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Two new dihydroamentoflavone glycosides from Cycas revoluta a
a
b
Abeer Moawad , Mona Hetta , Jordan K. Zjawiony , Daneel b
Ferreira & Mohamed Hifnawy
c
a
Department of Pharmacognosy , School of Pharmacy, Beni Suef University , Beni Suef , 62514 , Egypt b
Department of Pharmacognosy and Research Institute of Pharmaceutical Sciences , School of Pharmacy, University of Mississippi , University , MS , 38677 , USA c
Department of Pharmacognosy , School of Pharmacy, Cairo University , Cairo , 11561 , Egypt Published online: 09 Sep 2013.
To cite this article: Natural Product Research (2013): Two new dihydroamentoflavone glycosides from Cycas revoluta , Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2013.832675 To link to this article: http://dx.doi.org/10.1080/14786419.2013.832675
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Natural Product Research, 2013 http://dx.doi.org/10.1080/14786419.2013.832675
Two new dihydroamentoflavone glycosides from Cycas revoluta Abeer Moawada, Mona Hettaa*, Jordan K. Zjawionyb, Daneel Ferreirab and Mohamed Hifnawyc a
Department of Pharmacognosy, School of Pharmacy, Beni Suef University, Beni Suef 62514, Egypt; Department of Pharmacognosy and Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, MS 38677, USA; cDepartment of Pharmacognosy, School of Pharmacy, Cairo University, Cairo 11561, Egypt b
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(Received 21 March 2013; final version received 9 July 2013) Phytochemical investigation of the ethyl acetate extract of Cycas revoluta Thunb. leaflets afforded five compounds including two new dihydroamentoflavone glucosides, (2S)I-(2,3)-dihydro-I-7-O-b-D -glucopyranosylamentoflavone (1) and (2S)-I-(2,3)-dihydro-I-7, II-7-di-O-b-D -glucopyranosylamentoflavone (2), in addition to the known compounds prunin (3), vitexin-200 -rhamnoside (4) and protocatechuic acid (5). Compounds (3) and (4) being reported for the first time in this plant. The structures of these compounds were established by the detailed analysis of their spectroscopic data, mainly 1D NMR, 2D NMR, CD and HR-MSD-TOF. The ethyl acetate extract showed weak cytotoxicity against HepG2 (IC50 ¼ 207.6 mg/mL) and RAW 264.2 cells (IC50 ¼ 160.8 mg/mL). Compound 4 showed significant activity towards Leishmania donavani (IC50 ¼ 13.8 mM, IC90 ¼ 34.6 mM). The isolated compounds showed weak antimicrobial activity (IC50 . 10 mg/mL). Keywords: Cycadaceae; Cycas revoluta; amentoflavone glycosides; antimicrobial; antileishmanial; cytotoxicity
1. Introduction The cycad Cycas revoluta is the most widespread species of the genus Cycas and is involved in many patented Chinese and Japanese drug formulae for treatment of liver diseases (Han 1992; Wang 2009), AIDS (Wang 2007) stimulation of hair growth (Osamu et al. 2002) and prevention of development of grey hair (Kenji et al. 2001). In a previous communication (Moawad et al. 2010), we reported the isolation of 12 compounds from C. revoluta leaflets from the chloroform solubles of the methanolic extract. In this article, we report the isolation of two new dihydroamentoflavone glycosides, I-(2,3)dihydro-I-7-O-b-D -glucopyranosylamentoflavone (1) and I-(2,3)-dihydro-I-7,II-7-di-O-b-D glucopyranosylamentoflavone (2), as well as two other monoflavonoid glycosides from the ethyl acetate solubles of the same extract of C. revoluta. The structures of these compounds were established by the detailed analysis of their spectroscopic data, mainly 1D NMR, 2D NMR, CD and HR-MSD-TOF. The monoflavonoid glycosides are reported for the first time from C. revoluta. The antimicrobial and antileishmanial activities of the isolated compounds, as well as the cytotoxic activity of the EtOAc solubles, were performed. 2. Results and discussion The phytochemical investigation of the EtOAc solubles of the methanolic extract of C. revoluta leaflets (Figure S1) afforded three known and two new amentoflavone glucosides (cf. Dossaji
*Corresponding author. Email:
[email protected] q 2013 Taylor & Francis
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A. Moawad et al.
et al. 1973). These authors reported that the Cycadales species including C. revoluta leaflets were free from biflavone glycosides. It is worthwhile to mention that biflavone glycosides are rare natural products; only amentoflavone, ginkgetin and isoginkgetin glycosides were previously reported to occur in the Psilotaceae (Markham 1984), Ferns (Wallace & Markham 1978), Berberidaceae (Naokata et al. 1974) and Ginkgoaceae (Hyun et al. 2005). Thus, the presence of amentoflavone glucosides in C. revoluta may contribute to the chemotaxonomy of the family Cycadaceae. Of the three known compounds, prunin (3) and vitexin-200 -rhamnoside (4) are reported for the first time in C. revoluta. The structures of these compounds were established by the detailed analysis of their physical and spectroscopic data in comparison with reported data.
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2.1. Structure elucidation of compounds 1 –5 Compound 1: It was obtained as a yellow powder. Its molecular formula was determined as C36H30O15, on the basis of a pseudomolecular (M –H)2 ion at m/z 701.1617 in the negative mode and the [M þ H]þ ion at m/z 703.1601 in the positive mode of HR-MSD-TOF. The dihydrobiflavone structure of compound 1 was evident from the 1H NMR spectrum that showed two D2O exchangeable signals: one signal at dH 12.06 (characteristic for the 5-OH of a flavanone unit) and the other signal at dH 13.08 (characteristic for the 5-OH of a flavone unit). It also showed one ABX spin system for the heterocyclic protons at dH 5.54, 2.73 and another signal at dH 3.12 overlapped with sugar protons (could be detected from the HMQC and HMBC). The 13C NMR spectrum showed signals at dC 79.5 (I-C-2), 43.0 (I-C-3) and 197.3 (I-C-4) corresponding to ring I-C of the flavanone unit and others at dC 102.6 (II-C-3) and 182.0 (II-C-4) of ring II-C of the flavone unit. The 1H NMR spectrum also showed four singlets corresponding to H-6 and H-8 of I-A ring at dH 6.12 and 6.13, H-6 of II-A ring at dH 6.22 and H-3 of II-C ring at dH 6.72. The AA0 BB0 spin pattern of ring II-B resonated at dH 6.78 (d, J ¼ 8.7 Hz, H-30 , H-50 ) and 7.60 (d, J ¼ 8.7 Hz, H-20 , H-60 ). The ABM spin pattern of ring I-B resonated at dH 6.96 (d, J ¼ 8.3 Hz, H-50 ), 7.38 (d, J ¼ 8.3 Hz, H-60 ) and 7.45 (s, H-20 ). The assignment of these protons was confirmed by HMQC and HMBC experiments. The HMBC spectrum of 1 confirmed the involvement of C-30 (I-B ring) and C-8 (II-A ring) in the interflavanyl linkage via the 3JCH correlations of H-50 (I-B ring, dH 6.96) with C-30 (I-B ring, dC 120.8) and of H-6 (II-A ring, dH 6.22) and H-20 (I-B ring, dH 7.45) with C-8 (II-A ring, dC 106.2), thus indicating compound 1 as a member of the amentoflavone class of biflavonoids. The presence of one sugar moiety was evident from the presence of the anomeric proton at dH 4.93 and six carbon signals at dC 100.2, 77.3, 76.7, 73.5, 69.8, 60.9 which is consistent with O-glucose, the large coupling constant (J ¼ 7.2 Hz) of the anomeric proton-indicated b-orientation. The attachment site of the sugar was confirmed through HMBC since the anomeric proton at dH 4.93 showed 3JCH correlations to I-C-7 at dC 165.6 (Figure 1). Thus, compound 1 was identified as I-(2,3)-dihydro-I-7,II-7-di-O-b-D -glucopyranosylamentoflavone. Because the attachment site of the sugar moiety is distant from the interflavonoid bond, the signals were not duplicated and the rotational isomerism was minimal. Compound 2: It was obtained as a yellow powder. The HR-MSD-TOF showed a pseudomolecular ion peak at m/z 865.2223 [M þ H]þ in the positive mode and an [M – H]2 signal at m/z 863.2030 in the negative mode corresponding to a molecular formula C42H40O20. Compound 2 is also a dihydroamentoflavone glycoside but it contains two O-b-D glucopyranosyl moieties. The 1H and 13C NMR spectra of compound 2 at 294 K showed a pair of rotamers (1:1) due to presence of the sugar unit at II-7 which causes restricted rotation about the interflavanyl bond. At this temperature, some signals were duplicated especially for unit II (Figure S2). The attachment sites of the two sugars to the aglycone were confirmed through HMBC. Correlations were found between the duplicated anomeric protons at dH 5.10 and 4.97 and II-C-7 (dC 160.7) and the other anomeric proton at dH 4.92 and I-C-7
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(dC 165.7) (Figure 1). Compound 2 was thus identified as I-(2,3)-dihydro-I-7,II-7-di-O-b-D glucopyranosylamentoflavone. Compound 3: It was obtained as a white powder. The HR-MSD-TOF spectrum of 3 showed a pseudomolecular ion peak at m/z 435.1310 for the [M þ H]þ at the positive ESI mode (calcd 435.1291) which is consistent with a molecular formula C21H22O10. The 1H NMR spectrum showed an ABX spin system characteristic of ring C of a flavanone moiety and A2B2 spin system characteristic for 1,4 substituted B-ring, indicating that the flavanone aglycone is naringenin. The sugar moiety showed anomeric proton at dH 4.95 and anomeric carbon at dC 100, the carbon signals of the sugar moiety were typical for glucose. HMBC correlation was found between the anomeric proton (dH 4.95) and the dC 165.8 (C-7). Compound 3 was identified as naringenin-7O-glucopyranoside which was consistent with published data (Rahman et al. 1978) (Figure S3). Compound 4: It was obtained as a yellow powder. The HR-MSD-TOF of compound 4 showed a molecular ion peak at m/z 577.1523 [M – 1]2 (calcd 577.5108) corresponding to a molecular formula C27H30O14. The 1H NMR, 13C NMR and HMBC spectra indicated that compound 4 is an apigenin diglycoside in which b-D -glucose is attached to C-8 through CZC linkage and an a-rhamnose moiety attached to C-200 of the glucose moiety. The NMR data were typical for vitexin-200 -rhamnoside (Xu et al. 2009). Compound 5: HR-MSD-TOF of compound 5 showed a molecular ion peak at m/z 153.0284 [M þ 1]þ (calcd 153.1122) corresponding to a molecular formula C7H6O4. 1H and 13C NMR spectra of compound 5 indicated the presence of 1,3,4 substituted phenolic acid with the carboxylic acid carbon at dC 169.3 and two quaternary oxygenated signals at dC 144 and 149. Careful HMBC study of compound 5 allowed us to identify it as protocatechuic acid and not any other positional isomer of dihydroxy benzoic acids (Hang et al. 1998). 2.2. Antimicrobial activity All the tested compounds showed low activities as antibacterial or antifungal against all the selected strains (IC50 . 10 mg/mL).
OH 4' 5'
3' II-B 1'
OH
5'
8
O OH 1''
9 7
I-A
O
1'
2
2
OH
6'
O HO HO
2'
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Unit I
4' I-B
II-C
9
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3' 8
2'
I-C
O
10
II-A
3
6 5
10
OH
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RO
7
O
1 R= H 2 R= β-D-glucopyranosyl
Figure 1. Structures of compounds 1 and 2 with key HMBC correlations.
5 6
Unit II
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O
OH
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2.3. Antileishmanial activity Vitexin rhamnoside (4) showed significant activity towards Leishmania donavani (IC50 ¼ 13.8 mM, IC90 ¼ 34.6 mM) compared to pentamidine (IC50 ¼ 2.9 mM, IC90 ¼ 14.7 mM) and amphotericin B (IC50 ¼ 0.1 mM, IC90 ¼ 0.35 mM) as positive control.
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2.4. Cytotoxic activity Using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, the effect of the sample on the proliferation of Hep-G2 and RAW 264.7 cells was studied after 48 h of incubation. The tested sample showed weak cytotoxic effect against Hep-G2 cells (IC50 ¼ 207.9 mg/mL); however, it showed moderate cytotoxic effect against RAW 264.7 (IC50 ¼ 160.8 mg/mL). 3. Experimental 3.1. General experimental procedures NMR spectra were recorded on a Bruker DRX Ultra 400 NMR spectrometer (Billerica, MA, USA), and Varian Inova-600 NMR spectrometer (Palo Alto, CA, USA), using DMSO-d6 or methanol-d4. UV spectra were recorded on a Varian Cary 50 Bio UV-Vis spectrometer and CD on an Olis DSM 20 instrument, (Bogart, GA, USA), whereas optical rotations were acquired with a 589-546 Rudolph Research Analytical Autopol IV automatic polarimeter (Hackettstown, NJ, USA), and IR was measured on Bruker Tensor 27 (Billerica, MA, USA). HR-MSD-TOF data was obtained on an Agilent Series 1100 SL mass spectrometer (Palo Alto, CA, USA). HPLC was done using a Delta Prep 4000 (Waters Corporation, Milford, MA, USA) equipped with a dual wavelength detector Model 2487 adjusted at 210 and 330 nm. Column chromatography was performed using LC-MS (Bruker Daltonics micrOTOF, Leipzig, Germany) for the evaluation of purified samples’ silica gel (32–63 mm, Dynamic Adsorbents, Inc., Norcross, GA, USA) and Sephadex LH-20 (40–70 mm, GE Healthcare Bio-Science AB, Uppsala, Sweden). The preparative HPLC column was Symmetry C8 PrepTM (Waters Corp., Milford, MA, USA) (19 mm £ 300 mm, 7 mm). 3.2. Plant material Plant material (Figure S1) was collected and identified as mentioned before (Moawad et al. 2010). 3.3. Extraction and isolation The powdered leaflets (1.5 kg) was extracted with 80% MeOH (10 L) and fractionated successively as mentioned before (Moawad et al. 2010). The EtOAc solubles (E, 15 g) was subjected to CC on silica gel (450 g, 52 cm £ 5.5 cm) and eluted successively with gradient CHCl3 – MeOH (5 – 50% in 5% increments, and 50– 100% in 10% increments) to yield four fractions. The CHCl3 – MeOH (65:35– 50 –50) eluate (E.2, 4.5 g) was refractionated on silica column (65 g, 23 cm £ 3 cm) with gradient CHCl3 –MeOH in 5% increments to give three subfractions (E.2.1 –E.2.3). The CHCl3 – MeOH (80:20) eluate (E.2.1, 340 mg) was purified on Sephadex LH-20 with MeOH, and then on RP-HPLC using H2O þ 0.05% formic acid (A) and MeOH þ 0.05% formic acid (B) in a gradient mode: A/B 65/35; 10 min, 65/35 – 50/50; 10 min, 50/50 – 0/100; 20 min with a flow rate of 10 mL/min, UV detection at 210 and 254 nm to afford compound 3 (tR ¼ 26.9 min, 2 mg). The CHCl3 – MeOH (75:25) eluate (E.2.2, 2.5 g) was filtered through Sephadex LH-20 with MeOH and then with RP-HPLC to yield compounds 4 (tR ¼ 21.8 min, 2.5 mg) and 5 (tR ¼ 10.2 min, 2 mg). The CHCl3 – MeOH (60:40) eluate
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(E.2.3, 900 mg) was filtered through Sephadex LH-20 with MeOH and then with RP-HPLC to yield compounds 1 (tR ¼ 34.6 min, 2 mg) and 2 (tR ¼ 30.1 min, 2.5 mg).
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3.4. Characterisation Compound 1: I-(2,3)-dihydro-I-7-O-b-D -glucopyranosylamentoflavone yellow powder; ½a25 D 2 26.7 (MeOH, c ¼ 0.08); UV (MeOH) lmax 275, 330 nm; CD (MeOH) [u ]240 2 6.69 £ 102, [u ]246.3 2 2.54 £ 102, [u ]287.5 2 59.52 £ 102, [u ]319.4 2 60.08 £ 102; IR (NaCl) nmax 3424, 2253, 1648, 1027, 824, 762, 627 cm21; negative HR-MSD-TOF m/z: 701.1617 [M – H]2 (calcd 701.1506), positive HR-MSD-TOF m/z: 703.1601 [M þ H]þ ion (calcd 703.1663). 1 H NMR (400 MHz, DMSO-d6): Unit-I; 5.54 (d, J ¼ 11.7 Hz; H-2), 2.73 (d, J ¼ 15.2 Hz; H-3a), 12.06 (s, 5-OH), 6.12 (s, H-6), 6.13 (s, H-8), 7.45 (s, H-20 ), 6.96 (d, J ¼ 8.3 Hz; H-50 ), 7.38 (d, J ¼ 8.3 Hz; H-60 ), 4.93 (d, J ¼ 7.2 Hz; H-100 ). Unit-2; 6.72 (s, H-3), 13.08 (s, 5-OH), 6.22 (s, H-6), 6.78 (d, J ¼ 8.7, H-30 , H-50 ), 7.60 (d, J ¼ 8.7, H 20 , H-60 ). 13 C NMR (600 MHz, DMSO-d6): Unit-1; 79.5 (C-2), 43.0 (C-3), 197.3 (C-4), 163.34 (C-5), 95.8 (C-6), 165.6 (C-7), 95.9 (C-8), 163.29 (C-9), 103.5 (C-10), 128.3 (C-10 ), 131.8 (C-20 ), 120.8 (C-30 ), 157.3 (C-40 ), 117.3 (C-50 ), 127.7 (C-60 ), 100.2 (C-100 ), 73.5 (C-200 ), 76.7 (C-300 ), 69.8 (C-400 ), 77.3 (C-500 ), 60.9 (C-600 ). Unit-2; 163.38 (C-2), 102.6 (C-3), 182.0 (C-4), 160.7 (C-5), 99.8 (C-6), 160.5 (C-7), 106.2 (C-8), 155.0 (C-9), 102.6 (C-10), 122.3 (C-10 ), 128.6 (C-20 ), 116.2 (C-30 , C-50 ), 161.1 (C-40 ), 128.6 (C-20 , C-60 ). Compound 2: I-(2,3)-dihydro-I-7,II-7-di-O-b-D -glucopyranosyl-amentoflavone, yellow powder; UV (MeOH) lmax 275, 330 nm; CD (MeOH) [u ]221.9 2193.59 £ 102, [u ]235 25.629 £ 102, [u ]246.3 233.15 £ 102, [u ]289.4 274.05 £ 102; IR (NaCl) nmax 3361, 2916, 2848, 1590, 1351, 1274, 2 1071 cm21; ½a25 D 212 (c ¼ 0.05, MeOH); negative HR-MSD-TOF m/z: 863.2030 [M–H] (calcd þ 863.2035), positive HR-MSD-TOF m/z: 865.2223 [M þ H] ion (calcd 865.2191). 1 H NMR (400 MHz, DMSO-d6): Unit-I; 5.52 (t, J ¼ 14.3 Hz; H-2), 2.72 (d, J ¼ 16.2 Hz; H-3a), 12.09 (s, 5-OH), 6.11 (s, H-6), 6.13 (s, H-8), 7.48, 7.45 (s, H-20 ), 7.06 (t, J ¼ 7.1 Hz; H-50 ), 7.43, 7.41 (d, J ¼ 8.3 Hz; H-60 ), 4.92 (d, J ¼ 7.3 Hz; H-100 ). Unit-2; 6.85, 6.83 (s, H-3), 13.18 (s, 5-OH), 6.74, 6.70 (s, H-6), 6.79 (d, J ¼ 7.9, H-30 , H-50 ), 7.64 (d, J ¼ 8.7, H 20 , H-60 ), 7.61 (d, J ¼ 8.2, H 20 , H-60 ), 5.10 (d, J ¼ 7.6 Hz, H-100 ), 4.97 (d, J ¼ 7.4 Hz, H-100 ). 13 C NMR (600 MHz, DMSO-d6): Unit-1; 79.7 (C-2), 43.1 (C-3), 197.9 (C-4), 163.4a (C-5), 96.8 (C-6), 165.7 (C-7), 96.0 (C-8), 163.5a (C-9), 103.4 (C-10), 132.2 (C-10 ), 133.5 (C-20 ), 118.6 (C-30 ), 156.3 (C-40 ), 116.4 (C-50 ), 128.3 (C-60 ), 99.8 (C-100 ), 73.6 (C-200 ), 76.7 (C-300 ), 69.9c (C-400 ), 77.6 (C-500 ), 61.1 (C-600 ). Unit-2; 164.7 (C-2), 103.1 (C-3), 183.0 (C-4), 160.6b (C-5), 97.9 (C-6), 160.7b (C-7), 106.6 (C-8), 154.0 (C-9), 105.7 (C-10), 121.4 (C-10 ), 128.6 (C-20 ), 116.4 (C-30 , C-50 ), 161.8 (C-40 ), 128.6 (C-20 , C-60 ), 100.5 (C-100 ), 73.4 (C-200 ), 76.6 (C-300 ), 69.8c (C-400 ), 77.3 (C-500 ), 60.97, 60.98 (C-600 ). 3.5. Antimicrobial assay All organisms were obtained from the American Type Culture Collection (ATCC) and included the fungi Candida albicans ATCC90028, Candida glabrata ATCC 90030, Candida krusei ATCC 6258, Cryptococcus neoformans ATCC 90113 and Aspergillus fumigatus ATCC 204305, as well as the bacteria Staphylococcus aureus ATCC 29213, methicillin-resistant S. aureus ATCC 33591, Escherichia coli ATCC 35218, Pseudomonas aeruginosa ATCC 27853 and Mycobacterium intracellulare ATCC 23068. This activity was performed according to the method previously mentioned (Moawad et al. 2010). 3.6. Antileishmanial assay Antileishmanial activity of the compounds was tested in vitro against a culture of L. donavani promastigotes, grown in RPMI 1640 medium supplemented with 10% foetal calf serum (FCS,
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Gibco Chem. Co., Grand Island, NY, USA) at 268C. A 3-day-old culture was diluted to 5 £ 105 promastigotes/mL. Drug dilutions (3.1 –50 mg/mL) were prepared directly in cell suspension in 96-well plates. Plates were incubated at 268C for 48 h, and growth of Leishmania promastigotes was determined by the alamarBlue assay (Mikus and Steverding 2000). Standard fluorescence was measured on a Fluostar Galaxy plate reader (BMG Lab Technologies, Ortenberg, Germany) at an excitation wavelength of 544 nm and emission wavelength of 590 nm. Pentamidine and amphotericin B were used as the standard antileishmanial agents. Per cent growth was calculated and plotted versus test concentration for computing the IC50 and IC90 values.
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3.7. Cyototoxic assay Human hepatocarcinoma cell line (HepG2) and Raw murine macrophage (RAW 264.7) purchased from ATCC, Manassas, VA, USA, were used to evaluate the cytotoxic effects of the tested sample following the protocol reported by Hansen et al. (1989). All cell culture materials were obtained from Cambrex Bioscience (Copenhagen, Denmark). Chemicals were purchased from Sigma/Aldrich, St. Louis, MO, USA. All experiments were repeated four times, unless mentioned otherwise. The cytotoxicity of samples was measured against HepG2 and RAW 264.7 cells using the MTT Cell Viability Assay. The MTT assay is based on the ability of active mitochondrial dehydrogenase enzyme of living cells to cleave the tetrazolium rings of the yellow MTT and form dark blue insoluble formazan crystals that are largely impermeable to cell membranes, resulting in its accumulation within healthy cells. Solubilisation of the cells results in the liberation of crystals, which are then solubilised. The number of viable cells is directly proportional to the level of soluble formazan dark blue colour. The extent of the reduction of MTT was quantified by measuring the absorbance at 570 nm. Percentage of relative viability was calculated using the following equation: Absorbance of treated cells £ 100: Absorbance of control cells 4. Conclusion C. revoluta is a good and important source of dihydroamentoflavone class of compounds and could be useful as antileishmanial natural source. Supplementary material Supplementary material relating to this article is available online, alongside Figures S1 and S2 and 1D NMR, 2D NMR and HR-MSD-TOF charts of compounds 1 and 2. Acknowledgements A. Moawad would like to thank the Egyptian government for a fellowship through the Ministry of Higher Education and Scientific Research, Bharathi Avula, Frank Wiggers and Jannie Marais for recording the MS, CD and NMR data and Marsha Wright for the antimicrobial screening which is supported by the NIH, NIAID, Division of AIDS, Grant No. AI 27094 and the USDA Agricultural Research Service Specific Cooperative Agreement No. 58-6408-2-0009.
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