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Homoisoflavanones from Ledebouria floribunda
Fitoterapia 80 (2009) 96–101
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Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f i t o t e
Homoisoflavanones from Ledebouria floribunda María Isabel Calvo ⁎ Dpto. Farmacia y Tecnología Farmacéutica (Facultad de Farmacia), University of Navarra, 31008 Pamplona, Spain
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Article history: Received 16 September 2008 Accepted in revised form 9 October 2008 Available online 30 October 2008 Keywords: Ledebouria floribunda Hyacynthaceae Homoisoflavanones Antioxidant DPPH β-carotene/linoleic acid system
a b s t r a c t The bulbs of Ledebouria floribunda (Baker) Jessop have yielded two novel compounds, 7-O-[αrhamnopyranosyl-(1→6)-β-glucopiranosyl]-5-hydroxy-3-(4-methoxybenzyl)-chroman-4-one (1) and 7-O-[α-rhamnopyranosyl-(1→6)-β-glucopiranosyl]-5-hydroxy-3-(4′-hydroxybenzyl)chroman-4-one (2) along with five other known compounds, 5,7-dihydroxy-3-(4′methoxybenzyl)-chroman-4-one or 3,9-dihidroeucomin (3), 5,7-dihidroxy-6-methoxy-3-(4′methoxybenzyl)-chroman-4-one (4), 5,7-dihidroxy 3-(4′-hydroxybenzyl)-chroman-4-one or 4,4′-demethyl-3,9-dihydropuctatin (5), 5,7-dihidroxy-3-(4′-hydroxybenzyl)-6-methoxychroman-4-one or 3,9-dihydroeucomnalin (6) and 7-hydroxy-3-(4′-hydroxybenzyl)-5methoxy-chroman-4-one (7). Their structures were elucidated by spectra analysis. The seven homoisoflavanones were found to be antioxidant against DPPH radical and β-carotene/linoleic acid system. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Ledebouria is a genus of deciduous or weakly evergreen bulbs in the Hyacynthaceae. It is only recently established as a genus apart from Scilla. Ledebouria sp. is found in areas of summer rainfall in subtropical savannah and grasslands, in the eastern and northeastern parts of southern Africa, India and Madagascar. Ledebouria floribunda (Baker) Jessop is a large species and is used by traditional healers in the Eastern Cape, for a variety of ailments. It occurs in grassland. Several flowering spikes are produced in spring. Homoisoflavanones, which are distributed in the Hyacynthaceae family, have been showed several activities [1–5]. In our continuing study to search for natural antioxidants from medicinal plants, this report presents the isolation and structure elucidation of two new homoisoflavanone (1–2) along with five know compounds (3–7) from the most bioactive MeOH and DCM extracts of the bulbs of L. floribunda. This is the first report of the chemical composition of L. floribunda and antioxidant activity of the homoisoflava⁎ Tel.: +34 48425600 6239; fax: +34 48425649. E-mail address: [email protected]. 0367-326X/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2008.10.006
nones isolated against DPPH radical and β-carotene–linoleic system. 2. Experimental 2.1. General Dichloromethane, n-hexane, ethyl acetate, chloroform, and methanol were purchased from Merck (Darmstadt, Germany). DPPH, β-carotene, linoleic acid, BHA, BTA and αtocopherol were from Sigma (CA, USA). Sephadex LH-20 (Sigma) was used for all CC separations, while silica gel 60 PF254 (Merck) was used for analytical (0.50 mm) and preparative TLC (1.0 mm). 1 H and 13C NMR spectra were measured on a Bruker Avance 300 at 300 MHz and 75 MHz, respectively, with TMS as an internal standard and CDCl3 (Aldrich, CA, USA) as solvent. Chemical shifts were reported in δ units (ppm) and coupling constants (J) in Hz. D/CI mass spectra of the pure compounds were measured on a Finnigan-MatTSQ-700 triple stage quadrupole mass spectrometer, with the following parameters: source current 5 μA, vaporization temperature 450 ° C, sheath gas flow (N2) 80 psi, capillary temperature 150
M.I. Calvo / Fitoterapia 80 (2009) 96–101
to 200 °C. The optical rotations were recorded on a PerkinElmer 241 polarimeter at 25 °C. UV spectra were measured on a Perkin-Elmer UV–Vis-Lambda 200 spectrometer. 2.2. Plant material Fresh bulbs of L. floribunda were collected at Congo and voucher specimen deposited in the authors' laboratory (no. 96097). 2.3. Extraction and isolation Air-dried bulbs (250 g) were ground into fine powder and extracted with dichloromethane (DCM), methanol (MeOH) and water (H2O) at room temp. in a closed container several times. The extracts were concentrated under reduced pressure at 40 °C yielding 3.45 g, 1.07 and 8.79 g of dry extract, respectively.
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The methanol extract was adsorbed on highly polymer resin (Diaion HP-20) and eluted with H2O–MeOH gradient (H2O, 20%, 40%, 60%, 80%, and 100% MeOH). The 40% MeOH eluted fraction (2.61 g) was subjected to silica gel column using CH2Cl2–MeOH gradient (from 15:1 to 1:1), to give six fractions (1–6). Fraction 2 (0.97 g) and 4 (0.84 g) were over Sephadex LH-20 column with methanol to afford compounds 1 (17 mg) and 2 (21 mg), respectively. A part of the DCM extract (2.5 g) was chromatographed on a silica gel column (i.d. 2 × 30 cm) eluted with a gradient nhexane:ethyl acetate (9:1→1:9) yielding 10 fractions (1–10). The antioxidant testing of these fractions with DDPH radical permitted to select the active fractions for further investigation. Fractions 6 (94.1 mg), 7 (421.3 mg) and 9 (90 mg) were found to be active on TLC assay. Separation of fraction 6 through Sephadex LH-20 column (i.d. 1.5 × 25 cm) with CHCl3– MeOH (1:1), afforded five subfractions (6.1→6.5). Purification
Fig. 1. Structure of new homoisoflavanones (1, 2) isolated from Ledebouria floribunda.
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of subfraction 6.1 on prep. TLC using mobile phase n-hexane– EtOAc (1:1) yielded 3 (14 mg). Purification of subfractions 6.3, 6.4 and 6.5 on Sephadex LH-20 with EtOAc yielded 4 (24 mg). Separation of fraction 7 through silica gel column (i.d. 2.5 × 30 cm) with a gradient CHCl3–MeOH (9:1→1:9), afforded 19 subfractions (7.1→7.19). Purification of subfraction 9.7 and 9.19 on prep. TLC using mobile phase n-hexane–EtOAc (1:1) yielded 5 (31 mg) and 6 (27.1 mg), respectively. Similar purification of fraction 9 on prep. TLC yielded 7 (9 mg). These compounds showed a satisfactory purity, N95%, as evidenced by NMR and HPLC analysis. 7-O-[α-rhamnopyranosyl-(1→6)-β-glucopiranoside]-5hydroxy3-(4-methoxybenzyl)-chroman-4-one (1, Fig. 1). Pale yellow needles, 0.0068% of dried bulbs; [α]25 D — 23.7° (c 0.13, MeOH); UV max (MeOH): 210 (log ε 4.80), 295 (4.60); APCIMS m/z: 609.5 [M+H]+ (15), 608.4 (100), 463.3 (20), 301.8 (27), 272 (29), 267 (10), 245 (17), 213 (9), 208 (83), 192 (62), 179 (64), 167 (81), 150 (100). 1H (300 MHz, CD3OD) and 13C (75 MHz, CD3OD) NMR data: see Tables 1 and 2. 7-O-[α-rhamnopyranosyl-(1→6)-β-glucopiranoside]-5hydroxy-3-(3-hydroxy-4-methoxybenzyl)-chroman-4-one (2, Fig. 1). White crystals, 0.0084% of dried bulbs; [α]25 D —22.7° (c 0.11, MeOH); UV max (MeOH): 212 (log ε 4.38), 293 (4.33); APCI-MS m/z: 595.5 [M+H]+ (22), 594.4 (100), 449.6 (18), 287 (15), 210 (61), 179 (93), 167 (29), 158 (31), 107 (100). 1H (300 MHz, CD3OD) and 13C (75 MHz, CD3OD) NMR data: see Tables 1 and 2.
Table 1 1 H NMR assignments [δ (ppm), J (Hz)] of compounds 1–2 (in pyridine-d5) Position
Compound 1
Compound 2
2
4.16 dd (7.3, 11.3) 4.24 dd (11.3, 3.9) 2.81 m 5.87 d (1.8) – 5.85 d (1.8) 2.68 dd (9.3, 13.7) 3.12 dd (13.7, 4.5) 7.15 d (6.8) 6.87 d (6.8) 6.87 d (6.8) 7.15 d (6.8) 3.77 s – 12.25 s
4.08 dd (7.2, 11.4) 4.27 dd (11.4, 4.6) 2.82 m 6.01 d (1.8) – 5.95 s 2.68 dd (10.2, 13.8) 3.12 dd (13.8, 4.5) 7.07 d (8.7) 6.79 d (8.7) 6.79 d (8.7) 7.07 d (8.7) – 9.6 s 12.24 s
4.97 d (7.3) 3.27 u 3.22 m 3.12 t (9.3) 3.53 m 3.42 u 3.80 m
4.98 t (7.3) 3.27 u 3.22 m 3.13 dd (9;5) 3.53 m 3.42 u 3.80 d (10.3)
4.52 s 3.64 m 3.43 u 3.14 t (9.3) 3.40 u 1.08 d (6.3)
4.52 s 3.64 m 3.42 u 3.15 d (10.2) 3.40 u 1.09 d (6.3)
3 6 7 8 9 2′ 3′ 5′ 6′ OCH3 OH–4′ OH–5 Glc 1″ 2″ 3″ 4″ 5″ 6″
Rha 1‴ 2‴ 3‴ 4‴ 5‴ 6‴
Multiplicities: m: multiplet, s: singlet, d: doublet; u: multiplicity unclear due to overlapped signals.
Table 2 13 C NMR assignments [δ (ppm)] of compounds 1–2 (in pyridine-d5) Position
Compound 1
Compound 2
2 3 4 4a 5 6 7 8 8a 9 1′ 2′ 3′ 4′ 5′ 6′ OCH3
70.1 45.9 199.0 101.9 165.6 96.8 167.1 95.0 162.9 32.4 129,0 132.4 116.1 159.20 116.1 132.4 60.9
70.3 47.7 195.1 101.2 160.2 96.8 165.4 94.0 160.8 32.5 130.5 130.6 115.7 155 115.7 130.6
Glc 1″ 2″ 3″ 4″ 5″ 6″
99.61 76.42 73.13 69.73 75.58 66.18
100.20 77.02 73.50 70.51 76.22 66.80
Rha 1‴ 2‴ 3‴ 4‴ 5‴ 6‴
100.75 70.40 70.84 72.21 68.45 17.96
101.40 71.20 71.50 72.63 69.24 18.21
2.4. Acid hydrolysis Acidic hydrolysis was performed on compounds 1 and 2. 10 ml of HCl 2 N was added to 2 mg of each homoisoflavanone and heated during 3 h. The solution was neutralised with NaHCO3. The aglycone was extracted with EtOAc. The sugars were extracted from the aqueous phase with anhydrous pyridine; the pyridine was evaporated and the residue dissolved in water for identification by TLC analysis and detection with vanillin-sulphuric reagent [6]. 2.5. Antioxidant activity Antioxidant activity was studied by free radical scavenging activity by 2,2-diphenyl-1-picrylhydrazyl (DPPH) method [7,8] and β-carotene bleaching (BCB) method [9,10]. Assays were carried out in triplicate. 3. Results and discussion Repeated column chromatography (including normal silica gel and Sephadex LH-20) of the bulbs of L. floribunda led to the isolation of seven homoisoflavanones: 7- O-[α-rhamnopyranosyl-(1→6)-β-glucopiranosyl]-5hydroxy-3-(4-methoxybenzyl)-chroman-4-one (1) and 7-O[α-rhamnopyranosyl-(1→6)-β-glucopiranosyl]-5-hydroxy3-(4′-hydroxybenzyl)-chroman-4-one (2) (Fig. 1), and five known homoisoflavanones: 5,7-dihydroxy-3-(4′-
M.I. Calvo / Fitoterapia 80 (2009) 96–101
methoxy-benzyl)-chroman-4-one or 3,9-dihidroeucomin (3), 5,7-dihidroxy-6-methoxy-3-(4′-methoxybenzyl)-chroman-4one (4), 5,7-dihidroxy 3-(4′-hydroxybenzyl)-chroman-4-one or 4,4′-demethyl-3,9-dihydropuctatin (5), 5,7-dihidroxy-3-(4′hydroxybenzyl)-6-methoxy-chroman-4-one or 3,9-dihydroeucomnalin (6) and 7-hydroxy-3-(4′-hydroxybenzyl)-5-methoxychroman-4-one (7) (Fig. 2). Compounds 1 and 2 have not been reported previously but 3, 4, 5, 6 and 7 are known and their structures were confirmed by comparison of 1, 2-D NMR, APCIMS and other data against literature values. These compounds (3–7) have been described in various genus, but it is the first report in Ledebouria sp. [1,11–15]. The APCI-MS analysis in the positive ion mode displayed for 1 a protonated molecule at m/z 609.5 [M+H]+, with fragmentation showing the departure of two sugar units (ions at m/z 463.6 [M+H-146]+ and m/z 301.8 [M+H-308] +) suggesting the presence of a homoisoflavanone diglycoside. The 1H NMR spectrum of compound 1 indicated the presence of a –(2)CH2–(3)CH–(9)CH2-group typical of 3-benzyl-4chromanone homoisoflavonoids at δH 4.16 (J = 7.3, 11.3 Hz, H2a), δH 4.24 (J = 11.3, 3.9 Hz, H-2b), δH 2.81 (m, H-3), δH 2.68 (J = 9.3, 13.7 Hz, H-9a) and δH 3.12 (J = 13.7, 4.5 Hz, H-9b). These resonances were assigned by comparison with 3-benzyl-4chromanones [16]. The 1H and 13C NMR spectra further showed the presence of one hydroxyl (δH 12.25 s) and a methoxyl substituents (δΗ 3.77 s) on a homoisoflavonoid skeleton (Tables 1 and 2). The two ortho-related aromatic protons at δH 7.15 and 6.87 (d, J = 6.8, each 2H) suggested a symmetrical substituted aromatic ring B. The presence of two meta-related aromatic protons at δH 5.87 and 5.85 (d, J = 1.8) suggested ring A had two oxygenated functions. The UV spectrum, with a maximum at λ 295 nm (band II and a shoulder at about 330 nm (band I), rather indicated the presence of a homoisoflavanone. Use of shift reagents permitted to determine the substitution scheme [17]. A shift
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of +22 nm of band II was observed after addition of AlCl3, indicating the presence of a hydroxyl group in position C-5. Addition of a base (NaOAc or NaOMe) caused no effect, showing the absence of a free hydroxyl in C-7. The absence of o-dihydroxyl groups on A- or B-rings could be established. An acidic hydrolysis was performed. Observation of the shifts induced in the UV spectrum of the aglycone by addition of reagents enabled determination of the position of glycosylation. Unlike the glycoside, a bathochromic shift of 35 nm of band II was noted after addition of a strong (NaOMe) or weak base (NaOAc), indicating the presence of a free hydroxyl at C-7 [18]. The sugar moiety was thus determined to be attached to C-7. Finally, 2D NMR analyses, particularly HMBC correlations, located the methoxyl group at C-4′. Moreover, the glycosidic part could be identified as rutinose [rhamnosyl(α1→6-glucose)], as indicated by HMBC correlation between the proton at δ 4.52 (H-1‴) and the carbon at δ 66.18 (C-6″). A correlation between H-1″ and C-7 confirmed the position of the glycosylation. The coupling constant of the anomeric proton at δ 4.97 (1H, d, J = 7.3 Hz) indicated the β configuration of the glucose [19]. Resolution of the 1H NMR spectra was not good enough to reveal the coupling constant of the other anomeric proton. The α configuration of the rhamnose was determined by observing the chemical shifts of the carbons, which differ between α and β configuration [20,21]. Compound 1 was identified as the 7-O-[α-rhamnopyranosyl-(1→6)-βglucopyranosyl]-5-hydroxy-3-(4′-methoxy-benzyl)-chroman4-one or 3,9-dihidroeucomin-7-rutinoside. The APCI-MS analysis in the positive ion mode displayed for compound 2 a protonated molecule at m/z 595.5 [M+H]+, with fragmentation showing again the departure of two sugar units (ions at m/z 449.6 [M+H-146]+ and m/z 287.8 [M+H-308]+). The UV spectrum was identical to that of 1, showing 2 also be a homoisoflavanone. A bathochromic shift of 18 nm of band II caused by AlCl3 indicated the presence of a 5–OH. Observation
Fig. 2. Structure of known homoisoflavanones (3–7) isolated from Ledebouria floribunda.
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Table 3 Free radical scavenging capacities measured in DPPH assay Sample
IC50 (μg/ml)
DCM crude extract MeOH crude extract H2O crude extract Compound 1 Compound 2 Compound 3 Compound 4 Compound 5 Compound 6 Compound 7 α– tocoferol BHA BHT
101.0 ± 1.9 108.4 ± 2.3 N1000 273.1 ± 6.2 212.4 ± 3.8 84.3 ± 2.6 80.2 ± 4.1 31.1 ± 0.7 39.4 ± 0.5 138.6 ± 3.2 55.1 ± 0.9 98.7 ± 2.1 103 ± 1.6
Each value is presented as mean ± DS (n = 3).
of the 1H NMR spectrum indicated a great similarity with compound 1, the only difference was the absence of a methoxyl group at δ 3.77 and the presence of a hydroxyl group at δ 9.6. In the 13C spectrum, the absence of the corresponding methoxylic carbon at about δ 60.9 was also noted. The signal at δ 155.0 representing two oxygenated carbons indicated the presence of one hydroxyl group on ring B. This was in accordance with the preliminary UV and mass analyses, with a mass difference of 14 between compounds 1 (MW of 608) and 2 (MW of 594). Compound 2 was thus identified as 7-O-[α-rhamnopyranosyl(1→6)-β-glucopyranosyl]-3′,4′,5-trihydroxy-2,3-dihydro-4Hchroman-4-one. Similar resonances were present in other known compounds (3–7), indicating these compounds were also homoisoflavanones. The use of simplified model systems for quantifying the antioxidant action can be very helpful in clarifying the action of potential antioxidant [22–24]. The antioxidant activity of phenolic compounds is determined by their molecular structure and, more specifically, by the position and degree of hydroxylation of the rings. The hydroxyl groups influence the ability of the delocalization of unpaired electrons to stabilize the formed phenoxyl radical after reaction with the lipid radical [25]. All flavonoids with 3′,4′-dihydroxy configuration possess marked antioxidant activity [26–28]. This activity increases with the number of hydroxyl groups substituted on the A- and B-rings [29]. Flavanones and
flavones are less active than their corresponding chalcones, while the dihydrochalcones are more effective than their corresponding chalcones [26,29]. But there are no references about the antioxidant activity of homoisoflavanones in the scientific literature. In the present study, two different methods were used to examine the antioxidant activity of DCM, MeOH and H2O crude extracts of L. floribunda and their homoisoflavanones: DPPH radical scavenging method and β-carotene bleaching (BCB) method. The antioxidant activity was compared with the activity of natural (α-tocopherol) and synthetic antioxidants (butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA)). Among the radical scavenging assays, the one based on the utilisation of DPPH was chosen due to its simplicity and world-wide acceptance for comparative purposes [30,31]. DPPH method is independent of the substrate polarity. This method is based on the reduction of alcoholic DPPH solutions at 517 nm in the presence of an antioxidant that donate hydrogen or electron. Non-radical form DPPH-H is formed. The scavenging activity of the extract and homoisoflavanones 1–7 is shown in Table 3. The DPPH radical scavenging activity decreases in order DCMNMeOHNH20 extract. The antioxidant properties of the DCM and MeOH crude extracts (101.0 ± 1.9 and 108.4 ± 2.3 μg/ml, respectively) were found to be close or higher than BHA and BHT (98.7 ± 2.1 and 103 ± 1.6 μg/ml). The compounds 5 and 6 (IC50 = 31.1 and 39.4 μg/ml, respectively) had the highest hydrogen-donating capacity. The compounds 3 and 4 were weakly active (IC50 = 84.3 and 80.2 μg/ml, respectively), while compounds 1 and 2 were the less active of seven homoisoflavanones. The BCB method is usually used to evaluate the antioxidant activity of compounds in emulsions, accompanied with the coupled oxidation of β-carotene and linoleic acid. The mechanism of bleaching of β-carotene is a free-radicalmediated phenomenon resulting from the hydroperoxides formed from linoleic acid. β-carotene, in this model system, undergoes rapid discolouration in the absence of an antioxidant. The linoleic acid free radical, formed upon the abstraction of a hydrogen atom from one of its diallylic methylene groups, attacks the highly unsaturated β-carotene molecules. As β-carotene molecules lose their double bonds by oxidation, the compound loses its chromophore and characteristic orange colour, which can be monitored spectro-
Table 4 Percentage of antioxidant activity (%AA) by β-carotene–linoleate model system Sample
DCM extract MeOH extract H2O extract Compound 1 Compound 2 Compound 3 Compound 4 Compound 5 Compound 6 Compound 7 Tocopherol BHA BHT
Time (min) 0
15
45
60
90
120
0.00 ± 0.06 0.00 ± 0.01 0.00 ± 0.10 0.00 ± 0.01 0.00 ± 0.06 0.00 ± 0.03 0.00 ± 0.02 0.00 ± 0.10 0.00 ± 0.02 0.00 ± 0.07 0.00 ± 0.05 2.17 ± 0.02 2.17 ± 0.01
10.87 ± 0.21 9.45 ± 0.23 2.36 ± 0.09 9.21 ± 0.09 10.54 ± 0.10 17.39 ± 0.45 15.22 ± 0.85 19.57 ± 0.068 30.43 ± 0.18 2.17 ± 0.01 39.13 ± 0.06 34.78 ± 0.81 30.43 ± 0.41
22.73 ± 0.38 17.32 ± 0.37 4.65 ± 0.42 9.42 ± 0.42 14.65 ± 0.10 25.00 ± 0.02 25.00 ± 0.25 25.00 ± 0.27 40.91 ± 0.62 15.91 ± 0.26 50.00 ± 0.12 36.36 ± 0.71 36.36 ± 0.55
31.82 ± 0.24 26.89 ± 0.21 5.99 ± 0.24 10.05 ± 0.24 17.32 ± 0.34 40.91 ± 0.62 36.36 ± 0.21 40.91 ± 0.06 56.82 ± 0.45 20.45 ± 0.74 68.18 ± 0.23 54.55 ± 0.60 45.45 ± 0.32
40.00 ± 0.22 34.18 ± 0.31 8.11 ± 0.10 11.41 ± 0.10 21.77 ± 0.47 42.02 ± 0.71 42.22 ± 0.79 55.56 ± 0.03 64.44 ± 0.71 24.44 ± 0.14 77.78 ± 0.26 62.22 ± 0.43 55.56 ± 0.61
41.03 ± 0.12 35.61 ± 0.11 10.43 ± 0.08 11.42 ± 0.08 25.98 ± 0.13 41.92 ± 0.83 41.99 ± 0.19 57.01 ± 0.05 64.47 ± 0.84 26.17 ± 0.18 79.78 ± 0.41 61.82 ± 0.11 55.98 ± 0.13
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photometrically. The antioxidant activity of the three crude extracts and homoisoflavanones is presented in Table 4. As oxidation progressed, the absorbance of β-carotene al 470 nm decreased and its yellow colour faded. The %AA values calculated from the formula given in experimental part facilitate comparisons of the relative activity. The reference compounds, BHT, BHA and α-tocopherol exhibited a more powerful antioxidant activity than the DCM (41.03%), MeOH (35.61%) and H2O (10.43%) crude extracts of L. floribunda. Compounds 6 (64.47%) and 5 (57.01%) showed the most effective antioxidant activity among the homoisoflavonoids whereas the compounds 1 (11.42%) and 2 (25.98%) were less efficient. The antioxidant activity (expressed in percentage) in this model system was in following order (Table 4): α-tocopherol N compound 6N BHAN compound 5N BHTN DCM extractN compound 4 N c o mp ou nd 3 N MeOH extract N compound 7N compound 2N compound 1N H2O extract. This is the first report of the antioxidant activity of homoisoflavanones against DPPH radical and β-carotene– linoleic system Five homoisoflavanones (3–7) isolated of L. floribunda were found to be potent antioxidants, comparable in activity with α-tocopherol and the widely used synthetic antioxidants BHT and BHA. This activity increases with the number of hydroxyl groups substituted on the A- and B-rings and decreases with sugar units substituted. This should merit further investigation to assess the effectiveness of these compounds in other biological models, to compare these antioxidant capacities with other homoisoflavonoids as well as their possible synergistic effects. Acknowledgment The author wish to thank Dr. Aizpurua for his help and technical assistance.
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Homoisoflavanones from Ledebouria floribunda María Isabel Calvo ⁎ Dpto. Farmacia y Tecnología Farmacéutica (Facultad de Farmacia), University of Navarra, 31008 Pamplona, Spain
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Article history: Received 16 September 2008 Accepted in revised form 9 October 2008 Available online 30 October 2008 Keywords: Ledebouria floribunda Hyacynthaceae Homoisoflavanones Antioxidant DPPH β-carotene/linoleic acid system
a b s t r a c t The bulbs of Ledebouria floribunda (Baker) Jessop have yielded two novel compounds, 7-O-[αrhamnopyranosyl-(1→6)-β-glucopiranosyl]-5-hydroxy-3-(4-methoxybenzyl)-chroman-4-one (1) and 7-O-[α-rhamnopyranosyl-(1→6)-β-glucopiranosyl]-5-hydroxy-3-(4′-hydroxybenzyl)chroman-4-one (2) along with five other known compounds, 5,7-dihydroxy-3-(4′methoxybenzyl)-chroman-4-one or 3,9-dihidroeucomin (3), 5,7-dihidroxy-6-methoxy-3-(4′methoxybenzyl)-chroman-4-one (4), 5,7-dihidroxy 3-(4′-hydroxybenzyl)-chroman-4-one or 4,4′-demethyl-3,9-dihydropuctatin (5), 5,7-dihidroxy-3-(4′-hydroxybenzyl)-6-methoxychroman-4-one or 3,9-dihydroeucomnalin (6) and 7-hydroxy-3-(4′-hydroxybenzyl)-5methoxy-chroman-4-one (7). Their structures were elucidated by spectra analysis. The seven homoisoflavanones were found to be antioxidant against DPPH radical and β-carotene/linoleic acid system. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Ledebouria is a genus of deciduous or weakly evergreen bulbs in the Hyacynthaceae. It is only recently established as a genus apart from Scilla. Ledebouria sp. is found in areas of summer rainfall in subtropical savannah and grasslands, in the eastern and northeastern parts of southern Africa, India and Madagascar. Ledebouria floribunda (Baker) Jessop is a large species and is used by traditional healers in the Eastern Cape, for a variety of ailments. It occurs in grassland. Several flowering spikes are produced in spring. Homoisoflavanones, which are distributed in the Hyacynthaceae family, have been showed several activities [1–5]. In our continuing study to search for natural antioxidants from medicinal plants, this report presents the isolation and structure elucidation of two new homoisoflavanone (1–2) along with five know compounds (3–7) from the most bioactive MeOH and DCM extracts of the bulbs of L. floribunda. This is the first report of the chemical composition of L. floribunda and antioxidant activity of the homoisoflava⁎ Tel.: +34 48425600 6239; fax: +34 48425649. E-mail address: [email protected]. 0367-326X/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2008.10.006
nones isolated against DPPH radical and β-carotene–linoleic system. 2. Experimental 2.1. General Dichloromethane, n-hexane, ethyl acetate, chloroform, and methanol were purchased from Merck (Darmstadt, Germany). DPPH, β-carotene, linoleic acid, BHA, BTA and αtocopherol were from Sigma (CA, USA). Sephadex LH-20 (Sigma) was used for all CC separations, while silica gel 60 PF254 (Merck) was used for analytical (0.50 mm) and preparative TLC (1.0 mm). 1 H and 13C NMR spectra were measured on a Bruker Avance 300 at 300 MHz and 75 MHz, respectively, with TMS as an internal standard and CDCl3 (Aldrich, CA, USA) as solvent. Chemical shifts were reported in δ units (ppm) and coupling constants (J) in Hz. D/CI mass spectra of the pure compounds were measured on a Finnigan-MatTSQ-700 triple stage quadrupole mass spectrometer, with the following parameters: source current 5 μA, vaporization temperature 450 ° C, sheath gas flow (N2) 80 psi, capillary temperature 150
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to 200 °C. The optical rotations were recorded on a PerkinElmer 241 polarimeter at 25 °C. UV spectra were measured on a Perkin-Elmer UV–Vis-Lambda 200 spectrometer. 2.2. Plant material Fresh bulbs of L. floribunda were collected at Congo and voucher specimen deposited in the authors' laboratory (no. 96097). 2.3. Extraction and isolation Air-dried bulbs (250 g) were ground into fine powder and extracted with dichloromethane (DCM), methanol (MeOH) and water (H2O) at room temp. in a closed container several times. The extracts were concentrated under reduced pressure at 40 °C yielding 3.45 g, 1.07 and 8.79 g of dry extract, respectively.
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The methanol extract was adsorbed on highly polymer resin (Diaion HP-20) and eluted with H2O–MeOH gradient (H2O, 20%, 40%, 60%, 80%, and 100% MeOH). The 40% MeOH eluted fraction (2.61 g) was subjected to silica gel column using CH2Cl2–MeOH gradient (from 15:1 to 1:1), to give six fractions (1–6). Fraction 2 (0.97 g) and 4 (0.84 g) were over Sephadex LH-20 column with methanol to afford compounds 1 (17 mg) and 2 (21 mg), respectively. A part of the DCM extract (2.5 g) was chromatographed on a silica gel column (i.d. 2 × 30 cm) eluted with a gradient nhexane:ethyl acetate (9:1→1:9) yielding 10 fractions (1–10). The antioxidant testing of these fractions with DDPH radical permitted to select the active fractions for further investigation. Fractions 6 (94.1 mg), 7 (421.3 mg) and 9 (90 mg) were found to be active on TLC assay. Separation of fraction 6 through Sephadex LH-20 column (i.d. 1.5 × 25 cm) with CHCl3– MeOH (1:1), afforded five subfractions (6.1→6.5). Purification
Fig. 1. Structure of new homoisoflavanones (1, 2) isolated from Ledebouria floribunda.
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of subfraction 6.1 on prep. TLC using mobile phase n-hexane– EtOAc (1:1) yielded 3 (14 mg). Purification of subfractions 6.3, 6.4 and 6.5 on Sephadex LH-20 with EtOAc yielded 4 (24 mg). Separation of fraction 7 through silica gel column (i.d. 2.5 × 30 cm) with a gradient CHCl3–MeOH (9:1→1:9), afforded 19 subfractions (7.1→7.19). Purification of subfraction 9.7 and 9.19 on prep. TLC using mobile phase n-hexane–EtOAc (1:1) yielded 5 (31 mg) and 6 (27.1 mg), respectively. Similar purification of fraction 9 on prep. TLC yielded 7 (9 mg). These compounds showed a satisfactory purity, N95%, as evidenced by NMR and HPLC analysis. 7-O-[α-rhamnopyranosyl-(1→6)-β-glucopiranoside]-5hydroxy3-(4-methoxybenzyl)-chroman-4-one (1, Fig. 1). Pale yellow needles, 0.0068% of dried bulbs; [α]25 D — 23.7° (c 0.13, MeOH); UV max (MeOH): 210 (log ε 4.80), 295 (4.60); APCIMS m/z: 609.5 [M+H]+ (15), 608.4 (100), 463.3 (20), 301.8 (27), 272 (29), 267 (10), 245 (17), 213 (9), 208 (83), 192 (62), 179 (64), 167 (81), 150 (100). 1H (300 MHz, CD3OD) and 13C (75 MHz, CD3OD) NMR data: see Tables 1 and 2. 7-O-[α-rhamnopyranosyl-(1→6)-β-glucopiranoside]-5hydroxy-3-(3-hydroxy-4-methoxybenzyl)-chroman-4-one (2, Fig. 1). White crystals, 0.0084% of dried bulbs; [α]25 D —22.7° (c 0.11, MeOH); UV max (MeOH): 212 (log ε 4.38), 293 (4.33); APCI-MS m/z: 595.5 [M+H]+ (22), 594.4 (100), 449.6 (18), 287 (15), 210 (61), 179 (93), 167 (29), 158 (31), 107 (100). 1H (300 MHz, CD3OD) and 13C (75 MHz, CD3OD) NMR data: see Tables 1 and 2.
Table 1 1 H NMR assignments [δ (ppm), J (Hz)] of compounds 1–2 (in pyridine-d5) Position
Compound 1
Compound 2
2
4.16 dd (7.3, 11.3) 4.24 dd (11.3, 3.9) 2.81 m 5.87 d (1.8) – 5.85 d (1.8) 2.68 dd (9.3, 13.7) 3.12 dd (13.7, 4.5) 7.15 d (6.8) 6.87 d (6.8) 6.87 d (6.8) 7.15 d (6.8) 3.77 s – 12.25 s
4.08 dd (7.2, 11.4) 4.27 dd (11.4, 4.6) 2.82 m 6.01 d (1.8) – 5.95 s 2.68 dd (10.2, 13.8) 3.12 dd (13.8, 4.5) 7.07 d (8.7) 6.79 d (8.7) 6.79 d (8.7) 7.07 d (8.7) – 9.6 s 12.24 s
4.97 d (7.3) 3.27 u 3.22 m 3.12 t (9.3) 3.53 m 3.42 u 3.80 m
4.98 t (7.3) 3.27 u 3.22 m 3.13 dd (9;5) 3.53 m 3.42 u 3.80 d (10.3)
4.52 s 3.64 m 3.43 u 3.14 t (9.3) 3.40 u 1.08 d (6.3)
4.52 s 3.64 m 3.42 u 3.15 d (10.2) 3.40 u 1.09 d (6.3)
3 6 7 8 9 2′ 3′ 5′ 6′ OCH3 OH–4′ OH–5 Glc 1″ 2″ 3″ 4″ 5″ 6″
Rha 1‴ 2‴ 3‴ 4‴ 5‴ 6‴
Multiplicities: m: multiplet, s: singlet, d: doublet; u: multiplicity unclear due to overlapped signals.
Table 2 13 C NMR assignments [δ (ppm)] of compounds 1–2 (in pyridine-d5) Position
Compound 1
Compound 2
2 3 4 4a 5 6 7 8 8a 9 1′ 2′ 3′ 4′ 5′ 6′ OCH3
70.1 45.9 199.0 101.9 165.6 96.8 167.1 95.0 162.9 32.4 129,0 132.4 116.1 159.20 116.1 132.4 60.9
70.3 47.7 195.1 101.2 160.2 96.8 165.4 94.0 160.8 32.5 130.5 130.6 115.7 155 115.7 130.6
Glc 1″ 2″ 3″ 4″ 5″ 6″
99.61 76.42 73.13 69.73 75.58 66.18
100.20 77.02 73.50 70.51 76.22 66.80
Rha 1‴ 2‴ 3‴ 4‴ 5‴ 6‴
100.75 70.40 70.84 72.21 68.45 17.96
101.40 71.20 71.50 72.63 69.24 18.21
2.4. Acid hydrolysis Acidic hydrolysis was performed on compounds 1 and 2. 10 ml of HCl 2 N was added to 2 mg of each homoisoflavanone and heated during 3 h. The solution was neutralised with NaHCO3. The aglycone was extracted with EtOAc. The sugars were extracted from the aqueous phase with anhydrous pyridine; the pyridine was evaporated and the residue dissolved in water for identification by TLC analysis and detection with vanillin-sulphuric reagent [6]. 2.5. Antioxidant activity Antioxidant activity was studied by free radical scavenging activity by 2,2-diphenyl-1-picrylhydrazyl (DPPH) method [7,8] and β-carotene bleaching (BCB) method [9,10]. Assays were carried out in triplicate. 3. Results and discussion Repeated column chromatography (including normal silica gel and Sephadex LH-20) of the bulbs of L. floribunda led to the isolation of seven homoisoflavanones: 7- O-[α-rhamnopyranosyl-(1→6)-β-glucopiranosyl]-5hydroxy-3-(4-methoxybenzyl)-chroman-4-one (1) and 7-O[α-rhamnopyranosyl-(1→6)-β-glucopiranosyl]-5-hydroxy3-(4′-hydroxybenzyl)-chroman-4-one (2) (Fig. 1), and five known homoisoflavanones: 5,7-dihydroxy-3-(4′-
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methoxy-benzyl)-chroman-4-one or 3,9-dihidroeucomin (3), 5,7-dihidroxy-6-methoxy-3-(4′-methoxybenzyl)-chroman-4one (4), 5,7-dihidroxy 3-(4′-hydroxybenzyl)-chroman-4-one or 4,4′-demethyl-3,9-dihydropuctatin (5), 5,7-dihidroxy-3-(4′hydroxybenzyl)-6-methoxy-chroman-4-one or 3,9-dihydroeucomnalin (6) and 7-hydroxy-3-(4′-hydroxybenzyl)-5-methoxychroman-4-one (7) (Fig. 2). Compounds 1 and 2 have not been reported previously but 3, 4, 5, 6 and 7 are known and their structures were confirmed by comparison of 1, 2-D NMR, APCIMS and other data against literature values. These compounds (3–7) have been described in various genus, but it is the first report in Ledebouria sp. [1,11–15]. The APCI-MS analysis in the positive ion mode displayed for 1 a protonated molecule at m/z 609.5 [M+H]+, with fragmentation showing the departure of two sugar units (ions at m/z 463.6 [M+H-146]+ and m/z 301.8 [M+H-308] +) suggesting the presence of a homoisoflavanone diglycoside. The 1H NMR spectrum of compound 1 indicated the presence of a –(2)CH2–(3)CH–(9)CH2-group typical of 3-benzyl-4chromanone homoisoflavonoids at δH 4.16 (J = 7.3, 11.3 Hz, H2a), δH 4.24 (J = 11.3, 3.9 Hz, H-2b), δH 2.81 (m, H-3), δH 2.68 (J = 9.3, 13.7 Hz, H-9a) and δH 3.12 (J = 13.7, 4.5 Hz, H-9b). These resonances were assigned by comparison with 3-benzyl-4chromanones [16]. The 1H and 13C NMR spectra further showed the presence of one hydroxyl (δH 12.25 s) and a methoxyl substituents (δΗ 3.77 s) on a homoisoflavonoid skeleton (Tables 1 and 2). The two ortho-related aromatic protons at δH 7.15 and 6.87 (d, J = 6.8, each 2H) suggested a symmetrical substituted aromatic ring B. The presence of two meta-related aromatic protons at δH 5.87 and 5.85 (d, J = 1.8) suggested ring A had two oxygenated functions. The UV spectrum, with a maximum at λ 295 nm (band II and a shoulder at about 330 nm (band I), rather indicated the presence of a homoisoflavanone. Use of shift reagents permitted to determine the substitution scheme [17]. A shift
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of +22 nm of band II was observed after addition of AlCl3, indicating the presence of a hydroxyl group in position C-5. Addition of a base (NaOAc or NaOMe) caused no effect, showing the absence of a free hydroxyl in C-7. The absence of o-dihydroxyl groups on A- or B-rings could be established. An acidic hydrolysis was performed. Observation of the shifts induced in the UV spectrum of the aglycone by addition of reagents enabled determination of the position of glycosylation. Unlike the glycoside, a bathochromic shift of 35 nm of band II was noted after addition of a strong (NaOMe) or weak base (NaOAc), indicating the presence of a free hydroxyl at C-7 [18]. The sugar moiety was thus determined to be attached to C-7. Finally, 2D NMR analyses, particularly HMBC correlations, located the methoxyl group at C-4′. Moreover, the glycosidic part could be identified as rutinose [rhamnosyl(α1→6-glucose)], as indicated by HMBC correlation between the proton at δ 4.52 (H-1‴) and the carbon at δ 66.18 (C-6″). A correlation between H-1″ and C-7 confirmed the position of the glycosylation. The coupling constant of the anomeric proton at δ 4.97 (1H, d, J = 7.3 Hz) indicated the β configuration of the glucose [19]. Resolution of the 1H NMR spectra was not good enough to reveal the coupling constant of the other anomeric proton. The α configuration of the rhamnose was determined by observing the chemical shifts of the carbons, which differ between α and β configuration [20,21]. Compound 1 was identified as the 7-O-[α-rhamnopyranosyl-(1→6)-βglucopyranosyl]-5-hydroxy-3-(4′-methoxy-benzyl)-chroman4-one or 3,9-dihidroeucomin-7-rutinoside. The APCI-MS analysis in the positive ion mode displayed for compound 2 a protonated molecule at m/z 595.5 [M+H]+, with fragmentation showing again the departure of two sugar units (ions at m/z 449.6 [M+H-146]+ and m/z 287.8 [M+H-308]+). The UV spectrum was identical to that of 1, showing 2 also be a homoisoflavanone. A bathochromic shift of 18 nm of band II caused by AlCl3 indicated the presence of a 5–OH. Observation
Fig. 2. Structure of known homoisoflavanones (3–7) isolated from Ledebouria floribunda.
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Table 3 Free radical scavenging capacities measured in DPPH assay Sample
IC50 (μg/ml)
DCM crude extract MeOH crude extract H2O crude extract Compound 1 Compound 2 Compound 3 Compound 4 Compound 5 Compound 6 Compound 7 α– tocoferol BHA BHT
101.0 ± 1.9 108.4 ± 2.3 N1000 273.1 ± 6.2 212.4 ± 3.8 84.3 ± 2.6 80.2 ± 4.1 31.1 ± 0.7 39.4 ± 0.5 138.6 ± 3.2 55.1 ± 0.9 98.7 ± 2.1 103 ± 1.6
Each value is presented as mean ± DS (n = 3).
of the 1H NMR spectrum indicated a great similarity with compound 1, the only difference was the absence of a methoxyl group at δ 3.77 and the presence of a hydroxyl group at δ 9.6. In the 13C spectrum, the absence of the corresponding methoxylic carbon at about δ 60.9 was also noted. The signal at δ 155.0 representing two oxygenated carbons indicated the presence of one hydroxyl group on ring B. This was in accordance with the preliminary UV and mass analyses, with a mass difference of 14 between compounds 1 (MW of 608) and 2 (MW of 594). Compound 2 was thus identified as 7-O-[α-rhamnopyranosyl(1→6)-β-glucopyranosyl]-3′,4′,5-trihydroxy-2,3-dihydro-4Hchroman-4-one. Similar resonances were present in other known compounds (3–7), indicating these compounds were also homoisoflavanones. The use of simplified model systems for quantifying the antioxidant action can be very helpful in clarifying the action of potential antioxidant [22–24]. The antioxidant activity of phenolic compounds is determined by their molecular structure and, more specifically, by the position and degree of hydroxylation of the rings. The hydroxyl groups influence the ability of the delocalization of unpaired electrons to stabilize the formed phenoxyl radical after reaction with the lipid radical [25]. All flavonoids with 3′,4′-dihydroxy configuration possess marked antioxidant activity [26–28]. This activity increases with the number of hydroxyl groups substituted on the A- and B-rings [29]. Flavanones and
flavones are less active than their corresponding chalcones, while the dihydrochalcones are more effective than their corresponding chalcones [26,29]. But there are no references about the antioxidant activity of homoisoflavanones in the scientific literature. In the present study, two different methods were used to examine the antioxidant activity of DCM, MeOH and H2O crude extracts of L. floribunda and their homoisoflavanones: DPPH radical scavenging method and β-carotene bleaching (BCB) method. The antioxidant activity was compared with the activity of natural (α-tocopherol) and synthetic antioxidants (butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA)). Among the radical scavenging assays, the one based on the utilisation of DPPH was chosen due to its simplicity and world-wide acceptance for comparative purposes [30,31]. DPPH method is independent of the substrate polarity. This method is based on the reduction of alcoholic DPPH solutions at 517 nm in the presence of an antioxidant that donate hydrogen or electron. Non-radical form DPPH-H is formed. The scavenging activity of the extract and homoisoflavanones 1–7 is shown in Table 3. The DPPH radical scavenging activity decreases in order DCMNMeOHNH20 extract. The antioxidant properties of the DCM and MeOH crude extracts (101.0 ± 1.9 and 108.4 ± 2.3 μg/ml, respectively) were found to be close or higher than BHA and BHT (98.7 ± 2.1 and 103 ± 1.6 μg/ml). The compounds 5 and 6 (IC50 = 31.1 and 39.4 μg/ml, respectively) had the highest hydrogen-donating capacity. The compounds 3 and 4 were weakly active (IC50 = 84.3 and 80.2 μg/ml, respectively), while compounds 1 and 2 were the less active of seven homoisoflavanones. The BCB method is usually used to evaluate the antioxidant activity of compounds in emulsions, accompanied with the coupled oxidation of β-carotene and linoleic acid. The mechanism of bleaching of β-carotene is a free-radicalmediated phenomenon resulting from the hydroperoxides formed from linoleic acid. β-carotene, in this model system, undergoes rapid discolouration in the absence of an antioxidant. The linoleic acid free radical, formed upon the abstraction of a hydrogen atom from one of its diallylic methylene groups, attacks the highly unsaturated β-carotene molecules. As β-carotene molecules lose their double bonds by oxidation, the compound loses its chromophore and characteristic orange colour, which can be monitored spectro-
Table 4 Percentage of antioxidant activity (%AA) by β-carotene–linoleate model system Sample
DCM extract MeOH extract H2O extract Compound 1 Compound 2 Compound 3 Compound 4 Compound 5 Compound 6 Compound 7 Tocopherol BHA BHT
Time (min) 0
15
45
60
90
120
0.00 ± 0.06 0.00 ± 0.01 0.00 ± 0.10 0.00 ± 0.01 0.00 ± 0.06 0.00 ± 0.03 0.00 ± 0.02 0.00 ± 0.10 0.00 ± 0.02 0.00 ± 0.07 0.00 ± 0.05 2.17 ± 0.02 2.17 ± 0.01
10.87 ± 0.21 9.45 ± 0.23 2.36 ± 0.09 9.21 ± 0.09 10.54 ± 0.10 17.39 ± 0.45 15.22 ± 0.85 19.57 ± 0.068 30.43 ± 0.18 2.17 ± 0.01 39.13 ± 0.06 34.78 ± 0.81 30.43 ± 0.41
22.73 ± 0.38 17.32 ± 0.37 4.65 ± 0.42 9.42 ± 0.42 14.65 ± 0.10 25.00 ± 0.02 25.00 ± 0.25 25.00 ± 0.27 40.91 ± 0.62 15.91 ± 0.26 50.00 ± 0.12 36.36 ± 0.71 36.36 ± 0.55
31.82 ± 0.24 26.89 ± 0.21 5.99 ± 0.24 10.05 ± 0.24 17.32 ± 0.34 40.91 ± 0.62 36.36 ± 0.21 40.91 ± 0.06 56.82 ± 0.45 20.45 ± 0.74 68.18 ± 0.23 54.55 ± 0.60 45.45 ± 0.32
40.00 ± 0.22 34.18 ± 0.31 8.11 ± 0.10 11.41 ± 0.10 21.77 ± 0.47 42.02 ± 0.71 42.22 ± 0.79 55.56 ± 0.03 64.44 ± 0.71 24.44 ± 0.14 77.78 ± 0.26 62.22 ± 0.43 55.56 ± 0.61
41.03 ± 0.12 35.61 ± 0.11 10.43 ± 0.08 11.42 ± 0.08 25.98 ± 0.13 41.92 ± 0.83 41.99 ± 0.19 57.01 ± 0.05 64.47 ± 0.84 26.17 ± 0.18 79.78 ± 0.41 61.82 ± 0.11 55.98 ± 0.13
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photometrically. The antioxidant activity of the three crude extracts and homoisoflavanones is presented in Table 4. As oxidation progressed, the absorbance of β-carotene al 470 nm decreased and its yellow colour faded. The %AA values calculated from the formula given in experimental part facilitate comparisons of the relative activity. The reference compounds, BHT, BHA and α-tocopherol exhibited a more powerful antioxidant activity than the DCM (41.03%), MeOH (35.61%) and H2O (10.43%) crude extracts of L. floribunda. Compounds 6 (64.47%) and 5 (57.01%) showed the most effective antioxidant activity among the homoisoflavonoids whereas the compounds 1 (11.42%) and 2 (25.98%) were less efficient. The antioxidant activity (expressed in percentage) in this model system was in following order (Table 4): α-tocopherol N compound 6N BHAN compound 5N BHTN DCM extractN compound 4 N c o mp ou nd 3 N MeOH extract N compound 7N compound 2N compound 1N H2O extract. This is the first report of the antioxidant activity of homoisoflavanones against DPPH radical and β-carotene– linoleic system Five homoisoflavanones (3–7) isolated of L. floribunda were found to be potent antioxidants, comparable in activity with α-tocopherol and the widely used synthetic antioxidants BHT and BHA. This activity increases with the number of hydroxyl groups substituted on the A- and B-rings and decreases with sugar units substituted. This should merit further investigation to assess the effectiveness of these compounds in other biological models, to compare these antioxidant capacities with other homoisoflavonoids as well as their possible synergistic effects. Acknowledgment The author wish to thank Dr. Aizpurua for his help and technical assistance.
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