chemical shifts were obtained from HMQC, HMBC and DEPT 135. 4. Conclusions In conclusion, a phytochemical investigation of the extracts from the peel, leaves and branch bark of C. reticulata Blanco species, found eight polymethoxy flavonoids, six acridone alkaloids, four flavonoids, four flavonoid glycosides, four coumarins, two isocoumarins, two depsides, two resorcylic derivatives, one coumarin glucosides, one limonoid, one triterpenoid and three benzoic derivatives. This is the first report for the isolation of resorcylic derivatives
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and depside from Rutaceae. The chemical composition and their antioxidative, cytotoxic and antimicrobial results may contribute to the establishment of future use of this plant. Further investigation on their bioactivity may be worth in future studies for better exploiting the function of these substances. Supplementary material Supplementary material relating to this article is available online, alongside Table S1 and Figures S1 –S12.
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Acknowledgements We are grateful to the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program [grant number PHD/0129/2552] and PSU Ph.D. Scholarship for a scholarship to U. Phetkul. The Graduate School, Prince of Songkla University is gratefully thanked for partial financial support. Thanks to Dr Brian Hodgson for assistance with the English
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