DNA topoisomerase IIα inhibitory and anti-HIV-1 flavones from leaves and twigs of Gardenia carinata

Fitoterapia 83 (2012) 368–372

Contents lists available at SciVerse ScienceDirect

Fitoterapia journal homepage: www.elsevier.com/locate/fitote

DNA topoisomerase IIα inhibitory and anti-HIV-1 flavones from leaves and twigs of Gardenia carinata Naowarat Kongkum a, Patoomratana Tuchinda a,⁎, Manat Pohmakotr a, Vichai Reutrakul a, Pawinee Piyachaturawat b, Surawat Jariyawat b, Kanoknetr Suksen b, Chalobon Yoosook c, Jitra Kasisit c, Chanita Napaswad c a b c

Department of Chemistry and Center for Innovation in Chemistry (PERCH-CIC), Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand Department of Physiology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand Department of Microbiology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand

a r t i c l e

i n f o

Article history: Received 23 July 2011 Accepted in revised form 23 November 2011 Available online 3 December 2011 Keywords: Gardenia carinata Rubiaceae Flavones Cytotoxic activity DNA topoisomerase IIα inhibitory activity Anti-HIV-1 activity

a b s t r a c t Four new flavones, 5,2′-dihydroxy-7,3′,4′,5′-tetramethoxyflavone (1), 5,2′,5′-trihydroxy7,3′,4′-trimethoxyflavone (2), 5,7,2′,5′-tetrahydroxy-6,3′,4′-trimethoxyflavone (3) and 5,2′,5′-trihydroxy-6,7,3′,4′-tetramethoxyflavone (4), along with the known 5,3′-dihydroxy6,7,4′,5′-tetramethoxyflavone (5), 5,7,3′,5′-tetrahydroxy-6,4′-dimethoxyflavone (6), syringaldehyde, vanillic acid and scopoletin were isolated from the leaves and twigs of Gardenia carinata (Rubiaceae). Their structures were determined by spectroscopic methods. Flavone 2 exhibited cytotoxic activity against P-388 and MCF-7 cell lines, while 3, 5 and 6 were active only in P-388 cell line. All active compounds were found to inhibit DNA topoisomerase IIα activity, which may be responsible for the observed cytotoxicity. Flavones 1–3, 5 and 6 also exhibited anti-HIV-1 activity in the anti-syncytium assay using ΔTat/revMC99 virus and 1A2 cell line system; 2 was most potent. Only flavones 1 and 6 showed considerably activity against HIV-1 reverse transcriptase. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Plants of the genus Gardenia (Rubiaceae), which comprises more than 80 species, are widely distributed among the tropical forests of the world. In Thailand, fifteen species, including G. carinata Wall., were reported [1]. Gardenia carinata, known in Thai as ‘Phut nam but’ or ‘Rak na’, is classified as a tree found in the southern part of the country. Previous investigations of Gardenia species revealed that these plants are rich of flavonoids [2–12] and several of them exhibited cytotoxic [9–11] and anti-HIV-1 [9,10,12] activities. As part of our ongoing search for cytotoxic and anti-HIV-1 agents from plants [9–12], a crude active methanol extract obtained from leaves and twigs was further dissolved in EtOAc:MeOH (1:1) and

⁎ Corresponding author. Tel.: + 66 2 2015159; fax: + 66 2 3547151. E-mail address: [email protected] (P. Tuchinda). 0367-326X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2011.11.015

MeOH, respectively. The EtOAc:MeOH (1:1) soluble fraction was found to be active against P-388, KB and MCF-7 cancer cell lines with ED50 values b4, 14.5 and 12.7 μg/mL, respectively. This fraction was also active in the anti-syncytium assay (EC50 7.9 μg/mL, selectivity index, SI = 1.5), and exhibited potent activity with 100% inhibition at 200 μg/mL against HIV-1 reverse transcriptase. Separation of the EtOAc:MeOH (1:1) soluble fraction by chromatography and recrystallization led to the isolation of four new flavones, 5,2′-dihydroxy-7,3′,4′,5′tetramethoxyflavone (1), 5,2′,5′-trihydroxy-7,3′,4′-trimethoxyflavone (2), 5,7,2′,5′-tetrahydroxy-6,3′,4′-trimethoxyflavone (3) and 5,2′,5′-trihydroxy-6,7,3′,4′-tetramethoxyflavone (4), along with the known 5,3′-dihydroxy-6,7,4′,5′-tetramethoxyflavone (5) [10,13], 5,7,3′,5′-tetrahydroxy-6,4′-dimethoxyflavone (6) [14], syringaldehyde [15], vanillic acid [16] and scopoletin [17]. We herein describe the isolation, structure elucidation, cytotoxic, DNA topoisomerase IIα inhibitory and antiHIV-1 activities of the compounds with sufficient amounts.

N. Kongkum et al. / Fitoterapia 83 (2012) 368–372

2. Experimental procedure 2.1. General Melting points: digital melting point apparatus (Electrothermal). Optical rotations: JASCO DIP-370. UV: JASCO V-530. FT-IR: Perkin Elmer 2000. 1H NMR (500 MHz), 13C NMR (125 MHz), DEPT and 2D-NMR spectra: Bruker AV 500 in CD3SOCD3, otherwise stated. HR-TOF-MS: Micromass model VQ-Tof2. EIMS 70 eV: Thermo Finnigan Polaris Q. Column chromatography (CC): silica gel 60 (Merck, 70–230 mesh) or Sephadex LH 20 (Pharmacia). Preparative thin-layer chromatography (Prep-TLC): Kieselgel 60 PF254 (Merck, 0.5 mm). 2.2. Plant material The leaves and twigs of G. carinata Wall. were collected from Narathiwat province of Thailand in November 1997 and identified by T. Santisuk. A voucher specimen (BKF 147819) has been deposited at the Forest Herbarium, Royal Forest Department, Bangkok, Thailand. 2.3. Extraction and isolation Air-dried and powdered leaves and twigs (4.4 kg) were percolated with MeOH (5 × 13.5 L) at room temperature to give a crude MeOH extract (546 g). Sequential dissolving in MeOH–EtOAc (1:1, 2.5 L) and MeOH (1 L) followed by solvent removal yielded fraction 1 (412 g), fraction 2 (47.1 g), and the residue (67.1 g), respectively. The bioactive fraction 1 (411 g) was subjected to Si-gel CC (EtOAc–hexane and MeOH–EtOAc gradients) to yield fractions A1–A6. Separation of fr. A4 (51.5 g) by Si-gel CC (EtOAc–hexane gradient) gave frs. B1–B10. Fr. B8 (9.45 g) was further separated on a Sephadex LH-20 column (70% MeOH–CH2Cl2 as eluent) to afford frs. C1–C3. Fr. C3 (1.09 g) was rechromatographed by Si-gel CC (MeOH–CH2Cl2 gradient) to give frs. D1–D6. Fr. D1 (38.3 mg) after Si-gel prep-TLC (60% EtOAc–hexane) and recrystallization from CH2Cl2–hexane gave syringaldehyde (13.1 mg, Rf = 0.48). Fr. D2 (137.0 mg) after recrystallization from DMSO–CH2Cl2 provided 5,2′-dihydroxy-7,3′,4′,5′-tetramethoxyflavone (1) (15.2 mg). Fr. D3 (323.2 mg) after recrystallization from MeOH–CH2Cl2 afforded vanillic acid (33.6 mg). Fr. A5 (127.0 g) was further separated by Si-gel CC (EtOAc–hexane gradient) to give frs. E1–E11. Fr. E6 (15.6 g) was rechromatographed by Si-gel CC (MeOH–CH2Cl2 gradient) to give frs. F1–F4. Fr. F1 (2.17 g) was further purified by Sephadex LH-20 CC (50% MeOH–CH2Cl2) to give frs. G1–G4. Fr. G2 (323.3 mg) after recrystallization from MeOH–CH2Cl2 afforded 5,3′-dihydroxy-6,7,4′,5′-tetramethoxyflavone (5) (27.2 mg). Fr. G3 (425.8 mg) after Si-gel CC (1% MeOH– CH2Cl2) and recrystallization from DMSO–CH2Cl2 furnished 5,2′,5′-trihydroxy-7,3′,4′-trimethoxyflavone (2) (66.6 mg). Fr. E7 (8.86 g) was further purified by Sephadex LH-20 CC (MeOH as eluent) to give frs. H1–H7. Fr. H3 (737.2 mg) after Si-gel prep-TLC (5% MeOH–CH2Cl2) and recrystallization from MeOH provided scopoletin (34.4 mg, Rf = 0.46). Fr. H4 (306.6 mg) after Si-gel prep-TLC (5% MeOH–CH2Cl2) and recrystallization from MeOH afforded 5,7,2′,5′-tertrahydroxy6,3′,4′-trimethoxyflavone (3) (42.5 mg, Rf = 0.34). Fr. H5 (265.3 mg) after recrystallization from MeOH gave 5,7,3′,5′-

369

tetrahydroxy-6,4′-dimethoxyflavone (6) (28.5 mg). Fr. E8 (15.2 g) was further purified by Sephadex LH-20 CC (MeOH as eluent) to yield frs. I1–I4. Fr. I3 (317.6 mg) was further separated by Si-gel CC (MeOH–CH2Cl2) to give frs. J1–J8. Fr. J2 (30.5 mg) after recrystallization from MeOH afforded 5,2′,5′trihydroxy-6,7,3′,4′-tetramethoxyflavone (4) (6.0 mg). 5,2′-Dihydroxy-7,3′,4′,5′-tetramethoxyflavone (1): yellow needles; mp 258–259 °C (DMSO–CH2Cl2). UV (MeOH) λmax (log ε): 268 (4.87), 290 (4.72), 363 (4.74) nm; FT-IR (KBr): υmax 3446, 1664, 1615, 1585, 1494, 1463, 1446, 1419, 1371, 1343, 1270, 1257, 1206, 1161, 1110, 1089, 1052, 950, 918 cm − 1; 1H NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) data, see Table 1; HMBC correlation data, see Table 2; EIMS m/z 374 [M] + (100), 359 (54), 313 (25), 208 (3), 193 (3), 167 (16), 138 (4); HR-TOF-MS m/z 375.1071 [M+H] + (calcd for C19H19O8, 375.1080). 5,2′,5′-Trihydroxy-7,3′,4′-trimethoxyflavone (2): yellow amorphous solid; mp 264–265 °C (DMSO–CH2Cl2). UV (MeOH) λmax (log ε): 267 (3.23), 311 (4.01), 365 (4.09) nm; FT-IR (KBr): υmax 3527, 1663, 1610, 1590, 1497, 1456, 1438, 1425, 1385, 1338, 1308, 1267, 1198, 1157, 1074, 1023, 955 cm − 1; 1H NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) data, see Table 1; HMBC correlation data, see Table 2; EIMS m/z 360 [M] + (100), 345 (77), 317 (21), 179 (8), 167 (37), 166 (7), 123 (2); HR-TOF-MS m/z 361.0976 [M+H] + (calcd for C18H17O8, 361.0923). 5,7,2′,5′-Tetrahydroxy-6,3′,4′-trimethoxyflavone (3): yellow amorphous solid; mp 263–264 °C (MeOH). UV (MeOH) λmax (log ε): 272 (4.39), 322 (4.26), 360 (4.32) nm; FT-IR (KBr): υmax 3522, 3474, 1664, 1619, 1573, 1491, 1457, 1424, 1387, 1371, 1279, 1199, 1159, 1113, 1072, 1028, 1006, 943 cm − 1; 1H NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) data, see Table 1; HMBC correlation data, see Table 2; EIMS m/z 376 [M] + (100), 361 (37), 333 (29), 194 (2), 183 (1), 139 (3); HR-TOF-MS m/z 399.0606 [M+Na] + (calcd for C18H16O9 Na, 399.0692). 5,2′,5′-Trihydroxy-6,7,3′,4′-tetramethoxyflavone (4): yellow amorphous solid; mp 228–229 °C (MeOH). UV (MeOH) λmax (log ε): 273 (4.31), 316 (4.17), 360 (6.18) nm; FT-IR (KBr): υmax 3434, 1664, 1617, 1590, 1498, 1460, 1430, 1349, 1301, 1274, 1203, 1175, 1156, 1125, 1080, 1034, 1017, 954 cm− 1; 1H NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) data, see Table 1; HMBC correlation data, see Table 2; HR-TOFMS m/z 413.0818 [M+Na]+ (calcd for C19H18O9Na, 413.0848). 2.4. Cytotoxic assay The cytotoxic activities of the tested compounds were carried out using the in vitro sulforhodamine B (SRB) method as previously reported [18] and ellipticine was used as a positive control. Test samples were dissolved in DMSO as a stock concentration at 4 mg/mL and were tested in triplicate with a final concentration of DMSO at 0.1%. The cancer cell lines were grown in a 96-well plate in the following media: P-388, in RPMI-1640 with 5% fetal bovine serum (FBS). The KB, Col-2, MCF-7 and ASK cell lines were cultured in MEM (minimum essential medium with Earle's salt and L-glutamine) with 10% FBS, while Lu-1 was grown in MEM with 5% FBS. After drug exposure at 37 °C for 72 h (48 h for P-388) with 5% CO2 in air, and 100% relative humidity, cells were fixed with a final concentration of 10% trichloroacetic acid and stained with

370

N. Kongkum et al. / Fitoterapia 83 (2012) 368–372

Table 1 NMR data of flavones 1–4 in DMSO-d6. Position

2 3 4 4a 5 6 7 8 8a 1′ 2′ 3′ 4′ 5′ 6′ 5-OH 7-OH 2′-OH 5′-OH 6-OMe 7-OMe 3′-OMe 4′-OMe 5′-OMe a 1

δH mult, J (Hz)a

δCb

1

2

3

4

1

2

3

4

– 7.10 s – – – 6.37 d (2.2) – 6.82 d (2.2) – – – – – – 7.26 s 12.89 s – 9.87 br s – – 3.86 s 3.78 s 3.86 s 3.84 s

– 7.09 s – – – 6.36 d (2.2) – 6.65 d (2.2) – – – – – – 7.17 s 12.91 s – 9.63 s 9.21 s – 3.85 s 3.77 s 3.86 s –

– 7.03 s – – – – – 6.49 s – – – – – – 7.11 s 13.01 s 10.81 br s 9.60 br s 9.25 br s 3.73 s – 3.76 s 3.85 s –

– 7.11 s – – – – – 6.82 s – – – – – – 7.19 s 12.88 s – 9.63 s 9.17 s 3.72 s 3.92 s 3.77 s 3.86 s –

161.4 108.8 182.0 104.6 161.0 98.0 165.2 92.7 157.4 111.7 145.4 141.8 145.9 145.8 106.5 – – – – – 56.1 61.1 60.6 56.6

161.4 108.8 182.0 104.7 161.2 97.9 165.2 92.4 157.3 112.0 143.3 141.8 144.9 143.8 109.1 – – – – – 56.0 61.0 60.3 –

161.2 108.1 182.2 104.1 152.7 131.3 157.4 94.0 152.5 112.1 143.3 141.8 144.7 143.6 108.9 – – – – 60.0 – 61.0 60.3 –

161.4 108.4 182.3 105.0 152.0 131.8 158.7 91.3 152.7 112.0 143.7 141.8 144.8 143.2 109.1 – – – – 60.3 56.4 60.9 60.3 –

H NMR (500 MHz). C NMR (125 MHz).

b 13

0.4% sulforhodamine B in 1% acetic acid. The bound and dried stain was solubilized with 10 mM trizma base, after removal of the unbound dye by washing. The absorbance at wavelength 510 nm was read on a Fluostar optima BMG plate reader. The cytotoxic activity is expressed as 50% effective dose (ED50). 2.5. Topoisomerase IIα assay The assay was conducted according to the standard protocol of TopoGen, Inc. (San Diego, CA, USA). The total reaction mixture (20 μL), containing Tris–HCl (50 mM, pH 8.0), NaCl (150 mM), MgCl2 (10 mM), ATP (2 mM), DTT (0.5 mM) and BSA (30 μg/mL), human topoisomerase IIα (1 unit), plasmid pBR322 DNA (Glen Burnie, MD, USA, 0.5 mg) and tested compound (20 μM), was incubated for 1 h at 37 °C. The reaction was stopped by addition of a proteinase K solution (1 μL of 1 mg/mL) and further incubated at 50 °C for 1 h. The reaction mixture was then separated by 1% agarose gel electrophoresis, and restained with ethidium bromide (2 μg/mL). The DNA fragments were detected by using an ultraviolet transilluminator gel documentation. The inhibitory activity of human topoisomerase IIα on the relaxation of supercoiled pBR322 plasmid DNA was determined by the conversion of supercoiled pBR322 plasmid to relaxation form. 2.6. Anti-HIV-1 reverse transcriptase assay Compounds were dissolved in DMSO at the concentration of 20 mg/mL and processed further to remove tannin. The assay was carried out in duplicate in a 96-well microtiter plate using tannin-free supernatant of the compound as previously described [19]. An appropriate amount of HIV-1 reverse transcriptase (Amersham Pharmacia Biotech Asia

Pacific Ltd., Hong Kong) employed was standardized with fagaronine chloride. This compound and nevirapine were used as positive controls, while DMSO without the extract as a negative control. Test compounds were prescreened at 200 μg/mL and only those which were very active (≥70% inhibition) at this concentration were further determined for the dose, μM, that inhibited 50% HIV-1 RT activity (IC50). 2.7. Cell-based assay for anti-HIV-1 The syncytium assay was carried out in triplicate using MC99 virus and 1A2 cell line system [20,21], starting

ΔTat/rev

Table 2 HMBC correlations observed in flavones 1–4. Carbon

Correlated H 1

2

2 3 4 4a 5 6

3, 6′ 6′ 3 3, 6, 8, 5-OH 6, 5-OH 8, 5-OH

3, – 3, 3, 6, 8,

7 8 8a 1′ 2′ 3′ 4′

6, 8, 5-OH, 7-OMe 6 8 3, 6′ 6′ 3′-OMe 6′, 4′-OMe

5′ 6′

6′, 5′-OMe –

6, 8, 5-OH, 7-OMe 6 8 3, 6′, 2′-OH 6′, 2′-OH 2′-OH, 3′-OMe 6′, 4′-OMe, 5′-OH 6′, 5′-OH 5′-OH

6′ 8 6, 8, 5-OH 5-OH 5-OH

3

4

3, 6′ – 3, 8 3, 8, 5-OH 5-OH 8, 5-OH, 6-OMe 8, 5-OH

3, 6′ – 3, 8 3, 8, 5-OH 5-OH 8, 5-OH, 6-OMe 8, 5-OH, 7-OMe – 8 3, 6′, 2′-OH 6′, 2′-OH 3′-OMe, 2′-OH 6′, 4′-OMe, 5′-OH, 6′, 5′-OH 5′-OH

– 8 3, 6′ 6′ 3′-OMe 6′, 4′-OMe 6′ –

N. Kongkum et al. / Fitoterapia 83 (2012) 368–372

at the final concentrations of 3.9–125 μg/mL or higher, as indicated otherwise. Virus control and cell control wells contained neither the compound nor virus; cytotoxicity control wells containing cells with the compound and a positive control, i.e., azidothymidine, AZT, were included. The result was expressed as 50% effective concentration (EC50). Cytotoxicity of the compound was also carried out, in parallel and in duplicate, using colorimetric XTT assay. The result was expressed as the concentration that inhibited 50% formazan formation in uninfected cells (IC50). The selectivity index (SI) was calculated using the equation: SI = IC50/EC50. 3. Results and discussion Compounds 1–4 showed the UV maxima, together with the IR absorptions of phenolic and conjugated carbonyl groups typical for flavone derivatives (see Section 2). In general, the 1H NMR spectra of 1–4 in DMSO-d6 (Table 1) displayed a lowfield singlet of a chelated 5-OH and two singlets corresponding to the olefinic H-3 and aromatic H-6′, as confirmed by the observed HMBC correlations (Table 2) of 5-OH/C-4a, C-5, C-6 and C-7, H-3/C-2 and H-6′/C-2. The structures of 1 (C19H18O8) and 2 (C18H16O8) are closely similar. Ring-A of both compounds was proved to be the same with a chelated hydroxy group at C-5 and a methoxy group at C-7 (Fig. 1). The observed HMBC correlations of the 7-OMe signals (δ 3.86 in 1 and 3.85 in 2) to C-7 signal (δ 165.2 in both 1 and 2) confirmed the assignments, and supported by the NOE enhancements of H-6 (δ 6.37 in 1, 4.6% and δ 6.36 in 2, 6.3%) and H-8 (δ 6.82 in 1, 5.0% and δ 6.65 in 2, 6.3%) when irradiated 7-OMe signal. More evidence for the assignment of ring-A oxygenation pattern was obtained from the observed ion at m/z = 167 [C8H7O4] + derived from a retro-Diels-Alder fragmentation in the EIMS. Ring-B of 1 was identified to possess three methoxy groups, whereas only two were found in 2. The placement of one methoxy group at C-5′ in 1 was confirmed by an NOE

371

Fig. 2. Inhibitory activity of flavones 2, 3, 5 and 6 on DNA topoisomerase. IIα, using etoposide as a positive control. Lane 1, pBR322 DNA. Lane 2, pBR322 DNA with topoisomerase IIα enzyme. Lane 3, pBR322 DNA with topoisomerase IIα enzyme and etoposide. Lanes 4–7, pBR322 DNA with topoisomerase IIα enzyme and the tested flavone. S = supercoiled DNA, R = relaxed DNA.

enhancement of H-6′ signal by 9.5%, when irradiated the 5′OMe signal (δ 3.84). As no NOE enhancement of ring-B proton or H-3 was inspected upon irradiation of δ 3.78 or 3.86 signal in 1, these two methoxy groups were suggested to be flanked by two oxygenated functionalities and assigned to 3′-OMe and 4′-OMe, respectively. These assignments were confirmed by the HMBC correlations to the corresponding carbons. Consequently, the only hydroxy group of ring-B in 1 was assigned to substitute at C-2′ and its structures were established as 5,2′-dihydroxy-7,3′,4′,5′-tetramethoxyflavone (1). Similarly, the two methoxy groups in 2 were suggested to locate at C-3′ and C-4′, as no enhancement of ring-B proton or H-3 was observed when irradiated the signals at δ 3.77 (3′OMe) and 3.86 (4′-OMe), respectively. The chemical shift assignments of the two hydroxy signals in ring-B of 2 were due to the HMBC correlations of 2′-OH/C-1′, C-2′ and C-3′, together with 5′-OH/C-4′, C-5′ and C-6′ (see Table 2). Full assignments as shown in Table 1 were performed on the basis of 2D-NMR analyses, which supported the structure as 5,2′,5′-trihydroxy-7,3′,4′-trimethoxyflavone (2). Compounds 3 (C18H16O9) and 4 (C19H18O9) were identified as flavone derivatives, possessing three and four methoxy groups in their structures, respectively. The B-ring oxygenation pattern (2′-OH, 3′-OMe, 4′-OMe and 5′-OH) of 3 and 4 was proposed to be the same as 2, due to their similarities of ring-B chemical shifts in the 1H and 13C spectra of which the assignments were confirmed by HMBC correlations around the ring (see Tables 1 and 2). As a consequence, ring-A of 3 and 4 had to contain one and two methoxy groups, respectively. The only methoxy group in ring-A of 3 was assigned to be at C-6, because no NOE enhancement of H-8 (δ 6.49, s) was observed when irradiated this OMe signal (δ 3.73, s). In contrast, the H-8 signal (δ 6.82) of 4 was enhanced by 3.9% after irradiation of the methoxy singlet at δ 3.92; therefore this methoxy group was proposed to be at Table 3 Cytotoxicities of flavones 1–3, 5 and 6. Compound Cell line (ED50 μM)a

1 2 3 5 6 Ellipticine

Fig. 1. The structures of flavones 1–6 isolated from leaves and twigs of G. carinata.

P-388 KB

Col-2

MCF-7 Lu-1

ASK

Hek 293

52.8 6.1 4.2 7.0 8.7 1.9

>53.5 >55.5 28.7 39.0 >57.8 2.2

> 53.5 10.3 28.1 > 53.5 > 57.8 2.3

> 53.5 46.4 34.1 23.0 47.4 2.2

>53.5 8.4 9.9 17.6 27.4 2.0

>53.5 41.1 28.8 >53.5 >57.8 2.2

>53.5 30.0 28.4 >53.5 >57.8 1.1

a P-388: murine lymphocytic leukemia, KB: human epidermoid carcinoma, Col-2: human colon cancer, MCF-7: human breast cancer, Lu-1: human lung cancer, ASK: rat glioma, Hek 293: non-cancerous human embryonic kidney cell line.

372

N. Kongkum et al. / Fitoterapia 83 (2012) 368–372

Table 4 Anti-HIV-1 activities of flavones 1–3, 5 and 6, as determined by using cell based, ΔTat/RevMC99 virus and 1A2 cell line, and reverse transcriptase assays. Compound

1 2 3 5 6

Cytotoxic and syncytium assays IC50

EC50

(μM)

(μM)

412.0 43.2 61.5 560.9 105.7

162.8 3.4 24.6 308.3 36.6

SI

2.5 12.7 2.5 1.8 2.9

Activity

A A A A A

HIV-1 RT assay % Inhibition at

IC50

200 μg/mL

(μM)

82.2 0.83 1.4 37.7 88.1

204.5 ND ND ND 291.2

Cytotoxic assay: IC50 = dose of compound that inhibited 50% metabolic activity of uninfected 1A2 cells. AZT, averaged from three experiments, IC50 > 10− 2 μM (less than 50% inhibition at this concentration). Syncytium assay: EC50 = dose of compound that reduced 50% syncytium formation by ΔTat/RevMC99 virus in 1A2 cells. AZT, averaged from three experiments, EC50 3.42 × 10− 3 μM; A = active (inactive = less than 50% reduction of syncytium formation at the highest concentrations tested). SI, selectivity index: IC50/EC50. RT assay: ND = not determined. Positive controls, averaged from two experiments, IC50 fagaronine chloride, 26.5 μM,and nevirapine,7.9 μM. Note: The coefficients of determination, R2, were less than 0.80–0.99 in all assays for 50% end point.

C-7. As described earlier that the functionality at C-5 was a hydroxyl group; another methoxy group (δ 3.72) of ring-A in 4 had to be allocated at C-6. However, the presence of fragment ions at m/z 183 in 3 and at m/z 197 in 4, derived from retro Diels-Alder fragmentation, confirmed the substitution pattern in ring-A. Thus, their structures were elucidated as 5,7,2′,5′-tetrahydroxy-6,3′,4′-trimethoxyflavone (3) and 5,2′,5′trihydroxy-6,7,3′,4′-tetramethoxyflavone (4), respectively. The known compounds were identified as 5,3′-dihydroxy-6,7,4′,5′-tetramethoxyflavone (5) [10,13], 5,7,3′,5′tetrahydroxy-6,4′-dimethoxyflavone (6) [14], syringaldehyde [15], vanillic acid [16] and scopoletin [17] by direct comparison of their physical properties and spectroscopic data with those reported in the literature. Pure isolated flavones 1–3, 5 and 6 were evaluated for cytotoxic effects against a panel of cultured mammalian cancer cell lines [18] and non-cancerous human embryonic kidney cell line (Hek 293). The results are shown in Table 3. All tested flavones, except 1, exhibited cytotoxic activity against P388 cell line. Flavone 2 also showed cytotoxic activity against MCF-7 cell line. However, flavones 2 and 3 were found toxic against Hek 293 cell line. Based on the cytotoxic activities, the active compounds 2, 3, 5 and 6 were evaluated for their DNA topoisomerase-IIα inhibition by relaxation assay. All tested compounds exhibited similar inhibitory effects on the topoisomerase-IIα enzyme at a concentration of 20 μM. The relative rank order inhibitory potency of compounds was 3 > 6 > 2 > 5 as shown in Fig. 2. These data indicated that all tested compounds effectively inhibited relaxation activity of topoisomerase IIα

similar to that of the positive control etoposide at the same concentration. These inhibitory activities may be responsible for their observed cytotoxicities. The same set of compounds were also tested employing HIV-1 RT assay as described previously [19] and the syncytium assay using ΔTat/RevMC99 and 1A2 cell line system [20,21]. The results as shown in Table 4 indicated that all tested compounds were active in the syncytium assay, while flavone 2 was most potent (EC50 = 3.4 μM and SI, selectivity index, IC50/EC50 = 12.7. Flavones 1 and 6 were considered very active (82.2% and 88.1% inhibition at 200 μg/mL, with IC50 values of 204.5 and 291.2 μM, respectively) against HIV-1 reverse transcriptase.

Acknowledgements This research project is supported by Mahidol University, the Office of the Higher Education Commission and Mahidol University under the National Research Universities Initiative, the Commission on Higher Education (CHE-RES-RG) and Center for Innovation in Chemistry (PERCH-CIC).

References [1] Smitinand T. Thai plant names. Revised edition by The Forest Herbarium, Royal Forest Department. Bangkok: Pra Cha Chon Co., Ltd; 2001. p. 248. [2] Gunatilaka AAL, Sirimanne SR, Sotheeswaran S, Sriyani HTB. Phytochemistry 1982;21:805. [3] Chhabra SC, Gupta SR, Sharma ND. Phytochemistry 1977;16:399. [4] Chhabra SC, Gupta SR, Sharma CS, Sharma ND. Phytochemistry 1977;16:1109. [5] Krishnamurti M, Seshardi TR, Sharma ND. Indian J Chem 1972;10:23. [6] Krishnamurti M, Seshadi TR, Sharma ND. Indian J Chem 1971;9:189. [7] Rao AVR, Venkataraman K, Chakrabarti P, Sunyal AK, Bose PK. Indian J Chem 1970;8:398. [8] Rao AVR, Venkataraman K. Indian J Chem 1968;6:677. [9] Tuchinda P, Pompimon W, Reutrakul V, Pohmakotr M, Yoosook C, Kongyai N, et al. Tetrahedron 2002;58:8073. [10] Reutrakul V, Krachangchaeng C, Tuchinda P, Pohmakotr M, Jaipetch T, Yoosook C, et al. Tetrahedron 2004;60:1517. [11] Silva GL, Gil RR, Cui B, Chai H, Santisuk T, Srisook E, et al. Tetrahedron 1997;53:529. [12] Tuchinda P, Saiai A, Pohmakotr M, Yoosook C, Kasisit J, Napaswat C, et al. Planta Med 2004;70:366. [13] Iinuma M, Tanaka T, Matsuura S. Chem Pharm Bull 1984;32:2296. [14] Parmar VS, Taneja P, Singh S, Jain R, Sharma SK, Boll PM, et al. Indian J Chem 1994;33B:305. [15] Gutierrez AB, Herz W. Phytochemistry 1988;27:3871. [16] Christophoridou S, Dais P, Tseng LH, Spraul M. J Agric Food Chem 2005;53:4667. [17] Razdan TK, Qadri B, Harkar S, Waight ES. Phytochemistry 1987;26: 2063. [18] Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, et al. J Natl Cancer Inst 1990;82:1107. [19] Tan GT, Pezzuto JM, Kinghorn AD, Hughes SH. J Nat Prod 1991;54:143. [20] Kiser R, Makovsky S, Terpening SJ, Laing N, Clanton DJ. J Virol Meth 1996;58:99. [21] Nara PL, Hatch WC, Dunlop NM, Robey WG, Arthur LO, Gonda MA, et al. AIDS Res Hum Retrovirus 1987;3:283.