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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
A new prenylated biflavonoid from the leaves of Garcinia dulcis a
b
Arun Saelee , Souwalak Phongpaichit & Wilawan ac
Mahabusarakam 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
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Natural Products Center of Excellence, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand Published online: 12 Feb 2015.
To cite this article: Arun Saelee, Souwalak Phongpaichit & Wilawan Mahabusarakam (2015): A new prenylated biflavonoid from the leaves of Garcinia dulcis, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2015.1010087 To link to this article: http://dx.doi.org/10.1080/14786419.2015.1010087
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Natural Product Research, 2015 http://dx.doi.org/10.1080/14786419.2015.1010087
A new prenylated biflavonoid from the leaves of Garcinia dulcis Arun Saeleea, Souwalak Phongpaichitb 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
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(Received 8 December 2014; final version received 17 January 2015)
OH HO
O
OH HO
O
OH O OH O
A new prenylated biflavonoid, named dulcisbiflavonoid A, together with five biflavonoids were isolated from the leaves of Garcinia dulcis. Their structures were elucidated by analysing their spectroscopic data, especially 1D and 2D NMR. Keywords: Guttiferae; Garcinia dulcis; biflavonoids
1. Introduction Garcinia dulcis Kurz. (Guttiferae) grows mainly in Southeast Asia. The leaves and seeds have been used in traditional medicine against lymphatitis, parotitis, struma and other disease conditions (Kasahara & Henmi 1986). This plant produces a wide range of secondary metabolites including biflavonoids, chromones, flavonoids, xanthones and triterpenoids (Ansari et al. 1976; Harrison et al. 1994; Deachathai et al. 2005). These compounds showed various activities such as antibacterial, antioxidative, cytotoxic, anti-HIV and hypocholesterolemic activity (Lin et al. 1997; Deachathai et al. 2005, Tuansulong et al. 2011, Lee et al. 2012). Our groups have been studying on the phytochemistry of G. dulcis that was collected in the southern part of Thailand, locally known as Ma-Phut. Many new compounds have been isolated including glycosides, isoflavones and xanthones from fruits, xanthones and chromones from flowers, and xanthones from seeds (Deachathai et al. 2005; 2006; 2008). In continuing, the leaves of G. dulcis have been further investigated for chemical constituents and antibacterial activity as well.
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
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2. Results and discussion The leaves of G. dulcis were extracted with acetone. The crude extract was dissolved in CH2Cl2 to give CH2Cl2-soluble and insoluble fractions. Extensive chromatography of the insoluble fraction gave six biflavonoids. Structural elucidation by analysis of spectroscopic data, especially by 1D and 2D NMR and comparison of the data to those previously reported indicated that dulcisbiflavonoid A (1) is a new prenylated biflavonoid (Figure 1). The known compounds were GB-2a (2), amentoflavone (3), morelloflavone (4), morelloflavone-70 -sulfate (5) and volkensiflavone (6) (Ansari et al. 1976; Cao et al. 1997; Li et al. 2002). Dulcisbiflavonoid A (1) was a light yellow powder, m.p. 233 –2348C, ½a25 D 2 17.2 (c ¼ 0.1, MeOH). A molecular ion in the EI-MS at m/z 674.2146 (calcd. 674.2152) corresponded to a molecular formula of C40H34O10. The UV spectrum showed absorption bands at lmax (log 1) 203 (4.69), 269 (4.47) and 339 (4.42) nm, which is characteristic of a compound containing an extended aromatic chromophore. The IR spectrum showed the absorption bands of OZH stretching at 3421 and CvO stretching at 1653 cm21. The 1H NMR spectrum showed singlet resonances of the methine olefinic protons H-3 and H-300 of flavonylflavone at d 6.82 and d 6.77, respectively (Kuo et al. 2000). The resonances of a chelated hydroxyl proton at d 12.96 (5-OH), olefinic methine proton at d 6.82 (H-3), meta aromatic protons at d 6.16 and d 6.43 (d, J ¼ 1.8 Hz each, H-6 and H-8), and meta aromatic protons at d 7.87 and d 7.76 (d, J ¼ 2.1 Hz each, H-20 and H-60 ) were assigned for the first unit. In the HMBC correlations of H-8 and 5-OH to C-4a (d 104.2), C-6 (d 99.3); H-6 to C-4a (d 104.2), C-8 (d 94.5); H-3 to C-4a (d104.2), C-10 (d 121.6), and H-20 /H-60 to C-2 (d 164.4) confirmed the assignment of 5-OH, H-8, H-6, H-3 and tetrasubstituted aromatic ring. The resonances of a chelated hydroxyl proton at d 13.38 (500 -OH), olefinic proton at d 6.77 (H-300 ) and aromatic protons at d 7.52 and 6.70 (AA0 BB0 pattern, J ¼ 8.7 Hz, H-2000 /H-6000 and H-3000 /H-5000 ) were assigned for the second unit. The HMBC
Figure 1. Structure of biflavonoids from the leaves of G. dulcis.
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correlation of H-300 to C-1000 (d 121.9) and H-2000 /H-6000 to C-200 (d 163.8) confirmed the placement of H-300 and disubstituted aromatic ring. The remaining signals corresponded to characteristic signals of two prenyl groups. According to COSY and HMBC correlations, the resonances at d 3.48 (d, J ¼ 7.2 Hz, H-70 ), d 5.42 (t, J ¼ 7.2 Hz, H-80 ) and d 1.74 (s, H-100 and H-110 ) were for 30 -prenyl group, whereas those at d 3.35 (d, J ¼ 6.6 Hz, H-900 ), d 5.23 (t, J ¼ 6.6 Hz, H-1000 ) and d 1.63, d 1.74 (s, H-1200 and H-1300 ) were for 600 -prenyl group. The HMBC correlations of H-20 to C-70 (d 29.3) and H-900 to C-500 (d 158.4), C-600 (d 112.0), suggested the 30 -prenyl group at C-30 and the 600 -prenyl group at C-600 . The 50 -800 biflavones was established from the HMBC correlation of H-60 to C-800 (d 104.2). To complete the structure, the hydroxyl groups were substituted at C-7, C-40 , C-700 and C-4000 . The chemical shifts of C-7 (d 164.5), C-40 (d 158.4), C700 (d 161.9) and C-4000 (d 161.4) were indicated from the HMBC correlations of H-6, H-8 to C-7, H-20 / H-60 to C-40 , H-900 to C-700 , and H-2000 / H-6000 to C-4000 . Each flavone unit corresponded to yinyanghuo D (Chen et al. 1996) and 6-prenylapigenin (Lee et al. 1998). The 13C NMR spectrum exhibited 12 methine, 2 methylene, 4 methyl and 22 quaternary carbons. Dulcisbiflavonoid A (1) therefore was assigned as 8-(5-(5,7-dihydroxy-4-oxo-4H-chromen-2-yl)-2-hydroxy-3-(3methylbut-2-enyl)phenyl)-5,7-dihydroxy-2-(4-hydroxyphenyl)-6-(3-methylbut-2-enyl)-4Hchromen-4-one, or 30 ,600 -bis(3-methylbut-2-enyl) amenthoneflavone. Dulcisbiflavonoid A (1) and GB-2a (2) were tested for their antibacterial activity. GB-2a (2) was found to inhibit the growth of both the Staphylococcus aureus ATCC25923 and methicillinresistant S. aureus SK1 with minimum inhibitory concentration (MIC) values of 128 and 64 mg/ mL, respectively while the standard drugs vancomycin and gentamicin had a MIC values 0.5 mg/ mL. They could not inhibit the growth of Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC27853 at 200 mg/mL. However, dulcisbiflavonoid A (1) showed no antibacterial activity against four bacterial with MIC greater than 200 mg/mL. Amentoflavone (3), morelloflavone (4) and volkensiflavone (6) have been previously reported for their antibacterial activity, therefore, we did not retest. 3. Experimental 3.1. General experimental procedures Melting points were recorded with a digital electrothermal melting point apparatus (Electrothermal 9100). 1H and 13C NMR spectra were recorded with a FT-NMR Bruker Avance 300 MHz spectrometer in DMSO-d6, with TMS as the internal standard. Column chromatography was performed using columns of silica gel 100, silica gel 60H (Merck), Sephadex LH-20 and RP-18 (40-63 mm, Merck, Darmstadt, Germany). Precoated sheets of silica gel 60 F254 were used for TLC analysis. High-resolution mass spectra were recorded on a MAT 95 XL Mass Spectrometer. 3.2. Plant material The leaves of G. dulcis were collected from Songkhla province in the southern part of Thailand. The voucher specimen (Coll. No. 02, Herbarium No. 0012652) has been deposited at the Prince of Songkla University Herbarium, Biology Department, Faculty of Science, Prince of Songkla University, Thailand. 3.3. Extraction and isolation The leaves of G. dulcis (800 g) were extracted at room temperature with acetone. Removal of the solvent from the extract yielded the acetone extract (45.19 g). The extract was dissolved in CH2Cl2 to give CH2Cl2-soluble (A 23.96 g) and insoluble (B 21.23 g) fractions. The insoluble
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fraction (B) was subjected to CC on Sephadex LH-20 and eluted with 70% MeOHZCH2Cl2 to yield four fractions (B1– B4). Fraction B2 was concentrated and left at room temperature. A yellow solid 4 (40.0 mg) which was formed was collected by filtration. The filtrate (8.80 g) was further purified by CC on silica gel and eluted with CH2Cl2ZMeOH (7.0:3.0) to give nine fractions (C1 – C9). Fraction C2 (34.0 mg) was purified by CC on silica gel and eluted with C6H14ZCH2Cl2ZMe2CO (2.5:1.5:6.0) to give a light yellow powder 1 (4.3 mg). Fraction C5 (362.4 mg) was purified by CC on silica gel and eluted with C6H14ZCH2Cl2ZMe2CO (5:2:3) to give a yellow solid 2 (3.0 mg). Fraction C6 (730.5 mg) was purified by CC on silica gel and eluted with CH2Cl2ZMeOH (9.5:0.5) to give a yellow powder 3 (5.5 mg). Fraction B3 (374.0 mg) was purified by CC on silica gel and eluted with MeOHZCH2Cl2 (1.8:8.2) to give 12 fractions (D1 –D12). Fraction D7 (78.2 mg) was purified by CC silica gel and eluted with MeOHZCH2Cl2 (1.5:8.5) to give a yellow solid 5 (9.0 mg). Fraction B4 (1.53 g) was purified by CC on reversed-phase and eluted with MeOHZH2O (2:8) to give 15 fractions (E1 – E15). Fraction E14 (80.0 mg) was purified by CC on silica gel and eluted with MeOHZCH2Cl2 (7:3) to give a yellow solid 6 (1.2 mg). Dulcisbiflavonoid A (1), light yellow powder, m.p. 233 –2348C, ½a25 D 2 17.2 (c ¼ 0.1, MeOH). EI-MS m/z (% rel. int): [M]þ674.5, 44), 673.5 (100), 673.5 (46), 574.5 (55), 120.9 (49). HR-EI-MS at [M]þm/z 674.2146 for C40H34O10. UV (MeOH) lmax (nm) (log 1): 339 (4.42), 269 (4.47), 203 (4.69). IR (neat) nmax (cm21): 3421 (OZH stretching) and 1653 (CvO stretching). 1 H-NMR (DMSO-d6, 300 MHz): d ¼ 13.38 (s, 500 -OH), 12.96 (s, 5-OH), 7.87 (d, J ¼ 2.1 Hz, H20 ), 7.76 (d, J ¼ 2.1 Hz, H-60 ), 7.52 (d, J ¼ 8.7 Hz, H-2000 and H-6000 ) 6.82 (s, H-3), 6.77 (s, H-300 ), 6.70 (d, J ¼ 8.7 Hz, H-3000 and H-5000 ), 6.43 (d, J ¼ 1.8 Hz, H-8), 6.16 (d, J ¼ 1.8 Hz, H-6), 5.42 (t, J ¼ 7.2 Hz, H-80 ), 5.23 (t, J ¼ 6.6 Hz, H-1000 ), 3.48 (d, J ¼ 7.2 Hz, H-70 ), 3.35 (d, J ¼ 6.6 Hz, H-900 ), 1.74 (s, H-100 , H-110 and H-1300 ) and 1.63 (s, H-1200 ). 13C-NMR (DMSO-d6, 75 MHZ): d ¼ 182.7 (C-400 ), 182.1 (C-4), 164.5 (C-7), 164.4 (C-2), 163.8 (C-200 ), 161.9 (C-5 and C-700 ), 161.4 (C-4000 ), 158.4 (C-40 and C-500 ), 157.8 (C-8a), 153.3 (C-8a00 ), 132.5 (C-90 ), 131.1 (C-1100 ), 130.1 (C-30 ), 129.8 (C-60 ), 128.6 (C-2000 and C-6000 ), 128.0 (C-20 ), 122.9 (C-1000 ), 122.8 (C-80 ), 121.9 (C-1000 ), 121.6 (C-10 ), 120.1 (C-50 ), 116.2 (C-3000 and C-5000 ), 112.0 (C-600 ), 104.4 (C-4a00 ), 104.2 (C-4a and C-800 ), 103.5 (C-3), 102.9 (C-300 ), 99.3 (C-6), 94.5 (C-8), 29.3 (C-70 ), 26.0 (C1200 ), 26.1 (C-110 ), 21.9 (C-900 ), 18.3 (C-100 ), 18.2 C-1300 ).
3.4. Antibacterial activity The compounds were screened for antibacterial activity at a concentration of 200 mg/mL by a broth microdilution method against four bacteria: S. aureus ATCC25923, a clinical isolate of methicillin-resistant S. aureus SK1, E. coli ATCC25922, P. aeruginosa ATCC27853. Compounds having antibacterial activity were further determined for their MICs by broth microdilution methods according to a modification of the Clinical and Laboratory Standards Institute. Resazurin was added as viability indicator. Colorimetric MIC end-points were interpreted as the lowest concentration that remained blue (indicating no growth) or the first dilution that changed from blue to slightly purple (equivalent to prominent growth inhibition) (Rattanaburi et al. 2013).
4. Conclusion Biflavonoids with prenyl groups is a small class of biflavonoids in natures (Cao et al. 1997). This is the first report of prenylated biflavonoids and morelloflavone-700 -sulfate in G. dulcis. Phytochemical study of other parts of this plant and bioactivity would be further conducted.
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Supplementary material Table S1 and Figures S1 –S4 relating to this article are available online. Funding This research was supported by a scholarship from the National Research University (NRU), Commission on Higher Education, and the Graduate School, Prince of Songkla University.
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References Ansari WH, Rahman W, Barraclough D, Maynard MR, Scheinmann F. 1976. Biflavanoids and a flavanone-chromone from the leaves of Garcinia dulcis (Roxb.) Kurz. J Chem Soc Perkin Trans I. 13:1458– 1463. doi:10.1039/ p19760001458. Cao S-G, Sim K-Y, Goh S-H. 1997. Biflavonoids of Calophyllum venulosum. J Nat Prod. 60:1245– 1250. doi:10.1021/ np970303f. Chen C-C, Huang Y-L, Sun CM, Shen C-C, Ko F-N, Teng C-M. 1996. New prenylflavones from the leaves of Epimedium sagittatum. J Nat Prod. 59:412–414. doi:10.1021/np9601925. Deachathai S, Mahabusarakam W, Phongpaichit S, Taylor WC. 2005. Phenolic compounds from the fruit of Garcinia dulcis. Phytochemistry. 66:2368–2375. doi:10.1016/j.phytochem.2005.06.025. Deachathai S, Mahabusarakam W, Phongpaichit S, Taylor WC, Zhang Y-J, Yang C-R. 2006. Phenolic compounds from the flowers of Garcinia dulcis. Phytochemistry. 67:464–469. doi:10.1016/j.phytochem.2005.10.016. Deachathai S, Phongpaichit S, Mahabusarakam W. 2008. Phenolic compounds from the seeds of Garcinia dulcis. J Nat Prod. 22:1327–1332. doi:10.1080/14786410601130406. Harrison LJ, Leong L-S, Leong Y-W, Sia G-L, Sim K-Y, Tan HT-W. 1994. Xanthone and flavonoid constituents of Garcinia dulcis (Guttiferae). Nat Prod Lett. 5:111–116. doi:10.1080/10575639408044043. Kasahara S, Henmi S. 1986. Medicine herb index in Indonesia. Jakarta: Eisai Indonesia. Kuo Y-H, Lin C-H, Hwang SY, Shen Y-C, Lee YL, Li S-Y. 2000. A novel cytotoxic c-methylated biflavone from the stem of Cephalotaxus wilsoniana. Chem Pharm Bull. 48:440–441. doi:10.1248/cpb.48.440. Lee S-J, Wood AR, Maier CGA, Dixon RA, Mabry TJ. 1998. Prenylated flavonoids from Maclura pomifera. Phytochemistry. 49:2573–2577. doi:10.1016/S0031-9422(98)00380-X. Lee E, Shin S, Lee J-Y, Lee S, Kim J-K, Yoon D-Y, Woo E-R, Kim Y. 2012. Cytotoxic activities of amentoflavone against human breast and cervical cancers are mediated by increasing of PTEN expression levels due to peroxisome proliferator-activated receptor g activation. Bull Korean Chem Soc. 33:2219–2223. doi:10.5012/ bkcs.2012.33.7.2219. Li X-C, Joshi AS, Tan B, Elsohly HN, Walker LA, Zjawiony J K, Ferreira D. 2002. Absolute configuration, conformation, and chiral properties of flavanone-(3 ! 800 )-flavone biflavonoids from Rheedia acuminata. Tetrahedron. 58:8709–8717. doi:10.1016/S0040-4020(02)01096-7. Lin Y-M, Anderson H, Flavin MT, Pai Y-H, Mata-Greenwood E, Pengsuparp T, Pezzuto JM, Schinazi RF, Hughes SH, Chen F-C, Lin Y-M, Anderson H, Flavin MT, Pai Y-H. 1997. In vitro anti-HIV activity of biflavonoids isolated from Rhus succedanea and Garcinia multiflora. J Nat Prod. 60:884–888. doi:10.1021/np9700275. Rattanaburi S, Mahabusarakam W, Phongpaichit S, Carroll AR. 2013. Acylphloroglucinols from Callistemon lanceolatus DC. Tetrahedron. 69:6070–6075. doi:10.1016/j.tet.2013.05.085. Tuansulong K, Hutadilok-Towatana N, Mahabusarakam M, Pinkaew D, Fujise K. 2011. Morelloflavone from Garcinia dulcis as a novel biflavonoid inhibitor of HMG-CoA reductase. Phytother Res. 25:424–428. doi:doi./10.1002/ptr. 3286.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
A new prenylated biflavonoid from the leaves of Garcinia dulcis a
b
Arun Saelee , Souwalak Phongpaichit & Wilawan ac
Mahabusarakam 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
Click for updates
Natural Products Center of Excellence, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand Published online: 12 Feb 2015.
To cite this article: Arun Saelee, Souwalak Phongpaichit & Wilawan Mahabusarakam (2015): A new prenylated biflavonoid from the leaves of Garcinia dulcis, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2015.1010087 To link to this article: http://dx.doi.org/10.1080/14786419.2015.1010087
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
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Natural Product Research, 2015 http://dx.doi.org/10.1080/14786419.2015.1010087
A new prenylated biflavonoid from the leaves of Garcinia dulcis Arun Saeleea, Souwalak Phongpaichitb 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
Downloaded by [New York University] at 00:55 19 June 2015
(Received 8 December 2014; final version received 17 January 2015)
OH HO
O
OH HO
O
OH O OH O
A new prenylated biflavonoid, named dulcisbiflavonoid A, together with five biflavonoids were isolated from the leaves of Garcinia dulcis. Their structures were elucidated by analysing their spectroscopic data, especially 1D and 2D NMR. Keywords: Guttiferae; Garcinia dulcis; biflavonoids
1. Introduction Garcinia dulcis Kurz. (Guttiferae) grows mainly in Southeast Asia. The leaves and seeds have been used in traditional medicine against lymphatitis, parotitis, struma and other disease conditions (Kasahara & Henmi 1986). This plant produces a wide range of secondary metabolites including biflavonoids, chromones, flavonoids, xanthones and triterpenoids (Ansari et al. 1976; Harrison et al. 1994; Deachathai et al. 2005). These compounds showed various activities such as antibacterial, antioxidative, cytotoxic, anti-HIV and hypocholesterolemic activity (Lin et al. 1997; Deachathai et al. 2005, Tuansulong et al. 2011, Lee et al. 2012). Our groups have been studying on the phytochemistry of G. dulcis that was collected in the southern part of Thailand, locally known as Ma-Phut. Many new compounds have been isolated including glycosides, isoflavones and xanthones from fruits, xanthones and chromones from flowers, and xanthones from seeds (Deachathai et al. 2005; 2006; 2008). In continuing, the leaves of G. dulcis have been further investigated for chemical constituents and antibacterial activity as well.
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
Downloaded by [New York University] at 00:55 19 June 2015
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A. Saelee et al.
2. Results and discussion The leaves of G. dulcis were extracted with acetone. The crude extract was dissolved in CH2Cl2 to give CH2Cl2-soluble and insoluble fractions. Extensive chromatography of the insoluble fraction gave six biflavonoids. Structural elucidation by analysis of spectroscopic data, especially by 1D and 2D NMR and comparison of the data to those previously reported indicated that dulcisbiflavonoid A (1) is a new prenylated biflavonoid (Figure 1). The known compounds were GB-2a (2), amentoflavone (3), morelloflavone (4), morelloflavone-70 -sulfate (5) and volkensiflavone (6) (Ansari et al. 1976; Cao et al. 1997; Li et al. 2002). Dulcisbiflavonoid A (1) was a light yellow powder, m.p. 233 –2348C, ½a25 D 2 17.2 (c ¼ 0.1, MeOH). A molecular ion in the EI-MS at m/z 674.2146 (calcd. 674.2152) corresponded to a molecular formula of C40H34O10. The UV spectrum showed absorption bands at lmax (log 1) 203 (4.69), 269 (4.47) and 339 (4.42) nm, which is characteristic of a compound containing an extended aromatic chromophore. The IR spectrum showed the absorption bands of OZH stretching at 3421 and CvO stretching at 1653 cm21. The 1H NMR spectrum showed singlet resonances of the methine olefinic protons H-3 and H-300 of flavonylflavone at d 6.82 and d 6.77, respectively (Kuo et al. 2000). The resonances of a chelated hydroxyl proton at d 12.96 (5-OH), olefinic methine proton at d 6.82 (H-3), meta aromatic protons at d 6.16 and d 6.43 (d, J ¼ 1.8 Hz each, H-6 and H-8), and meta aromatic protons at d 7.87 and d 7.76 (d, J ¼ 2.1 Hz each, H-20 and H-60 ) were assigned for the first unit. In the HMBC correlations of H-8 and 5-OH to C-4a (d 104.2), C-6 (d 99.3); H-6 to C-4a (d 104.2), C-8 (d 94.5); H-3 to C-4a (d104.2), C-10 (d 121.6), and H-20 /H-60 to C-2 (d 164.4) confirmed the assignment of 5-OH, H-8, H-6, H-3 and tetrasubstituted aromatic ring. The resonances of a chelated hydroxyl proton at d 13.38 (500 -OH), olefinic proton at d 6.77 (H-300 ) and aromatic protons at d 7.52 and 6.70 (AA0 BB0 pattern, J ¼ 8.7 Hz, H-2000 /H-6000 and H-3000 /H-5000 ) were assigned for the second unit. The HMBC
Figure 1. Structure of biflavonoids from the leaves of G. dulcis.
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correlation of H-300 to C-1000 (d 121.9) and H-2000 /H-6000 to C-200 (d 163.8) confirmed the placement of H-300 and disubstituted aromatic ring. The remaining signals corresponded to characteristic signals of two prenyl groups. According to COSY and HMBC correlations, the resonances at d 3.48 (d, J ¼ 7.2 Hz, H-70 ), d 5.42 (t, J ¼ 7.2 Hz, H-80 ) and d 1.74 (s, H-100 and H-110 ) were for 30 -prenyl group, whereas those at d 3.35 (d, J ¼ 6.6 Hz, H-900 ), d 5.23 (t, J ¼ 6.6 Hz, H-1000 ) and d 1.63, d 1.74 (s, H-1200 and H-1300 ) were for 600 -prenyl group. The HMBC correlations of H-20 to C-70 (d 29.3) and H-900 to C-500 (d 158.4), C-600 (d 112.0), suggested the 30 -prenyl group at C-30 and the 600 -prenyl group at C-600 . The 50 -800 biflavones was established from the HMBC correlation of H-60 to C-800 (d 104.2). To complete the structure, the hydroxyl groups were substituted at C-7, C-40 , C-700 and C-4000 . The chemical shifts of C-7 (d 164.5), C-40 (d 158.4), C700 (d 161.9) and C-4000 (d 161.4) were indicated from the HMBC correlations of H-6, H-8 to C-7, H-20 / H-60 to C-40 , H-900 to C-700 , and H-2000 / H-6000 to C-4000 . Each flavone unit corresponded to yinyanghuo D (Chen et al. 1996) and 6-prenylapigenin (Lee et al. 1998). The 13C NMR spectrum exhibited 12 methine, 2 methylene, 4 methyl and 22 quaternary carbons. Dulcisbiflavonoid A (1) therefore was assigned as 8-(5-(5,7-dihydroxy-4-oxo-4H-chromen-2-yl)-2-hydroxy-3-(3methylbut-2-enyl)phenyl)-5,7-dihydroxy-2-(4-hydroxyphenyl)-6-(3-methylbut-2-enyl)-4Hchromen-4-one, or 30 ,600 -bis(3-methylbut-2-enyl) amenthoneflavone. Dulcisbiflavonoid A (1) and GB-2a (2) were tested for their antibacterial activity. GB-2a (2) was found to inhibit the growth of both the Staphylococcus aureus ATCC25923 and methicillinresistant S. aureus SK1 with minimum inhibitory concentration (MIC) values of 128 and 64 mg/ mL, respectively while the standard drugs vancomycin and gentamicin had a MIC values 0.5 mg/ mL. They could not inhibit the growth of Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC27853 at 200 mg/mL. However, dulcisbiflavonoid A (1) showed no antibacterial activity against four bacterial with MIC greater than 200 mg/mL. Amentoflavone (3), morelloflavone (4) and volkensiflavone (6) have been previously reported for their antibacterial activity, therefore, we did not retest. 3. Experimental 3.1. General experimental procedures Melting points were recorded with a digital electrothermal melting point apparatus (Electrothermal 9100). 1H and 13C NMR spectra were recorded with a FT-NMR Bruker Avance 300 MHz spectrometer in DMSO-d6, with TMS as the internal standard. Column chromatography was performed using columns of silica gel 100, silica gel 60H (Merck), Sephadex LH-20 and RP-18 (40-63 mm, Merck, Darmstadt, Germany). Precoated sheets of silica gel 60 F254 were used for TLC analysis. High-resolution mass spectra were recorded on a MAT 95 XL Mass Spectrometer. 3.2. Plant material The leaves of G. dulcis were collected from Songkhla province in the southern part of Thailand. The voucher specimen (Coll. No. 02, Herbarium No. 0012652) has been deposited at the Prince of Songkla University Herbarium, Biology Department, Faculty of Science, Prince of Songkla University, Thailand. 3.3. Extraction and isolation The leaves of G. dulcis (800 g) were extracted at room temperature with acetone. Removal of the solvent from the extract yielded the acetone extract (45.19 g). The extract was dissolved in CH2Cl2 to give CH2Cl2-soluble (A 23.96 g) and insoluble (B 21.23 g) fractions. The insoluble
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A. Saelee et al.
fraction (B) was subjected to CC on Sephadex LH-20 and eluted with 70% MeOHZCH2Cl2 to yield four fractions (B1– B4). Fraction B2 was concentrated and left at room temperature. A yellow solid 4 (40.0 mg) which was formed was collected by filtration. The filtrate (8.80 g) was further purified by CC on silica gel and eluted with CH2Cl2ZMeOH (7.0:3.0) to give nine fractions (C1 – C9). Fraction C2 (34.0 mg) was purified by CC on silica gel and eluted with C6H14ZCH2Cl2ZMe2CO (2.5:1.5:6.0) to give a light yellow powder 1 (4.3 mg). Fraction C5 (362.4 mg) was purified by CC on silica gel and eluted with C6H14ZCH2Cl2ZMe2CO (5:2:3) to give a yellow solid 2 (3.0 mg). Fraction C6 (730.5 mg) was purified by CC on silica gel and eluted with CH2Cl2ZMeOH (9.5:0.5) to give a yellow powder 3 (5.5 mg). Fraction B3 (374.0 mg) was purified by CC on silica gel and eluted with MeOHZCH2Cl2 (1.8:8.2) to give 12 fractions (D1 –D12). Fraction D7 (78.2 mg) was purified by CC silica gel and eluted with MeOHZCH2Cl2 (1.5:8.5) to give a yellow solid 5 (9.0 mg). Fraction B4 (1.53 g) was purified by CC on reversed-phase and eluted with MeOHZH2O (2:8) to give 15 fractions (E1 – E15). Fraction E14 (80.0 mg) was purified by CC on silica gel and eluted with MeOHZCH2Cl2 (7:3) to give a yellow solid 6 (1.2 mg). Dulcisbiflavonoid A (1), light yellow powder, m.p. 233 –2348C, ½a25 D 2 17.2 (c ¼ 0.1, MeOH). EI-MS m/z (% rel. int): [M]þ674.5, 44), 673.5 (100), 673.5 (46), 574.5 (55), 120.9 (49). HR-EI-MS at [M]þm/z 674.2146 for C40H34O10. UV (MeOH) lmax (nm) (log 1): 339 (4.42), 269 (4.47), 203 (4.69). IR (neat) nmax (cm21): 3421 (OZH stretching) and 1653 (CvO stretching). 1 H-NMR (DMSO-d6, 300 MHz): d ¼ 13.38 (s, 500 -OH), 12.96 (s, 5-OH), 7.87 (d, J ¼ 2.1 Hz, H20 ), 7.76 (d, J ¼ 2.1 Hz, H-60 ), 7.52 (d, J ¼ 8.7 Hz, H-2000 and H-6000 ) 6.82 (s, H-3), 6.77 (s, H-300 ), 6.70 (d, J ¼ 8.7 Hz, H-3000 and H-5000 ), 6.43 (d, J ¼ 1.8 Hz, H-8), 6.16 (d, J ¼ 1.8 Hz, H-6), 5.42 (t, J ¼ 7.2 Hz, H-80 ), 5.23 (t, J ¼ 6.6 Hz, H-1000 ), 3.48 (d, J ¼ 7.2 Hz, H-70 ), 3.35 (d, J ¼ 6.6 Hz, H-900 ), 1.74 (s, H-100 , H-110 and H-1300 ) and 1.63 (s, H-1200 ). 13C-NMR (DMSO-d6, 75 MHZ): d ¼ 182.7 (C-400 ), 182.1 (C-4), 164.5 (C-7), 164.4 (C-2), 163.8 (C-200 ), 161.9 (C-5 and C-700 ), 161.4 (C-4000 ), 158.4 (C-40 and C-500 ), 157.8 (C-8a), 153.3 (C-8a00 ), 132.5 (C-90 ), 131.1 (C-1100 ), 130.1 (C-30 ), 129.8 (C-60 ), 128.6 (C-2000 and C-6000 ), 128.0 (C-20 ), 122.9 (C-1000 ), 122.8 (C-80 ), 121.9 (C-1000 ), 121.6 (C-10 ), 120.1 (C-50 ), 116.2 (C-3000 and C-5000 ), 112.0 (C-600 ), 104.4 (C-4a00 ), 104.2 (C-4a and C-800 ), 103.5 (C-3), 102.9 (C-300 ), 99.3 (C-6), 94.5 (C-8), 29.3 (C-70 ), 26.0 (C1200 ), 26.1 (C-110 ), 21.9 (C-900 ), 18.3 (C-100 ), 18.2 C-1300 ).
3.4. Antibacterial activity The compounds were screened for antibacterial activity at a concentration of 200 mg/mL by a broth microdilution method against four bacteria: S. aureus ATCC25923, a clinical isolate of methicillin-resistant S. aureus SK1, E. coli ATCC25922, P. aeruginosa ATCC27853. Compounds having antibacterial activity were further determined for their MICs by broth microdilution methods according to a modification of the Clinical and Laboratory Standards Institute. Resazurin was added as viability indicator. Colorimetric MIC end-points were interpreted as the lowest concentration that remained blue (indicating no growth) or the first dilution that changed from blue to slightly purple (equivalent to prominent growth inhibition) (Rattanaburi et al. 2013).
4. Conclusion Biflavonoids with prenyl groups is a small class of biflavonoids in natures (Cao et al. 1997). This is the first report of prenylated biflavonoids and morelloflavone-700 -sulfate in G. dulcis. Phytochemical study of other parts of this plant and bioactivity would be further conducted.
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Supplementary material Table S1 and Figures S1 –S4 relating to this article are available online. Funding This research was supported by a scholarship from the National Research University (NRU), Commission on Higher Education, and the Graduate School, Prince of Songkla University.
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