Research article Received: 5 December 2013
Revised: 10 March 2014
Accepted: 12 March 2014
Published online in Wiley Online Library: 3 April 2014
(wileyonlinelibrary.com) DOI 10.1002/mrc.4071
Complete NMR assignments of bioactive rotameric (3 → 8) biflavonoids from the bark of Garcinia hombroniana Nargis Jamila,a* Melati Khairuddean,a Sadiq Noor Khanb and Naeem Khanc The genus Garcinia is reported to possess antimicrobial, anti-inflammatory, anticancer, hepatoprotective and anti-HIV activities. Garcinia hombroniana in Malaysia is used to treat itching and as a protective medicine after child birth. This study was aimed to isolate the chemical constituents from the bark of G. hombroniana and explore their possible pharmacological potential. Ethyl acetate extract afforded one new (1) and six (2–7) known 3 → 8 rotameric biflavonoids. Their structures were elucidated by UV, IR and NMR (1D and 2D) spectroscopy together with electron ionization/ESI mass spectrometric techniques and were identified as (2R, 3S) volkensiflavone-7-O-rhamnopyranoside (1), volkensiflavone (2), 4″-O-methyl-volkensiflavone (3), volkensiflavone-7-O-glucopyranoside (4), morelloflavone (5), 3″-O-methyl-morelloflavone (6) and morelloflavone-7-Oglucopyranoside (7). The absolute configuration of compound 1 was assigned by circular dichroism spectroscopy as 2R, 3S. The coexistence of conformers of isolated biflavonoids in solution at 25 °C in different solvents was confirmed by variable temperature NMR studies. At room temperature (25 °C), compounds 1–7 exhibited duplicate NMR signals, while at elevated temperature (90 °C), a single set of signals was obtained. Compound 5 showed significant in vitro antioxidant activities against 1,1-diphenyl-2-picrylhydrazyl and 2,2′-azino-bis-3-ethyl benzthiazoline-6-sulfonic acid radicals. The antibacterial studies showed that compounds 5 and 6 are the most active against Staphylococcus aureus, Bacillus subtilis and Escherichia coli. Compounds 3 and 6 also showed moderate antituberculosis activity against H38Rv. Based on the research findings, G. hombroniana could be concluded as a rich source of flavanone–flavone (3 → 8) biflavonoids that exhibit rotameric behaviour at room temperature and display significant antioxidant and antibacterial activities. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: NMR; 1H NMR; antituberculosis
13
C NMR; variable temperature NMR; circular dichroism; Garcinia hombroniana; biflavonoids; antioxidant;
Introduction
Magn. Reson. Chem. 2014, 52, 345–352
* Correspondence to: Nargis Jamila, School of Chemical Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia. E-mail: [email protected] a School of Chemical Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia b Department of Medical Lab Technology, University of Haripur, 22060 Haripur, Pakistan c Department of Chemistry, Kohat University of Science and Technology, 26000 Kohat, Khyber Pakhtunkhwa, Pakistan
Copyright © 2014 John Wiley & Sons, Ltd.
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Garcinia, the largest genus of the family Clusiaceae, is native to the regions of Asia, northeast Australia, southern Africa and tropical America.[1] This genus is found to be a rich source of bioactive secondary metabolites, including oxygenated prenylated xanthones and benzophenones, flavonoids, biflavonoids and triterpenes.[2–5] These chemical constituents are well known for their pharmacological potential including antimicrobial, antiHIV, anticancer, anti-inflammatory and hepatoprotective activities.[6–8] Garcinia hombroniana, a seashore mangosteen in Malaysia, is used as a protective medicine after child birth and to cure skin allergies.[9,10] Biflavonoids are dimers of flavanone–flavone, flavone–flavone and flavone–flavonol combinations, possessing significant pharmacological activities such as antimicrobial, antiallergic, anti-inflammatory, hepatoprotective, anti-HIV and antioxidant.[11–16] They are reported to occur in several species of plants, but only a few of them such as Ginkgo biloba and Garcinia kola are found to be their rich sources.[17,18] Biflavonoids from G. kola have shown potent hypoglycaemic, hypolipidaemic and antioxidant effects compared with simple monomeric flavonoids.[19–21] Among the 450 species of Garcinia, G. hombroniana Pierre appeared to be one of the rarely studied. The previous phytochemical investigations of its stem wood, pericarp, leaves and twigs revealed the presence of xanthones, flavonoids and triterpenes.[22–24] The phenolic and triterpenoid constituents from
the twigs of G. hombroniana demonstrated copper-mediated low density lipoprotein antioxidation, antiplatelet aggregation and antibacterial activities.[25] To the best of our knowledge, so far, there is no authentic chemical and pharmacological investigation on the bark of G. hombroniana. In view of the limited data, traditional uses and possible pharmacological applications of G. hombroniana, this study was designed to investigate the biochemical constituents from the bark fractions of G. hombroniana. In our ongoing research, the ethyl acetate bark extract over silica gel column chromatography has resulted in the isolation of one new (1) and six known (2–7) flavanone–flavone 3 → 8 rotameric biflavonoids (Fig. 1). Their structures were elucidated using spectral techniques of NMR, EI/ESI-MS and circular dichroism (CD). The NMR data were recorded at variable temperature (90, 80, 70, 60, 50, 40, 30, 25, 0, 10, 20, 30, 40, 50 and 70 °C) and in three different solvents [dimethyl sulfoxide-d6 (DMSO-d6),
N. Jamila et al.
Figure 1. Chemical structures of compounds 1–7.
methanol-d4 and acetone-d6]. The antioxidant potential of the purified compounds (>92%) employing 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis-3-ethyl benzthiazoline-6-sulfonic acid (ABTS) and ferric reducing ability of plasma (FRAP) assays; antibacterial capacities against Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa and Escherichia coli and antituberculosis activities against H38Rv were also determined.
Results and Discussion
346
Compound 1 was obtained as a yellow solid. The molecular formula C36H30O14 was deduced from low resolution ESI-MS (positive mode) and HRESI-MS (negative mode) techniques (Fig. S1 (a) and (b), Supporting Information). Molecular ions appeared at m/z 687.9 [M + H]+ and 685.1572 [M H]+ (calcd. 685.1635) respectively. The TLC and ESI-MS data showed that it is a pure compound. However, the 1H and 13C NMR spectra exhibited double sets of signals. Therefore, it was a bit difficult to identify
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this compound by the NMR data recorded at ambient temperature. The duplicate signals in the NMR spectra of biflavonoids at room temperature are because of the presence of conformations that arise from the restricted free rotation of 3 → 8 interflavanyl bond.[26] These baffling NMR data were converted to a single set of signals at higher temperature because at elevated temperature, the hindered rotation becomes faster and faster, and the molecules do not exist in a preferred stable conformation any more.[27] The 1H NMR spectrum at 25 °C (Fig. S2, Supporting Information) demonstrated duplicate signals indicating two conformers. The 1H NMR resonances of the major conformer were displayed in the deshielded aromatic and shielded saccharadic region. A set of two doublets of two protons each at δH 8.01 (H-2″/6″) and 6.97 (H-3″/5″) and another set of two doublets of two integrals at δH 7.08 (H-2′/6′) and 6.36 (H-3′/5′) with J(s) of 8.0–9.0 Hz indicated a para substitution pattern in rings II-E and I-B respectively. Another pair of doublets of two protons that appeared at δH 6.02 and 5.96 (H-6/8) with a J value of 2.0 Hz was a clue to a meta substitution in ring A. Two singlets at δH 6.88 and 6.66 were assigned to H-II-3 and H-II-6 of rings F and D respectively. Furthermore, another pair of doublets in a non-aromatic region at δH 5.51 and 5.15 with a J of 12.0 Hz was respectively assigned to H-2 and H-3 of the ring I-C in the flavanone unit. The coupling constant (12.0 Hz) of these two protons verified their diaxial (trans) configuration. In addition, the 1H NMR spectrum also displayed signals, characteristic of a sugar moiety (rhamnopyranosyl) containing resonances of anomeric protons at δH 5.35 (H-1‴) and a complex mutiplet of non-anomeric protons in the chemical shift regions of δH 3.73–3.40 and a doublet of methyl protons at δH 1.18 ppm. The 13C NMR spectrum at 25 °C (Fig. S3, Supporting Information) also showed duplicate signals of 36 carbons that could be assigned to 1 methyl, 19 methine and 16 quaternary carbons, deduced from DEPT 135, 90 and Q NMR spectra (Fig. S4, Supporting Information). The complete assignments of 1H and 13C NMR of the two conformers at 25 °C are given in Tables 1 and 2 respectively. The 1H and 13C NMR spectra (Figs S5 and S6, Supporting Information) recorded at elevated temperature (90 °C) showed changes in resonances and appeared as a single set of signals. The 1H–1H COSY spectrum (Fig. S7, Supporting Information) showed correlations of the protons H-2′/6′ to protons H-3′/5′ of the flavanone unit and cross peaks of H-2″/6″ with the protons H-3″/5″ of the flavone unit, both of the major as well as the minor conformers, which further confirmed the para substituted B and E rings of the flavanone and flavone units respectively. The vicinal protons H-2 and H-3 on the C ring of the flavanone unit were also verified by their COSY correlations. The 1H–13C HSQC spectrum (Fig. S8, Supporting Information) helped in assigning the protonated carbons by showing cross peaks of the protons to their respective carbons. The rhamnopyranosyl moiety was located at C-7 of ring D in the flavone unit as evidenced by the 1H–13C HMBC correlations (Fig. S9, Supporting Information) of the anomeric proton at δH 5.35 with C-7 (δC 159.8) and C-6 (δC 97.9) of the D ring. The spatial associations of protons were determined by 1H–1H NOESY spectrum (Fig. S10, Supporting Information), in which the protons at δH 8.01 (H2″/6″) showed cross peaks to the protons at δH 6.97 (H-3″/5″), δH 7.08 (H-2′/6′), δH 6.66 (H-6 of the D ring) and δH 5.15 (H-3 of the C ring). The NOESY spectrum also showed correlations of the protons at δH 8.01 (II-2″/6″) of the major conformer to the protons at δH 7.66 II-2″/6″ of the minor conformer. This abnormal NOE happened because of the exchange of
Copyright © 2014 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2014, 52, 345–352
1
Copyright © 2014 John Wiley & Sons, Ltd.
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A
12.1 (s) 5.66 (d, 12.5) 5.04 (d, 12.5) 5.96 (d, 2.0) 5.96 (d, 2.0) 7.08 (d, 8.0) 6.61 (m) 6.61 (m) 7.08 (d, 8.0)
B
b
5.27 (br. s) Overlapped With H2O peak 3.30 (m) 3.30 (m) 1.07 (d, 7.0)
B
b
A
a
(MeOD-d4) 5.63 (d, 6.0) 4.0 (br. s) 3.81 (m) 3.44 (br. s) 3.51-3.48 (m) 1.31 (d, 6.0)
A
a
B
b
5.49 (d, 6.0) 3.94 (br. s) 3.62 (m) 3.39 (d, 3.0) 3.42 (br. s) 1.22 (d, 6.0)
B
b
12.0 (s) 5.46 (d, 12.0) 5.07 (d, 12.0) 6.02 (br. s) 6.02 (br. s) 7.02 (d, 8.3) 6.66 (d, 7.9) 6.66 (d, 7.9) 7.02 (d, 8.3)
(MeOD-d4)
12.1 (s) 5.69 (d, 12.5) 5.32 (d, 12.0) 5.92 (br. s) 5.92 (br. s) 7.02 (d, 8.3) 6.35 (d, 8.3) 6.35 (d, 8.3) 7.02 (d, 8.3)
Rhamnopyranoside unit
(DMSO-d6)
5.35 (s) 3.58 (br. s) 3.73 (br. s) 3.40 (m) 3.49 (m) 1.18 (d, 7.0)
a
12.2 (s) 5.51 (d, 12.0) 5.15 (d, 12.0) 6.02 (d, 2.0) 6.06 (d, 2.0) 7.08 (d, 8.0) 6.36 (d, 8.5) 6.36 (d, 8.5) 7.08 (d, 8.0)
A
(DMSO-d6)
DMSO-d6 and MeOD-d4, 500 MHz. Sets A and B are respectively in an intensity ratio of (~1 : 0.62). a A represents major conformer at 25 °C. b B represents minor conformer at 25 °C.
1‴ 2‴ 3‴ 4‴ 5‴ 6‴
H no.
I-5-OH I-2 I-3 I-6 I-8 I-2′ I-3′ I-5′ I-6′
H no
347
Magn. Reson. Chem. 2014, 52, 345–352
a
Flavanone unit (I)
H NMR data of the major and minor conformers of 1 at 25 °C
δH in ppm (multiplicity, J in Hz)
Table 1.
II-5-OH II-3 II-6 II-2″ II-3″ II-5″ II-6″
H no.
A
-
B
b
13.0 (s) 6.73 (s) 6.45 (s) 7.66 (d, 9.0) 6.64 (m) 6.64 (m) 7.66 (d, 9.0)
(DMSO-d6)
13.1 (s) 6.88 (s) 6.66 (m) 8.01 (d, 9.0) 6.97 (d, 9.0) 6.97 (d, 9.0) 8.01 (d, 9.0)
a
a
A
B
b
12.1 (s) 6.62 (s) 6.42 (br. s) 7.56 (d, 8.5) 6.68 (d, 8.5) 6.68 (d, 8.5) 7.56 (d, 8.5)
(MeOD-d4)
12.2 (s) 6.72 (s) 6.52 (s) 7.72 (d, 8.5) 6.83 (d, 8.5) 6.83 (d, 8.5) 7.72 (d, 8.5)
Flavone unit (II)
Rotameric biflavonoids from Garcinia hombroniana
N. Jamila et al. Table 2.
13
C NMR data of the major and minor conformers of 1 at 25 °C
δC (C-type)* Flavanone unit (I) C no. I-2 I-3 I-4 I-5 I-6 I-7 I-8 I-9 I-10 I-1′ I-2′ I-3′ I-4′ I-5′ I-6′
a
A
81.6 (CH) 48.1 (CH) 196.0 (C) 163.9 (C) 96.4 (CH) 167.1 (C) 96.4 (CH) 162.7 (C) 101.7 (C) 127.8 (C) 128.3 (CH) 114.5 (CH) 157.5 (C) 114.5 (CH) 128.3 (CH)
Flavone unit (II) b
B
C no.
81.3 47.9 195.8 163.8 95.4 166.7 95.4 162.5 101.3 127.5 128.2 114.4 157.3 114.4 128.2
II-2 II-3 II-4 II-5 II-6 II-7 II-8 II-9 II-10 II-1″ II-2″ II-3″ II-4″ II-5″ II-6″
a
Rhamnopyranoside unit b
A
164.2 (C) 103.2 (CH) 181.9 (C) 161.3 (C) 97.9 (CH) 159.8 (C) 102.4 (C) 154.5 (C) 105.0 (C) 120.9 (C) 129.0 (CH) 115.9 (CH) 160.9 (C) 115.9 (CH) 129.0 (CH)
B
C no.
164.0 102.4 181.8 161.1 97.2 159.5 102.2 153.7 104.5 120.5 128.6 115.6 160.5 115.6 128.6
1‴ 2‴ 3‴ 4‴ 5‴ 6‴
a
A
100.2 (CH) 71.8 (CH) 69.6 (CH) 70.2 (CH) 70.3 (CH) 17.8 (CH3)
b
B
100.0 71.4 69.4 70.0 70.2 17.8
DMSO-d6, 125 MHz. * C-type was deduced from DEPT experiments. a A represents major conformer at 25 °C. b B represents minor conformer at 25 °C.
rotational conformations of the two conformers,[28] and it revealed that 3 → 8 biflavonoids are not a simple mixture of two conformers but are in a dynamic system. The absolute configuration of 1 was examined by CD spectrum (Fig. 2), in which a positive Cotton effect was observed at 287 nm (π → π* transition), suggesting a 2β-orientation of the B-ring and hence a 2R absolute configuration of C-2 in the flavanone unit. According to Gaffield (1970),[29] the more reliable Cotton effect for the determination of C-2 stereochemistry is near 290 nm (π → π* transition) than the Cotton effect at a longer wavelength (n → π* transition) as the latter becomes smaller when the amount of the opposite enantiomer is increased. The diagnostic absorption at 287 nm is characteristic of the flavanone-type chromophore. The stereochemistry of the C-3 relative to C-2 of the flavanone unit was then determined by applying Cahn–Ingold–Prelog
348
Figure 2. CD spectrum of 1 at 25 °C.
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sequence rules according to which C-3 was assigned as 3S. This configuration was also supported by the coupling constants (12.0 Hz) of H-2 and H-3 in a 1H NMR experiment that suggested a trans relative configuration of C-2 and C-3. The previously mentioned spectral evidence established the structure of 1 as (2R, 3S) volkensiflavone-7-O-rhamnopyranoside. We also recorded and compared the 1H and 13C NMR spectra at variable temperatures ranging from 80 to 70 °C (Figs S11 and S12, Supporting Information) and in three different solvents, dimethyl sulfoxide-d6, methanol-d4 and acetone-d6. At elevated temperature (90 °C), the duplicate signals were collapsed and appeared as a clear single set of signals. The 1H NMR spectra at lower temperature (0 to 70 °C) also exhibited changes and converted to a single set of signals at 50 to 70 °C. At lower temperature, the hindered rotation of 3 → 8 interflavanyl bond becomes slower and slower and is low enough to produce a preferred stable conformation. These observed results of different temperature NMR studies revealed the rotameric behaviour of 3 → 8 biflavonoids and close association of their NMR with temperature. The duplicate NMR data of 3 → 8 biflavonoids at room temperature were first reported by Jackson et al. (1971).[30] The NMR spectra were recorded in dimethyl sulfoxide-d6, methanold4 and acetone-d6 at 25 °C to investigate the effect of solvents on the rotameric behaviour of 3 → 8-type biflavonoids. The 13C NMR spectra in these three solvents (Figs S3, S13 and S14, Supporting Information) at 25 °C exhibited the same spectral complexity of duplicate signals, which revealed that the solvents do not affect the rotamerism of 3 → 8-type biflavonoids. Based on the comparison of 1D and 2D NMR data of the compounds 2–7 with the published literature, they were identified as volkensiflavone (2), 4″-O-methyl-volkensiflavone (3), volkensiflavone-7-O-glucopyranoside (4), morelloflavone (5), 3″-O-methyl-morelloflavone (6) and morelloflavone-7-O-
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Magn. Reson. Chem. 2014, 52, 345–352
Rotameric biflavonoids from Garcinia hombroniana glucopyranoside (7).[31–33] Their 1H and 13C NMR spectra and complete assignments given in Figs S15–S20 and Tables S1–S6 are shown in the Supporting Information. In antioxidant studies, compounds 5, 6 and 7 showed the highest potential in DPPH, ABTS and FRAP assays with the most active compound 5 displaying a half maximal inhibitory concentration (IC50) of 4.08 μM in DPPH and 3.90 μM in ABTS respectively, even higher than the standards, trolox (IC50 24.8 μM in DPPH and 12.6 μM in ABTS) and gallic acid (IC50 7.79 μM in DPPH). However, in the ABTS assay, compound 5 was found equipotent to gallic acid. Compound 5 also showed higher reducing ability [298.8 μM trolox equivalent (TE)] in the FRAP assay. The complete inhibition of the DPPH free radical by the compounds and trolox was attained at 50 μM except for the compound 5 and gallic acid that was attained completely at 20 μM. On the other hand, compounds 2, 3, 5 and 6 and trolox were found to have complete inhibition of ABTS+ at 30 μM, while compounds 1, 4 and 7 inhibited ABTS+ at a concentration of 50 μM. Gallic acid was found to be the most active inhibitor of ABTS+ (99%) at a concentration of about 10 μM. The detailed results of the antioxidant assays and DPPH and ABTS inhibition profiles of the compounds 1–7 and standards at various concentrations are respectively shown in Table 3 and Fig. 3. In regard to the compounds’ properties as an antibacterial, compounds 5 and 6 showed the highest activity with the minimum inhibitory concentration (MIC) of 62.5 μM each against S. aureus, B. subtilis and E. coli, followed by compounds 2 and 3. The rest of the compounds were found moderately active. In antituberculosis studies, only compounds 3 and 6 showed a moderate inhibition with the MIC of 108.5 and 102.3 μM respectively, while the other compounds were inactive (Table 4). A new biflavonoid (1) did not significantly contribute to all the tested biological activities.
Figure 3. DPPH and ABTS radical inhibition profiles of compounds 1–7 and standards (trolox and gallic acid).
Chemicals and bacterial strains
Materials and Methods Plant materials Plant materials were collected from Penang Botanical Garden, Malaysia, and verified by Mr Saul Hamid Pakir Mohamed. A voucher specimen (PBGK12) has been deposited at the Herbarium of Penang Botanical Garden, Malaysia. Table 3. Antioxidant activities of compounds 1–7 Compounds
1 2 3 4 5 6 7 Trolox Gallic acid
DPPH assay
ABTS assay
FRAP assaya
(IC50, μM)
(IC50, μM)
(μM TE)
i
37.3 ± 3.49 f 17.3 ± 2.8 g 23.27 ± 2.91 h 28.5 ± 1.54 a 4.08 ± 0.74 b 5.70 ± 0.69 d 11.9 ± 0.78 e 24.1 ± 0.62 c 7.79 ± 0.45
i
35.5 ± 0.93 f 16.6 ± 0.25 g 20.8 ± 1.28 h 29.1 ± 0.31 b 3.90 ± 0.51 c 5.87 ± 1.35 d 11.8 ± 1.96 e 12.6 ± 0.08 a 3.41 ± 0.03
d
12.6 ± 1.24 c 10.9 ± 0.96 b 7.50 ± 1.29 a 4.34 ± 0.17 d 293.5 ± 5.16 f 206.1 ± 4.35 e 187.2 ± 2.57 — —
General experimental procedure Silica gel 60 (0.040–0.063 mm) was used as an adsorbent for column chromatography. The semi-pure compounds were finally purified over Sephadex LH-20 (bead size 25–100 μ, Sigma-Aldrich) with MeOH (100%). Merck TLC plates (silica gel 60F254) were used for the detection of the purity of compounds. Melting points were determined with a Stuart Scientific Melting Point SMP 1 (UK). UV spectra were recorded using a PerkinElmer Lambda 25 UV/Vis spectrometer. IR spectra were recorded in KBr on a PerkinElmer 2000 FT-IR spectrophotometer. EI/ESI mass spectra were recorded on an Agilent 5975C MSD and ThermoFinnigan MAT95XL mass spectrometers at National University of Singapore,
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349
a FRAP values are expressed as μM TE at 20 μM concentration. Superscript lowercase letters (a–i) represent significant differences (p < 0.05) of the antioxidant activities of the compounds 1–7.
Magn. Reson. Chem. 2014, 52, 345–352
All the solvents were of analytical or HPLC grade. The chemicals of antioxidant activities, DPPH (96%), ABTS (>98%), 2,4,6Tripyridyl-s-Triazine (>98%), trolox (97%) and gallic acid (97.5%), were purchased from Sigma-Aldrich (Steinheim, Germany) and Merck (Darmstadt, Germany). Two gram-positive bacteria, S. aureus (ATCC 29213) and B. subtilis (ATCC 19659), and two gram-negative bacteria, P. aeruginosa (ATCC 17588) and E. coli (ATCC 25922), Mueller Hinton Broth, nutrient agar, piodonitrotetrazolium chloride, vincomycin (99%), gentamicin (99%) and streptomycin (98%) were purchased from Oxoid (England), Sigma-Aldrich (USA) and Duchefa Biochemie (the Netherlands). Mycobacterium tuberculosis H38Rv was purchased from American Type Culture Collection (Rockville, USA).
N. Jamila et al. Table 4. Antibacterial and antituberculosis activities of compounds 1–7 Antibacterial activity Compounds
1 2 3 4 5 6 7 Vancomycin* Streptomycin* Gentamicin*
Antituberculosis activity (μM) Minimum inhibitory concentration (MIC), μM
Disc diffusion, mm S. aureus
B. subtilis
P. aeruginosa
E. coli
b 12.0 ± 0.1 c 13.5 ± 0.3 a 7.5 ± 0.2 a 8.0 ± 0.1 a 7.0 ± 0.2 d 16.0 ± 0.0 d 16.0 ± 0.1 e 21.0 ± 0.0
a 12.5 ± 0.2 a 13.0 ± 0.2 b 15.0 ± 0.3 b 15.0 ± 0.2 d 26.0 ± 0.1 c 23.0 ± 0.0 c 22.0 ± 0.1
a 8.0 ± 0.1 a 8.0 ± 0.2 b 10.0 ± 0.3 a 8.0 ± 0.1 c 19.5 ± 0.0
8.0 ± 0.2 b 12.0 ± 0.1 c 14.5 ± 0.2 a 7.0 ± 0.1 d 15.5 ± 0.0 f 16.0 ± 0.1 c 14.5 ± 0.2 b 12.0 ± 0.0 d 15.0 ± 0.1 g 25.0 ± 0.1
a
S. aureus
B. subtilis
P. aeruginosa
E. coli
d 250 c 125 b 62.5 b 62.5 e 500 e 500 a 15.25
d 250 c 125 b 62.5 b 62.5 e 500 e 500 a 15.25
b 62.5 c 250 c 250 c 250 d 500 a 31.5
250 c 250 c 250 b 62.5 b 62.5 d 500 a 7.5
c
H38Rv d 108.5 c 102.3 ND a 0.60 b 12.5
Results are mean values of triplicate (n = 3). Superscript lowercase letters (a–g) represent significant differences at p < 0.05. - = no inhibition observed; * = positive control
Singapore. The absolute configuration of 1 was determined using a Jasco-815 spectrometer at 25 °C (scan range λ = 200–350 nm, cell length = 10 mm).
NMR spectra
350
The NMR experiments of compounds 1–7 were performed at variable temperatures ranging from 90 to 70 °C in DMSO-d6, MeOD-d4 and acetone-d6 using a Bruker Ascend 500 MHz (1H) and 125 MHz (13C) spectrometer equipped with a Bruker 5 mm Broadband probe. Chemical shifts (δ) in ppm are referenced to tetramethylsilane at 0.00 ppm for 1H and 13C. Coupling constants are expressed in hertz (Hz). Pulse programme (pulprog) for 1H at 25 °C = zg30, spectrometer frequency (SF) = 500.13 MHz, spectral width (SWH) = 10 330.57 Hz, f1 channel-90° high power pulse (P1) = 13.20 μs, size of the real spectrum (SI) = 65 536, line broadening factor for em (LB) = 0 Hz, acquisition time (AQ) = 3.17 s, relaxation delay 1–5 * T1 (D1) = 1.00 s and number of dummy scan (DS) = 2. Pulprog for 1H at 90 °C is same as for the 1H at 25 °C. Pulprog for 13C in DMSO-d6, MeOD-d4 and Me2CO-d6 at 25 °C = zgpg30, SF = 125.75 MHz, SWH = 29 761.90 Hz, f1 channel-90° high power pulse (P1) = 10.90 μs, SI = 32768, LB = 1.00 Hz, AQ = 1.10 s, relaxation delay 1–5 * T1 (D1) = 2.00 s and DS = 4. Pulprog for 13C in DMSO-d6 at 90 °C = zgpg30, SF = 125.75 MHz, SWH = 29 761.90 Hz, f1 channel-90° high power pulse (P1) = 10.90 μs, SI = 32 768, LB = 3.00 Hz, AQ = 1.10 s, relaxation delay 1–5 * T1 (D1) = 2.00 s and DS = 4. Pulprog for DEPT135 = deptsp135, SF = 125.75 MHz, SWH = 25 252.52 Hz, f1 channel-90° high power pulse (P1) = 9.70 μs, SI = 32768, LB = 1.00 Hz, AQ = 1.29 s, relaxation delay 1–5 * T1 (D1) = 2.00 s and DS = 4. Pulprog for DEPT90 = deptsp90, SF = 125.75 MHz, SWH = 29 761.90 Hz, f1 channel-90° high power pulse (P1) = 10.90 μs, SI = 32 768, LB = 3.00 Hz, AQ = 1.10 s, relaxation delay 1–5 * T1 (D1) = 2.00 s and DS = 4. Pulprog for 1H–1H COSY = cosygpppqf, SF = 500.13 MHz, SWH = 7936.50 Hz, f1 channel-90° high power pulse (P1) = 13.20 μs,
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SI = 1024, LB = 0.00 Hz, AQ = 0.129 s, relaxation delay 1–5 * T1 (D1) = 2.02 s and DS = 8. Pulprog for 1H–13C HSQC = hsqcetgpsi2, SF for 1H = 500.13 MHz and SF for 13C = 125.75 MHz, SWH = 4587.15 Hz, f1 channel-90° high power pulse (P1) for 1H = 13.20 μs, f2 channel-90° high power pulse (P3) for 13C = 10.90 μs, SI for 1H and 13C = 1024, LB for 1H and 13C = 0.00 Hz, AQ = 0.171 s, relaxation delay 1–5 * T1 (D1) = 1.50 s and DS = 16. Pulprog for 1H–13C HMBC = hmbcgplpndqf, SF for 1H = 500.13 MHz and SF for 13C = 125.75 MHz, SWH = 7142.85 Hz, f1 channel90° high power pulse (P1) = 13.20 μs, f2 channel-90° high power pulse (P3) for 13C = 10.90 μs, SI for 1H = 2048 and SI for 13 C = 1024, LB for 1H and 13C = 0.00 Hz, AQ = 0.143 s, relaxation delay 1–5 * T1 (D1) = 1.51 s and DS = 16. Pulprog) for 1H–1H NOESY = noesyphpp.2, SF = 500.13 MHz, SWH = 7002.80 Hz, f1 channel-90° high power pulse (P1) = 13.20 μs, SI = 1024, LB = 0.00 Hz, AQ = 0.146 s, relaxation delay 1–5 * T1 (D1) = 2.00 s and DS = 32. Extraction and isolation The air-dried ground bark (5.2 kg) of G. hombroniana was extracted using a Soxhlet extractor with solvents of increasing polarity: n-hexane (C6H14), dichloromethane, ethyl acetate (EtOAc) and methanol (MeOH) in a successive manner. The extracts obtained were concentrated in vacuo at 40 °C. An ethyl acetate extract (18.0 g) was chromatographed over silica gel (0.040–0.060 mm, Merck, 6 100 cm). The elution was carried out using CHCl3/EtOAc 10 : 0, 9 : 1, 8 : 2, 7 : 3, 6 : 4, 4 : 6, 2 : 8 to 0 : 10 (1 l each) and EtOAc/MeOH 9 : 1 (2 l).The eluents (250 ml each) were collected in 40 fractions EF1 to EF40 and checked by TLC (C6H14/Me2CO 7 : 3 and C6H14/EtOAc 2 : 8, SiO2, 95% methanolic H2SO4). They were combined into seven fractions EFA1 to EFA7 on the basis of their similar TLC profiles. The TLC analysis of fraction EFA7 (3.2 g) with C6H14/Me2CO 7 : 3 that indicated few prominent yellow spots on its TLC plate was rechromatographed over silica gel with EtOAc/MeOH, yielding fractions EFA7c1 to EFA7c25. The sub-fractions ESFA7c1 to ESFA7c4 upon column chromatography (0.040–0.060 mm,
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Rotameric biflavonoids from Garcinia hombroniana 3 60 cm, EtOAc/MeOH 9.5 : 0.5) afforded compound 2 (10.0 mg, 1.92 104% of the dried bark). Fractions ESFA7c10 to ESFA7c16 afforded compound 1 (15.0 mg, 2.86 104% of the dried bark) with EtOAc/MeOH 9 : 1, ESFA7c17 to ESFA7c25 afforded compounds 4 (10.0 mg, 1.92 104% of the dried bark) with EtOAc/ MeOH 7.5 : 2.5 and 5 (40.0 mg, 7.96 104% of the dried bark) with EtOAc/MeOH 9. : 0.5. Sub-fraction EFA6 (4.0 g) was rechromatographed and fractionated using silica gel column chromatography (0.040–0.060 mm, 5 80 cm) with CHCl3/MeOH. Fractions EFA6b1 to EFA6b35 were collected and examined by TLC. Fractions EFA6b3 to EFA6b9 (CHCl3/MeOH 9.7 : 0.3), EFA6b13 to EFA6b20 (CHCl3/MeOH 9.5 : 0.5) and EFA6b27 to EFA6b33 (CHCl3/MeOH 9.0 : 1.0) were combined on the basis of same TLC profile of yellow spots. These fractions were subjected to chromatographic purification with CHCl3 and MeOH (100 : 0 to 0 : 100) that afforded compounds 3 (12.0 mg, 2.30 104% of the dried bark), 6 (15.0 mg, 2.86 104% of the dried bark) and 7 (15.0 mg, 2.86 104% of the dried bark) eluted with CHCl3/MeOH 9.0 : 1.0, 8.5 : 1.5 and 8.0 : 2.0 respectively. All the compounds were finally purified over Sephadex LH-20 (bead size 25–100 μ, Sigma-Aldrich) with MeOH (100%). Antioxidant activities The antioxidant activities of compounds 1–7 were evaluated by free radical scavenging of DPPH[34] and ABTS[35] and reducing power FRAP assays.[34] Compounds 1–7 and the standard solutions were prepared in the concentration range of 2.0–50 μM. The inhibition percentages in DPPH and ABTS assays were calculated according to the formula given in the succeeding texts. % inhibition ¼
1 Asample =Acontrol
100
The results of DPPH and ABTS assays were expressed as IC50, while those of the FRAP assay were represented in micromole TE (μM TE). Antibacterial activities Antibacterial activities of the compounds 1–7 were evaluated by Kirby-Bauer disc diffusion[36] and MIC[37] methods against two gram-positive and two gram-negative bacteria. Antituberculosis activities The antituberculosis activities of compounds 1–7 were evaluated against M. tuberculosis H38Rv according to the method of Collins’s BACTEC 460 system (1997).[38] Statistical analyses The data were analyzed and expressed as means ± standard deviation of three replicates (n = 3). The differences between assayed values were analysed using one-way analysis of variance, followed by Tukey’s honestly significant difference test at 95 and 99% confidence intervals using SPSS software, version 19.0 (SPSS Inc., Chicago, USA). Results with p < 0.05 were considered significant, while those with p < 0.01 were regarded as very significant. Volkensiflavone 7-O-rhamnopyranoside (1)
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Acknowledgements This study was financially supported by a Research University Grant (RU1001/PKIMIA/811129). Financial support of Nargis Jamila as a TWAS (Third World Academy of Sciences)-USM (Universiti Sains Malaysia) fellow is also acknowledged. The authors also thank Mr Zahari Othman for NMR measurements.
Conflict of Interest The authors declare no conflict of interest.
References [1] L. Xiwen, L. Jie, N. K. B. Robson, P. F. Stevens, in Flora of China, Science Press Beijing and Missouri Botanical Garden, China, (Revised) 2013-2014, 13, pp. 40–47. [2] V. Peres, T. J. Nagem, F. F. de Oliveira. Phytochemistry 2000, 55, 683–710. [3] J. J. Magadula. Fitoterapia 2010, 81, 420–423. [4] S. Harati, H. B. Wang, L. B. S. Kardon, S. Kosela, G. W. Qin. Zhongguo Tianran Yaowu (Chin. J. Nat. Med.) 2007, 5, 272–276. [5] V. Rukachaisirikul, S. Saelim, P. Karnsomchoke, S. Phongpaichit. J. Nat. Prod. 2005, 68, 1222–1225. [6] K. R. Gustafson, J. W. Blunt, H. G. M. Munro, R. W. Fuller, C. T. McKee, J. H. Cardellina II, J. B. McMahon, G. M. Cragg, M. R. Boyd. Tetrahedron 1992, 48, 10093–10102. [7] A. E. Hay, J. J. Helesbeux, O. Duval, M. Labaied, P. Grellier, P. Richomme. Life Sci. 2004, 75, 3077–3085. [8] L. A. Savi, T. Caon, A. P. de Oliveira, A. M. Sobottka, W. Werner, F. H. Reginatto, E. P. Schenkel, C. R. Barardi, C. M. Simões. Fitoterapia 2010, 81, 1142–1146. [9] I. H. Burkill, A Dictionary of the Economic Products of the Malay Peninsula, Published on behalf of the governments of the Straits settlements and federated Malay states by the Crown agents for the colonies, London, 1935. [10] J. B. L. Pierre. Flore Forestiere de la Cochinchine Paris 1882–1885, 12, 54–98. [11] Y. M. Lin, M. T. Flavin, C. S. Cassidy, A. Mar, F. C. Chen. Bioorg. Med. Chem. Lett. 2001, 11, 2101–2104. [12] M. J. Son, T. C. Moon, E. K. Lee, K. H. Son, H. P. Kim, S. S. Kang, H. W. Chang, J. K. Son, S. H. Lee. Arch. Pharmacal. Res. 2006, 29, 282–286. [13] J. C. Castardo, A. S. Prudente, J. Ferreira, C. L. Guimarães, F. D. Monache, M. F. Otuki, D. A. Cabrini. J. Ethnopharmacol. 2008, 118, 405–411. [14] O. A. Adaramoye, I. Awogbindin, J. O. Okusaga. J. Med. Food 2009, 12, 584–590. [15] Y. M. Lin, H. Anderson, M. T. Flavin, Y. H. Pai, E. Mata-Greenwood, T. Pengsuparp, J. M. Pezzuto, R. F. Schinazi, S. H. Hughes, F. C. Chen. J. Nat. Prod. 1997, 60, 884–888. [16] H. P. Kim, H. Park, K. H. Son, H. W. Chong, S. S. Kang. Arch. Pharm. Res. 2008, 31, 265–273. [17] P. C. Chan, Q. Xia, P. P. Fu. J. Environ. Sci. Health Part C 2007, 25, 211–244. [18] I. A. Adedara, E. O. Farombi. Hum. Exp. Toxicol. 2012, 31, 506–517. [19] O. A. Adaramoye, E. O. Adeyemi. J. Pharm. Pharmacol. 2006, 58, 121–128. [20] E. O. Farombi, B. F. Adepoju, O. E. Ola-Davies, G. O. Emerole. Eur. J. Cancer Prev. 2005, 14, 207–214. [21] M. Iwu, in Plant Flavonoids in Biology and Medicine, (Eds: V. Cody, E. Middleton, J. B. Harborne), Alan R. Liss, New York, 1986, pp. 485–488. [22] W. D. Ollis, B. T. Redman, I. O. Sutherland, K. Jewers. J. Chem. Soc. D Chem. Commun. 1969, 879–880. doi:10.1039/C29690000879. [23] V. Ruachaisirikul, A. Adair, P. Dampawan, W. C. Taylor, P. C. Turner. Phytochemistry 2000, 55, 183–188. [24] S. Klaiklay, Y. Sukpondma, V. Rukachaisirikul, S. Phongpaichit. Phytochemistry 2013, 85, 161–166. [25] F. C. Saputri, I. Jantan. Phytother. Res. 2012, 26, 1845–1850. doi:10.1002/ptr.4667.
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Yellow amorphous solid; mp 275–278 °C; UV λmax nm: (MeOH) 288, (NaOMe) 328, 392, (H3BO3) 293, (NaOAc) 329, 407, (AlCl3)
307, (AlCl3 + HCl) 305, 341; CD (DMSO) [θ]287.4 8.75199; IR (KBr) vmax: 3255, 1637, 1591 cm1, 1H and 13C NMR, refer to Tables 1 and 2 respectively; +/ESI-MS at m/z 687.9 and 685.1572 [M–H]+ (calcd. 685.1635).
N. Jamila et al. [26] Q. B. Han, S. F. Lee, C. F. Qiao, Z. D. He, J. Z. Song, H. D. Sun, H. X. Xu. Chem. Pharm. Bull. 2005, 53, 1034–1036. [27] X. C. Li, A. S. Joshi, B. Tan, H. N. ElSohly, L. A. Walker, J. K. Zjawiony, D. Ferreira. Tetrahedron 2002, 58, 8709–8717. [28] T. Hatano, R. W. Hemingway. J. Chem. Soc. Perkin Trans. 1997, 2, 1035–1043. [29] W. Gaffield. Tetrahedron 1970, 26, 4093–4108. doi:10.1016/S00404020(01)93050-9. [30] B. Jackson, H. D. Locksley, F. Scheinmann, W. A. Wolstenholme. J. Chem. Soc. 1971, C, 3791–3804. [31] R. S. Compagnone, A. C. Suarez, S. G. Leitao, M. F. Delle. Rev. Bras. de Farmacog 2008, 18, 6–10. [32] P. G. Waterman, E. G. Crichton. Phytochemistry 1980, 19, 2723–2726. [33] E. Elfita, M. Muharni, L. Madyawati, D. Darwati, W. Ari, S. Supriyatna, H. H. Bhatti, D. Dachriyanus, P. Cos, L. Maes, K. Foubert, S. Aspers, L. Pieters. Phytochemistry 2009, 70, 907–912.
[34] K. Thaipong, B. Unaroj, C. Kevin, C. Z. Luis, H. B. David. J. Food. Compos. Anal. 2006, 19, 669–675. [35] N. Jamila, M. Khairuddean, C. S. Lai, O. Hasnah, K. C. Wong, M. Vikneswaran, K. Y. Khaw. Afr. J. Pharm. Pharmacol. 2013, 28, 454–459. [36] J. Hudzicki. http://www.microbelibrary. org/library/laboratory-test/ 3189-kirby-bauer-disk-diffusion-susceptibility-test-protocol 2009 (Accessed 24 July 2012). [37] J. N. Eloff. J. Ethnopharmacol. 1998, 60, 1–8. [38] L. Collins, S. G. Franzblau. Antimicrob. Agents. Ch. 1997, 41, 1004–1009.
Supporting Information Additional supporting information may be found in the online version of this article at the publisher’s website.
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Copyright © 2014 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2014, 52, 345–352
Revised: 10 March 2014
Accepted: 12 March 2014
Published online in Wiley Online Library: 3 April 2014
(wileyonlinelibrary.com) DOI 10.1002/mrc.4071
Complete NMR assignments of bioactive rotameric (3 → 8) biflavonoids from the bark of Garcinia hombroniana Nargis Jamila,a* Melati Khairuddean,a Sadiq Noor Khanb and Naeem Khanc The genus Garcinia is reported to possess antimicrobial, anti-inflammatory, anticancer, hepatoprotective and anti-HIV activities. Garcinia hombroniana in Malaysia is used to treat itching and as a protective medicine after child birth. This study was aimed to isolate the chemical constituents from the bark of G. hombroniana and explore their possible pharmacological potential. Ethyl acetate extract afforded one new (1) and six (2–7) known 3 → 8 rotameric biflavonoids. Their structures were elucidated by UV, IR and NMR (1D and 2D) spectroscopy together with electron ionization/ESI mass spectrometric techniques and were identified as (2R, 3S) volkensiflavone-7-O-rhamnopyranoside (1), volkensiflavone (2), 4″-O-methyl-volkensiflavone (3), volkensiflavone-7-O-glucopyranoside (4), morelloflavone (5), 3″-O-methyl-morelloflavone (6) and morelloflavone-7-Oglucopyranoside (7). The absolute configuration of compound 1 was assigned by circular dichroism spectroscopy as 2R, 3S. The coexistence of conformers of isolated biflavonoids in solution at 25 °C in different solvents was confirmed by variable temperature NMR studies. At room temperature (25 °C), compounds 1–7 exhibited duplicate NMR signals, while at elevated temperature (90 °C), a single set of signals was obtained. Compound 5 showed significant in vitro antioxidant activities against 1,1-diphenyl-2-picrylhydrazyl and 2,2′-azino-bis-3-ethyl benzthiazoline-6-sulfonic acid radicals. The antibacterial studies showed that compounds 5 and 6 are the most active against Staphylococcus aureus, Bacillus subtilis and Escherichia coli. Compounds 3 and 6 also showed moderate antituberculosis activity against H38Rv. Based on the research findings, G. hombroniana could be concluded as a rich source of flavanone–flavone (3 → 8) biflavonoids that exhibit rotameric behaviour at room temperature and display significant antioxidant and antibacterial activities. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: NMR; 1H NMR; antituberculosis
13
C NMR; variable temperature NMR; circular dichroism; Garcinia hombroniana; biflavonoids; antioxidant;
Introduction
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* Correspondence to: Nargis Jamila, School of Chemical Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia. E-mail: [email protected] a School of Chemical Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia b Department of Medical Lab Technology, University of Haripur, 22060 Haripur, Pakistan c Department of Chemistry, Kohat University of Science and Technology, 26000 Kohat, Khyber Pakhtunkhwa, Pakistan
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345
Garcinia, the largest genus of the family Clusiaceae, is native to the regions of Asia, northeast Australia, southern Africa and tropical America.[1] This genus is found to be a rich source of bioactive secondary metabolites, including oxygenated prenylated xanthones and benzophenones, flavonoids, biflavonoids and triterpenes.[2–5] These chemical constituents are well known for their pharmacological potential including antimicrobial, antiHIV, anticancer, anti-inflammatory and hepatoprotective activities.[6–8] Garcinia hombroniana, a seashore mangosteen in Malaysia, is used as a protective medicine after child birth and to cure skin allergies.[9,10] Biflavonoids are dimers of flavanone–flavone, flavone–flavone and flavone–flavonol combinations, possessing significant pharmacological activities such as antimicrobial, antiallergic, anti-inflammatory, hepatoprotective, anti-HIV and antioxidant.[11–16] They are reported to occur in several species of plants, but only a few of them such as Ginkgo biloba and Garcinia kola are found to be their rich sources.[17,18] Biflavonoids from G. kola have shown potent hypoglycaemic, hypolipidaemic and antioxidant effects compared with simple monomeric flavonoids.[19–21] Among the 450 species of Garcinia, G. hombroniana Pierre appeared to be one of the rarely studied. The previous phytochemical investigations of its stem wood, pericarp, leaves and twigs revealed the presence of xanthones, flavonoids and triterpenes.[22–24] The phenolic and triterpenoid constituents from
the twigs of G. hombroniana demonstrated copper-mediated low density lipoprotein antioxidation, antiplatelet aggregation and antibacterial activities.[25] To the best of our knowledge, so far, there is no authentic chemical and pharmacological investigation on the bark of G. hombroniana. In view of the limited data, traditional uses and possible pharmacological applications of G. hombroniana, this study was designed to investigate the biochemical constituents from the bark fractions of G. hombroniana. In our ongoing research, the ethyl acetate bark extract over silica gel column chromatography has resulted in the isolation of one new (1) and six known (2–7) flavanone–flavone 3 → 8 rotameric biflavonoids (Fig. 1). Their structures were elucidated using spectral techniques of NMR, EI/ESI-MS and circular dichroism (CD). The NMR data were recorded at variable temperature (90, 80, 70, 60, 50, 40, 30, 25, 0, 10, 20, 30, 40, 50 and 70 °C) and in three different solvents [dimethyl sulfoxide-d6 (DMSO-d6),
N. Jamila et al.
Figure 1. Chemical structures of compounds 1–7.
methanol-d4 and acetone-d6]. The antioxidant potential of the purified compounds (>92%) employing 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis-3-ethyl benzthiazoline-6-sulfonic acid (ABTS) and ferric reducing ability of plasma (FRAP) assays; antibacterial capacities against Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa and Escherichia coli and antituberculosis activities against H38Rv were also determined.
Results and Discussion
346
Compound 1 was obtained as a yellow solid. The molecular formula C36H30O14 was deduced from low resolution ESI-MS (positive mode) and HRESI-MS (negative mode) techniques (Fig. S1 (a) and (b), Supporting Information). Molecular ions appeared at m/z 687.9 [M + H]+ and 685.1572 [M H]+ (calcd. 685.1635) respectively. The TLC and ESI-MS data showed that it is a pure compound. However, the 1H and 13C NMR spectra exhibited double sets of signals. Therefore, it was a bit difficult to identify
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this compound by the NMR data recorded at ambient temperature. The duplicate signals in the NMR spectra of biflavonoids at room temperature are because of the presence of conformations that arise from the restricted free rotation of 3 → 8 interflavanyl bond.[26] These baffling NMR data were converted to a single set of signals at higher temperature because at elevated temperature, the hindered rotation becomes faster and faster, and the molecules do not exist in a preferred stable conformation any more.[27] The 1H NMR spectrum at 25 °C (Fig. S2, Supporting Information) demonstrated duplicate signals indicating two conformers. The 1H NMR resonances of the major conformer were displayed in the deshielded aromatic and shielded saccharadic region. A set of two doublets of two protons each at δH 8.01 (H-2″/6″) and 6.97 (H-3″/5″) and another set of two doublets of two integrals at δH 7.08 (H-2′/6′) and 6.36 (H-3′/5′) with J(s) of 8.0–9.0 Hz indicated a para substitution pattern in rings II-E and I-B respectively. Another pair of doublets of two protons that appeared at δH 6.02 and 5.96 (H-6/8) with a J value of 2.0 Hz was a clue to a meta substitution in ring A. Two singlets at δH 6.88 and 6.66 were assigned to H-II-3 and H-II-6 of rings F and D respectively. Furthermore, another pair of doublets in a non-aromatic region at δH 5.51 and 5.15 with a J of 12.0 Hz was respectively assigned to H-2 and H-3 of the ring I-C in the flavanone unit. The coupling constant (12.0 Hz) of these two protons verified their diaxial (trans) configuration. In addition, the 1H NMR spectrum also displayed signals, characteristic of a sugar moiety (rhamnopyranosyl) containing resonances of anomeric protons at δH 5.35 (H-1‴) and a complex mutiplet of non-anomeric protons in the chemical shift regions of δH 3.73–3.40 and a doublet of methyl protons at δH 1.18 ppm. The 13C NMR spectrum at 25 °C (Fig. S3, Supporting Information) also showed duplicate signals of 36 carbons that could be assigned to 1 methyl, 19 methine and 16 quaternary carbons, deduced from DEPT 135, 90 and Q NMR spectra (Fig. S4, Supporting Information). The complete assignments of 1H and 13C NMR of the two conformers at 25 °C are given in Tables 1 and 2 respectively. The 1H and 13C NMR spectra (Figs S5 and S6, Supporting Information) recorded at elevated temperature (90 °C) showed changes in resonances and appeared as a single set of signals. The 1H–1H COSY spectrum (Fig. S7, Supporting Information) showed correlations of the protons H-2′/6′ to protons H-3′/5′ of the flavanone unit and cross peaks of H-2″/6″ with the protons H-3″/5″ of the flavone unit, both of the major as well as the minor conformers, which further confirmed the para substituted B and E rings of the flavanone and flavone units respectively. The vicinal protons H-2 and H-3 on the C ring of the flavanone unit were also verified by their COSY correlations. The 1H–13C HSQC spectrum (Fig. S8, Supporting Information) helped in assigning the protonated carbons by showing cross peaks of the protons to their respective carbons. The rhamnopyranosyl moiety was located at C-7 of ring D in the flavone unit as evidenced by the 1H–13C HMBC correlations (Fig. S9, Supporting Information) of the anomeric proton at δH 5.35 with C-7 (δC 159.8) and C-6 (δC 97.9) of the D ring. The spatial associations of protons were determined by 1H–1H NOESY spectrum (Fig. S10, Supporting Information), in which the protons at δH 8.01 (H2″/6″) showed cross peaks to the protons at δH 6.97 (H-3″/5″), δH 7.08 (H-2′/6′), δH 6.66 (H-6 of the D ring) and δH 5.15 (H-3 of the C ring). The NOESY spectrum also showed correlations of the protons at δH 8.01 (II-2″/6″) of the major conformer to the protons at δH 7.66 II-2″/6″ of the minor conformer. This abnormal NOE happened because of the exchange of
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Magn. Reson. Chem. 2014, 52, 345–352
1
Copyright © 2014 John Wiley & Sons, Ltd.
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A
12.1 (s) 5.66 (d, 12.5) 5.04 (d, 12.5) 5.96 (d, 2.0) 5.96 (d, 2.0) 7.08 (d, 8.0) 6.61 (m) 6.61 (m) 7.08 (d, 8.0)
B
b
5.27 (br. s) Overlapped With H2O peak 3.30 (m) 3.30 (m) 1.07 (d, 7.0)
B
b
A
a
(MeOD-d4) 5.63 (d, 6.0) 4.0 (br. s) 3.81 (m) 3.44 (br. s) 3.51-3.48 (m) 1.31 (d, 6.0)
A
a
B
b
5.49 (d, 6.0) 3.94 (br. s) 3.62 (m) 3.39 (d, 3.0) 3.42 (br. s) 1.22 (d, 6.0)
B
b
12.0 (s) 5.46 (d, 12.0) 5.07 (d, 12.0) 6.02 (br. s) 6.02 (br. s) 7.02 (d, 8.3) 6.66 (d, 7.9) 6.66 (d, 7.9) 7.02 (d, 8.3)
(MeOD-d4)
12.1 (s) 5.69 (d, 12.5) 5.32 (d, 12.0) 5.92 (br. s) 5.92 (br. s) 7.02 (d, 8.3) 6.35 (d, 8.3) 6.35 (d, 8.3) 7.02 (d, 8.3)
Rhamnopyranoside unit
(DMSO-d6)
5.35 (s) 3.58 (br. s) 3.73 (br. s) 3.40 (m) 3.49 (m) 1.18 (d, 7.0)
a
12.2 (s) 5.51 (d, 12.0) 5.15 (d, 12.0) 6.02 (d, 2.0) 6.06 (d, 2.0) 7.08 (d, 8.0) 6.36 (d, 8.5) 6.36 (d, 8.5) 7.08 (d, 8.0)
A
(DMSO-d6)
DMSO-d6 and MeOD-d4, 500 MHz. Sets A and B are respectively in an intensity ratio of (~1 : 0.62). a A represents major conformer at 25 °C. b B represents minor conformer at 25 °C.
1‴ 2‴ 3‴ 4‴ 5‴ 6‴
H no.
I-5-OH I-2 I-3 I-6 I-8 I-2′ I-3′ I-5′ I-6′
H no
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a
Flavanone unit (I)
H NMR data of the major and minor conformers of 1 at 25 °C
δH in ppm (multiplicity, J in Hz)
Table 1.
II-5-OH II-3 II-6 II-2″ II-3″ II-5″ II-6″
H no.
A
-
B
b
13.0 (s) 6.73 (s) 6.45 (s) 7.66 (d, 9.0) 6.64 (m) 6.64 (m) 7.66 (d, 9.0)
(DMSO-d6)
13.1 (s) 6.88 (s) 6.66 (m) 8.01 (d, 9.0) 6.97 (d, 9.0) 6.97 (d, 9.0) 8.01 (d, 9.0)
a
a
A
B
b
12.1 (s) 6.62 (s) 6.42 (br. s) 7.56 (d, 8.5) 6.68 (d, 8.5) 6.68 (d, 8.5) 7.56 (d, 8.5)
(MeOD-d4)
12.2 (s) 6.72 (s) 6.52 (s) 7.72 (d, 8.5) 6.83 (d, 8.5) 6.83 (d, 8.5) 7.72 (d, 8.5)
Flavone unit (II)
Rotameric biflavonoids from Garcinia hombroniana
N. Jamila et al. Table 2.
13
C NMR data of the major and minor conformers of 1 at 25 °C
δC (C-type)* Flavanone unit (I) C no. I-2 I-3 I-4 I-5 I-6 I-7 I-8 I-9 I-10 I-1′ I-2′ I-3′ I-4′ I-5′ I-6′
a
A
81.6 (CH) 48.1 (CH) 196.0 (C) 163.9 (C) 96.4 (CH) 167.1 (C) 96.4 (CH) 162.7 (C) 101.7 (C) 127.8 (C) 128.3 (CH) 114.5 (CH) 157.5 (C) 114.5 (CH) 128.3 (CH)
Flavone unit (II) b
B
C no.
81.3 47.9 195.8 163.8 95.4 166.7 95.4 162.5 101.3 127.5 128.2 114.4 157.3 114.4 128.2
II-2 II-3 II-4 II-5 II-6 II-7 II-8 II-9 II-10 II-1″ II-2″ II-3″ II-4″ II-5″ II-6″
a
Rhamnopyranoside unit b
A
164.2 (C) 103.2 (CH) 181.9 (C) 161.3 (C) 97.9 (CH) 159.8 (C) 102.4 (C) 154.5 (C) 105.0 (C) 120.9 (C) 129.0 (CH) 115.9 (CH) 160.9 (C) 115.9 (CH) 129.0 (CH)
B
C no.
164.0 102.4 181.8 161.1 97.2 159.5 102.2 153.7 104.5 120.5 128.6 115.6 160.5 115.6 128.6
1‴ 2‴ 3‴ 4‴ 5‴ 6‴
a
A
100.2 (CH) 71.8 (CH) 69.6 (CH) 70.2 (CH) 70.3 (CH) 17.8 (CH3)
b
B
100.0 71.4 69.4 70.0 70.2 17.8
DMSO-d6, 125 MHz. * C-type was deduced from DEPT experiments. a A represents major conformer at 25 °C. b B represents minor conformer at 25 °C.
rotational conformations of the two conformers,[28] and it revealed that 3 → 8 biflavonoids are not a simple mixture of two conformers but are in a dynamic system. The absolute configuration of 1 was examined by CD spectrum (Fig. 2), in which a positive Cotton effect was observed at 287 nm (π → π* transition), suggesting a 2β-orientation of the B-ring and hence a 2R absolute configuration of C-2 in the flavanone unit. According to Gaffield (1970),[29] the more reliable Cotton effect for the determination of C-2 stereochemistry is near 290 nm (π → π* transition) than the Cotton effect at a longer wavelength (n → π* transition) as the latter becomes smaller when the amount of the opposite enantiomer is increased. The diagnostic absorption at 287 nm is characteristic of the flavanone-type chromophore. The stereochemistry of the C-3 relative to C-2 of the flavanone unit was then determined by applying Cahn–Ingold–Prelog
348
Figure 2. CD spectrum of 1 at 25 °C.
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sequence rules according to which C-3 was assigned as 3S. This configuration was also supported by the coupling constants (12.0 Hz) of H-2 and H-3 in a 1H NMR experiment that suggested a trans relative configuration of C-2 and C-3. The previously mentioned spectral evidence established the structure of 1 as (2R, 3S) volkensiflavone-7-O-rhamnopyranoside. We also recorded and compared the 1H and 13C NMR spectra at variable temperatures ranging from 80 to 70 °C (Figs S11 and S12, Supporting Information) and in three different solvents, dimethyl sulfoxide-d6, methanol-d4 and acetone-d6. At elevated temperature (90 °C), the duplicate signals were collapsed and appeared as a clear single set of signals. The 1H NMR spectra at lower temperature (0 to 70 °C) also exhibited changes and converted to a single set of signals at 50 to 70 °C. At lower temperature, the hindered rotation of 3 → 8 interflavanyl bond becomes slower and slower and is low enough to produce a preferred stable conformation. These observed results of different temperature NMR studies revealed the rotameric behaviour of 3 → 8 biflavonoids and close association of their NMR with temperature. The duplicate NMR data of 3 → 8 biflavonoids at room temperature were first reported by Jackson et al. (1971).[30] The NMR spectra were recorded in dimethyl sulfoxide-d6, methanold4 and acetone-d6 at 25 °C to investigate the effect of solvents on the rotameric behaviour of 3 → 8-type biflavonoids. The 13C NMR spectra in these three solvents (Figs S3, S13 and S14, Supporting Information) at 25 °C exhibited the same spectral complexity of duplicate signals, which revealed that the solvents do not affect the rotamerism of 3 → 8-type biflavonoids. Based on the comparison of 1D and 2D NMR data of the compounds 2–7 with the published literature, they were identified as volkensiflavone (2), 4″-O-methyl-volkensiflavone (3), volkensiflavone-7-O-glucopyranoside (4), morelloflavone (5), 3″-O-methyl-morelloflavone (6) and morelloflavone-7-O-
Copyright © 2014 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2014, 52, 345–352
Rotameric biflavonoids from Garcinia hombroniana glucopyranoside (7).[31–33] Their 1H and 13C NMR spectra and complete assignments given in Figs S15–S20 and Tables S1–S6 are shown in the Supporting Information. In antioxidant studies, compounds 5, 6 and 7 showed the highest potential in DPPH, ABTS and FRAP assays with the most active compound 5 displaying a half maximal inhibitory concentration (IC50) of 4.08 μM in DPPH and 3.90 μM in ABTS respectively, even higher than the standards, trolox (IC50 24.8 μM in DPPH and 12.6 μM in ABTS) and gallic acid (IC50 7.79 μM in DPPH). However, in the ABTS assay, compound 5 was found equipotent to gallic acid. Compound 5 also showed higher reducing ability [298.8 μM trolox equivalent (TE)] in the FRAP assay. The complete inhibition of the DPPH free radical by the compounds and trolox was attained at 50 μM except for the compound 5 and gallic acid that was attained completely at 20 μM. On the other hand, compounds 2, 3, 5 and 6 and trolox were found to have complete inhibition of ABTS+ at 30 μM, while compounds 1, 4 and 7 inhibited ABTS+ at a concentration of 50 μM. Gallic acid was found to be the most active inhibitor of ABTS+ (99%) at a concentration of about 10 μM. The detailed results of the antioxidant assays and DPPH and ABTS inhibition profiles of the compounds 1–7 and standards at various concentrations are respectively shown in Table 3 and Fig. 3. In regard to the compounds’ properties as an antibacterial, compounds 5 and 6 showed the highest activity with the minimum inhibitory concentration (MIC) of 62.5 μM each against S. aureus, B. subtilis and E. coli, followed by compounds 2 and 3. The rest of the compounds were found moderately active. In antituberculosis studies, only compounds 3 and 6 showed a moderate inhibition with the MIC of 108.5 and 102.3 μM respectively, while the other compounds were inactive (Table 4). A new biflavonoid (1) did not significantly contribute to all the tested biological activities.
Figure 3. DPPH and ABTS radical inhibition profiles of compounds 1–7 and standards (trolox and gallic acid).
Chemicals and bacterial strains
Materials and Methods Plant materials Plant materials were collected from Penang Botanical Garden, Malaysia, and verified by Mr Saul Hamid Pakir Mohamed. A voucher specimen (PBGK12) has been deposited at the Herbarium of Penang Botanical Garden, Malaysia. Table 3. Antioxidant activities of compounds 1–7 Compounds
1 2 3 4 5 6 7 Trolox Gallic acid
DPPH assay
ABTS assay
FRAP assaya
(IC50, μM)
(IC50, μM)
(μM TE)
i
37.3 ± 3.49 f 17.3 ± 2.8 g 23.27 ± 2.91 h 28.5 ± 1.54 a 4.08 ± 0.74 b 5.70 ± 0.69 d 11.9 ± 0.78 e 24.1 ± 0.62 c 7.79 ± 0.45
i
35.5 ± 0.93 f 16.6 ± 0.25 g 20.8 ± 1.28 h 29.1 ± 0.31 b 3.90 ± 0.51 c 5.87 ± 1.35 d 11.8 ± 1.96 e 12.6 ± 0.08 a 3.41 ± 0.03
d
12.6 ± 1.24 c 10.9 ± 0.96 b 7.50 ± 1.29 a 4.34 ± 0.17 d 293.5 ± 5.16 f 206.1 ± 4.35 e 187.2 ± 2.57 — —
General experimental procedure Silica gel 60 (0.040–0.063 mm) was used as an adsorbent for column chromatography. The semi-pure compounds were finally purified over Sephadex LH-20 (bead size 25–100 μ, Sigma-Aldrich) with MeOH (100%). Merck TLC plates (silica gel 60F254) were used for the detection of the purity of compounds. Melting points were determined with a Stuart Scientific Melting Point SMP 1 (UK). UV spectra were recorded using a PerkinElmer Lambda 25 UV/Vis spectrometer. IR spectra were recorded in KBr on a PerkinElmer 2000 FT-IR spectrophotometer. EI/ESI mass spectra were recorded on an Agilent 5975C MSD and ThermoFinnigan MAT95XL mass spectrometers at National University of Singapore,
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349
a FRAP values are expressed as μM TE at 20 μM concentration. Superscript lowercase letters (a–i) represent significant differences (p < 0.05) of the antioxidant activities of the compounds 1–7.
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All the solvents were of analytical or HPLC grade. The chemicals of antioxidant activities, DPPH (96%), ABTS (>98%), 2,4,6Tripyridyl-s-Triazine (>98%), trolox (97%) and gallic acid (97.5%), were purchased from Sigma-Aldrich (Steinheim, Germany) and Merck (Darmstadt, Germany). Two gram-positive bacteria, S. aureus (ATCC 29213) and B. subtilis (ATCC 19659), and two gram-negative bacteria, P. aeruginosa (ATCC 17588) and E. coli (ATCC 25922), Mueller Hinton Broth, nutrient agar, piodonitrotetrazolium chloride, vincomycin (99%), gentamicin (99%) and streptomycin (98%) were purchased from Oxoid (England), Sigma-Aldrich (USA) and Duchefa Biochemie (the Netherlands). Mycobacterium tuberculosis H38Rv was purchased from American Type Culture Collection (Rockville, USA).
N. Jamila et al. Table 4. Antibacterial and antituberculosis activities of compounds 1–7 Antibacterial activity Compounds
1 2 3 4 5 6 7 Vancomycin* Streptomycin* Gentamicin*
Antituberculosis activity (μM) Minimum inhibitory concentration (MIC), μM
Disc diffusion, mm S. aureus
B. subtilis
P. aeruginosa
E. coli
b 12.0 ± 0.1 c 13.5 ± 0.3 a 7.5 ± 0.2 a 8.0 ± 0.1 a 7.0 ± 0.2 d 16.0 ± 0.0 d 16.0 ± 0.1 e 21.0 ± 0.0
a 12.5 ± 0.2 a 13.0 ± 0.2 b 15.0 ± 0.3 b 15.0 ± 0.2 d 26.0 ± 0.1 c 23.0 ± 0.0 c 22.0 ± 0.1
a 8.0 ± 0.1 a 8.0 ± 0.2 b 10.0 ± 0.3 a 8.0 ± 0.1 c 19.5 ± 0.0
8.0 ± 0.2 b 12.0 ± 0.1 c 14.5 ± 0.2 a 7.0 ± 0.1 d 15.5 ± 0.0 f 16.0 ± 0.1 c 14.5 ± 0.2 b 12.0 ± 0.0 d 15.0 ± 0.1 g 25.0 ± 0.1
a
S. aureus
B. subtilis
P. aeruginosa
E. coli
d 250 c 125 b 62.5 b 62.5 e 500 e 500 a 15.25
d 250 c 125 b 62.5 b 62.5 e 500 e 500 a 15.25
b 62.5 c 250 c 250 c 250 d 500 a 31.5
250 c 250 c 250 b 62.5 b 62.5 d 500 a 7.5
c
H38Rv d 108.5 c 102.3 ND a 0.60 b 12.5
Results are mean values of triplicate (n = 3). Superscript lowercase letters (a–g) represent significant differences at p < 0.05. - = no inhibition observed; * = positive control
Singapore. The absolute configuration of 1 was determined using a Jasco-815 spectrometer at 25 °C (scan range λ = 200–350 nm, cell length = 10 mm).
NMR spectra
350
The NMR experiments of compounds 1–7 were performed at variable temperatures ranging from 90 to 70 °C in DMSO-d6, MeOD-d4 and acetone-d6 using a Bruker Ascend 500 MHz (1H) and 125 MHz (13C) spectrometer equipped with a Bruker 5 mm Broadband probe. Chemical shifts (δ) in ppm are referenced to tetramethylsilane at 0.00 ppm for 1H and 13C. Coupling constants are expressed in hertz (Hz). Pulse programme (pulprog) for 1H at 25 °C = zg30, spectrometer frequency (SF) = 500.13 MHz, spectral width (SWH) = 10 330.57 Hz, f1 channel-90° high power pulse (P1) = 13.20 μs, size of the real spectrum (SI) = 65 536, line broadening factor for em (LB) = 0 Hz, acquisition time (AQ) = 3.17 s, relaxation delay 1–5 * T1 (D1) = 1.00 s and number of dummy scan (DS) = 2. Pulprog for 1H at 90 °C is same as for the 1H at 25 °C. Pulprog for 13C in DMSO-d6, MeOD-d4 and Me2CO-d6 at 25 °C = zgpg30, SF = 125.75 MHz, SWH = 29 761.90 Hz, f1 channel-90° high power pulse (P1) = 10.90 μs, SI = 32768, LB = 1.00 Hz, AQ = 1.10 s, relaxation delay 1–5 * T1 (D1) = 2.00 s and DS = 4. Pulprog for 13C in DMSO-d6 at 90 °C = zgpg30, SF = 125.75 MHz, SWH = 29 761.90 Hz, f1 channel-90° high power pulse (P1) = 10.90 μs, SI = 32 768, LB = 3.00 Hz, AQ = 1.10 s, relaxation delay 1–5 * T1 (D1) = 2.00 s and DS = 4. Pulprog for DEPT135 = deptsp135, SF = 125.75 MHz, SWH = 25 252.52 Hz, f1 channel-90° high power pulse (P1) = 9.70 μs, SI = 32768, LB = 1.00 Hz, AQ = 1.29 s, relaxation delay 1–5 * T1 (D1) = 2.00 s and DS = 4. Pulprog for DEPT90 = deptsp90, SF = 125.75 MHz, SWH = 29 761.90 Hz, f1 channel-90° high power pulse (P1) = 10.90 μs, SI = 32 768, LB = 3.00 Hz, AQ = 1.10 s, relaxation delay 1–5 * T1 (D1) = 2.00 s and DS = 4. Pulprog for 1H–1H COSY = cosygpppqf, SF = 500.13 MHz, SWH = 7936.50 Hz, f1 channel-90° high power pulse (P1) = 13.20 μs,
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SI = 1024, LB = 0.00 Hz, AQ = 0.129 s, relaxation delay 1–5 * T1 (D1) = 2.02 s and DS = 8. Pulprog for 1H–13C HSQC = hsqcetgpsi2, SF for 1H = 500.13 MHz and SF for 13C = 125.75 MHz, SWH = 4587.15 Hz, f1 channel-90° high power pulse (P1) for 1H = 13.20 μs, f2 channel-90° high power pulse (P3) for 13C = 10.90 μs, SI for 1H and 13C = 1024, LB for 1H and 13C = 0.00 Hz, AQ = 0.171 s, relaxation delay 1–5 * T1 (D1) = 1.50 s and DS = 16. Pulprog for 1H–13C HMBC = hmbcgplpndqf, SF for 1H = 500.13 MHz and SF for 13C = 125.75 MHz, SWH = 7142.85 Hz, f1 channel90° high power pulse (P1) = 13.20 μs, f2 channel-90° high power pulse (P3) for 13C = 10.90 μs, SI for 1H = 2048 and SI for 13 C = 1024, LB for 1H and 13C = 0.00 Hz, AQ = 0.143 s, relaxation delay 1–5 * T1 (D1) = 1.51 s and DS = 16. Pulprog) for 1H–1H NOESY = noesyphpp.2, SF = 500.13 MHz, SWH = 7002.80 Hz, f1 channel-90° high power pulse (P1) = 13.20 μs, SI = 1024, LB = 0.00 Hz, AQ = 0.146 s, relaxation delay 1–5 * T1 (D1) = 2.00 s and DS = 32. Extraction and isolation The air-dried ground bark (5.2 kg) of G. hombroniana was extracted using a Soxhlet extractor with solvents of increasing polarity: n-hexane (C6H14), dichloromethane, ethyl acetate (EtOAc) and methanol (MeOH) in a successive manner. The extracts obtained were concentrated in vacuo at 40 °C. An ethyl acetate extract (18.0 g) was chromatographed over silica gel (0.040–0.060 mm, Merck, 6 100 cm). The elution was carried out using CHCl3/EtOAc 10 : 0, 9 : 1, 8 : 2, 7 : 3, 6 : 4, 4 : 6, 2 : 8 to 0 : 10 (1 l each) and EtOAc/MeOH 9 : 1 (2 l).The eluents (250 ml each) were collected in 40 fractions EF1 to EF40 and checked by TLC (C6H14/Me2CO 7 : 3 and C6H14/EtOAc 2 : 8, SiO2, 95% methanolic H2SO4). They were combined into seven fractions EFA1 to EFA7 on the basis of their similar TLC profiles. The TLC analysis of fraction EFA7 (3.2 g) with C6H14/Me2CO 7 : 3 that indicated few prominent yellow spots on its TLC plate was rechromatographed over silica gel with EtOAc/MeOH, yielding fractions EFA7c1 to EFA7c25. The sub-fractions ESFA7c1 to ESFA7c4 upon column chromatography (0.040–0.060 mm,
Copyright © 2014 John Wiley & Sons, Ltd.
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Rotameric biflavonoids from Garcinia hombroniana 3 60 cm, EtOAc/MeOH 9.5 : 0.5) afforded compound 2 (10.0 mg, 1.92 104% of the dried bark). Fractions ESFA7c10 to ESFA7c16 afforded compound 1 (15.0 mg, 2.86 104% of the dried bark) with EtOAc/MeOH 9 : 1, ESFA7c17 to ESFA7c25 afforded compounds 4 (10.0 mg, 1.92 104% of the dried bark) with EtOAc/ MeOH 7.5 : 2.5 and 5 (40.0 mg, 7.96 104% of the dried bark) with EtOAc/MeOH 9. : 0.5. Sub-fraction EFA6 (4.0 g) was rechromatographed and fractionated using silica gel column chromatography (0.040–0.060 mm, 5 80 cm) with CHCl3/MeOH. Fractions EFA6b1 to EFA6b35 were collected and examined by TLC. Fractions EFA6b3 to EFA6b9 (CHCl3/MeOH 9.7 : 0.3), EFA6b13 to EFA6b20 (CHCl3/MeOH 9.5 : 0.5) and EFA6b27 to EFA6b33 (CHCl3/MeOH 9.0 : 1.0) were combined on the basis of same TLC profile of yellow spots. These fractions were subjected to chromatographic purification with CHCl3 and MeOH (100 : 0 to 0 : 100) that afforded compounds 3 (12.0 mg, 2.30 104% of the dried bark), 6 (15.0 mg, 2.86 104% of the dried bark) and 7 (15.0 mg, 2.86 104% of the dried bark) eluted with CHCl3/MeOH 9.0 : 1.0, 8.5 : 1.5 and 8.0 : 2.0 respectively. All the compounds were finally purified over Sephadex LH-20 (bead size 25–100 μ, Sigma-Aldrich) with MeOH (100%). Antioxidant activities The antioxidant activities of compounds 1–7 were evaluated by free radical scavenging of DPPH[34] and ABTS[35] and reducing power FRAP assays.[34] Compounds 1–7 and the standard solutions were prepared in the concentration range of 2.0–50 μM. The inhibition percentages in DPPH and ABTS assays were calculated according to the formula given in the succeeding texts. % inhibition ¼
1 Asample =Acontrol
100
The results of DPPH and ABTS assays were expressed as IC50, while those of the FRAP assay were represented in micromole TE (μM TE). Antibacterial activities Antibacterial activities of the compounds 1–7 were evaluated by Kirby-Bauer disc diffusion[36] and MIC[37] methods against two gram-positive and two gram-negative bacteria. Antituberculosis activities The antituberculosis activities of compounds 1–7 were evaluated against M. tuberculosis H38Rv according to the method of Collins’s BACTEC 460 system (1997).[38] Statistical analyses The data were analyzed and expressed as means ± standard deviation of three replicates (n = 3). The differences between assayed values were analysed using one-way analysis of variance, followed by Tukey’s honestly significant difference test at 95 and 99% confidence intervals using SPSS software, version 19.0 (SPSS Inc., Chicago, USA). Results with p < 0.05 were considered significant, while those with p < 0.01 were regarded as very significant. Volkensiflavone 7-O-rhamnopyranoside (1)
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Acknowledgements This study was financially supported by a Research University Grant (RU1001/PKIMIA/811129). Financial support of Nargis Jamila as a TWAS (Third World Academy of Sciences)-USM (Universiti Sains Malaysia) fellow is also acknowledged. The authors also thank Mr Zahari Othman for NMR measurements.
Conflict of Interest The authors declare no conflict of interest.
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Yellow amorphous solid; mp 275–278 °C; UV λmax nm: (MeOH) 288, (NaOMe) 328, 392, (H3BO3) 293, (NaOAc) 329, 407, (AlCl3)
307, (AlCl3 + HCl) 305, 341; CD (DMSO) [θ]287.4 8.75199; IR (KBr) vmax: 3255, 1637, 1591 cm1, 1H and 13C NMR, refer to Tables 1 and 2 respectively; +/ESI-MS at m/z 687.9 and 685.1572 [M–H]+ (calcd. 685.1635).
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