Antioxidant and cytotoxic activity of polyphenolic compounds isolated from the leaves of Leucenia leucocephala

Abstract

Context: Cancer is a serious clinical problem to the health care system. Anticancer drugs have been extracted from plants containing phenolic compounds. Leucenia species (Fabaceae) contain a variety of bioactive components of numerous biological and pharmacological properties.

Objective: This study explored the constitutive polyphenols of Leucenia leucocephala Lam. growing in Egypt and evaluated the antioxidant and cytotoxic activity.

Materials and methods: Chemical structures of the isolated compounds from the leaves of L. leucocephala were established by spectral techniques (UV, ¹H, and ¹³C NMR, MS).

Results: Chromatographic separation of 80% MeOH extract of the leaves of L. leucocephala have resulted in a novel flavonoid-galloyl glycoside [myricetin 3-O-(2′,3′4′-tri-O-galloyl)-α-l-rhamnopyranoside] with three known polyphenolic compounds isolated for the first time from this species (apigenin 7-O-β-d-glucuronopyranoside methyl ester, luteolin 7-O-β-d-glucuronopyranoside methyl ester, and 1,3,6-tri-O-galloyl-β-d-glucopyranose) and seven known previously isolated compounds. Also, 80% methanol extract exhibited high antioxidant activity (SC₅₀ = 3.94 µg/ml), which is correlated with its phenolic content. The extract also showed cytotoxic activity against Hep G2 (IC₅₀ value 1.41 µg/ml) confirming its anticancer activity against hepatocellular carcinoma. Among the tested compounds (4–8) for antioxidant property, compound 7 was the most active compound (SC₅₀ = 2.49 µg/ml). Also compounds 7 and 8 exhibited high cytotoxic activity (IC₅₀ = 2.41 and 2.81 µg/ml, respectively).

Discussion and conclusion: These findings demonstrate that the leaves of L. leucocephala contain a considerable amount of polyphenolic compounds with high antioxidant properties, thus it has great potential as a source for natural health products.

Keywords::

• Fabaceae
• Acacia
• flavonoid glycosides
• tannins
• free radical scavenger
• anticancer activity

Introduction

Cancer is a serious clinical problem that poses significant social and economic changes to the health care system. Despite the improved imaging and molecular diagnostic techniques, cancer continues to affect millions of people globally (Eisenberg et al., 1998). Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third most common cause of cancer related death worldwide (El-Serag, 2002). The synchronous occurrence of HCC might be due to different risk factors such as chronic viral infection with hepatitis B virus (HBV) and hepatitis C virus (HCV), aflatoxin exposure, alcohol consumption or iron overload (Röcken & Carl-McGrath, 2001; Ohata et al., 2004). In most cases, the recovery rate from HCC is low and current conventional and modified therapies are rarely beneficial (Kapadia et al., 2000; Thomas & Zhu, 2005). Thus, there is an urgent need for new therapeutic agents for HCC patients. A common means of drug discovery is the ethno-medical approach, in which the selection of a plant is based on its use as folk medicine. A number of anticancer drugs have been extracted from plants containing phenolic compounds as flavonoids, tannins, terpenoids and steroid, etc. (Saetung et al., 2005; Rajkapoor et al., 2007). These plants received considerable attention in recent years for their antioxidant and free radical scavenging activities (Hsu et al., 2005; Marzouk et al., 2006; Prasad et al., 2009; Ayoub, 2010). The family Leguminoseae (Fabaceae) is particularly rich in flavonoids and related compounds (Wu et al., 2005). Among the Fabaceae is the genus Leucenia which comprises about 1350 species and is distributed in tropical and subtropical regions (Seigler, 2003). Leucenia species contain variety of bioactive components such as phenolic acid, alkaloids, tannins and flavonoids which have numerous biological and pharmacological properties as hypoglycemic, analgesic, anti-inflammatory, antihypertensive, antiatherosclerotic, anthelmintic, antibacterial and anticancer (Berlin et al., 1995; Muhammad et al., 1998; Kalsom et al., 2001; Ademola et al., 2005; Andrade-Cetto & Heinrich, 2005; Khatab et al., 2006; Ramli et al., 2008; Singh et al., 2009a; Tung et al., 2009). Few studies report on the constitutive polyphenols of Leucenia leucocephala Lam. (Lowry et al., 1984; Wheeler et al., 1994). Thus, it was deemed necessary to carry out this phytochemical and biological study to throw light on this important species native to Central America and Mexico and naturalized in over 150 countries including Egypt (Walton, 2003). The present study deals with the isolation and identification of some polyphenolic constituents of L. leucocephala species growing in Egypt and evaluation of the antioxidant, free radical scavenging and cytotoxic activities of its different extracts as well as some of its pure isolated compounds. Synonyms for the plant include Acacia leucocephala (Lamark) Link, Acacia glauca Willd, Mimosa leucocephala Lamark, Mimosa glauca sensu L., Leucaena glauca (sensu L.) Benth, Leucaena glabrata Rose and Leucaena latisiliqu (L.) Benth (Walton, 2003).

Materials and methods

instruments and materials

NMR (¹H- and ¹³C NMR) spectra were recorded at 300 MHz for ¹H and 75 MHz for ¹³C on a Varian Mercury 300. The δ-values are reported as ppm relative to TMS in DMSO-d₆ and J-values are in Hz. ESI-MS spectra were measured on mass spectrometer connected to an ESI-II ion source (Finnigan, Lc-MSLCQ^deca. Advantage MAX, Finnigan Surveyor LC pump) (Department of Biological Genetics, National Research Center, Cairo, Egypt). The UV analysis for pure samples were recorded on a Shimadzu UV 240 spectrophotometer, separately as solutions in methanol and with different diagnostic UV shift reagents (Mabry et al., 1970) and with sprayed Naturstoff reagent (Brasseur & Angenot, 1986). UV-VS spectrophotometer (Milton Roy 601) was used in for determination of total phenolic content. Fractionation of the extracts was done by column chromatography using polyamide 6S (Riedel-De Hän Ag, Seelze Hannover, Germany) and Sephadex (Fluka, Switzerland), isolation and purification of compounds was done on either cellulose LH-20 (Pharmacia, Uppsala, Sweden) or Sephadex columns of different dimensions and eluted with different solvent systems. Separation processes were followed up by 2D-PC and CoPC using Whatmann No. 1 paper with (S₁) n-BuOH-AcOH-H₂O (BAW) (4:1:5, top layer) and (S₂) 15% aqueous AcOH as solvent systems. Purity of the isolated compounds was tested by HPLC/DAD (Hewlett Packard, Agilent 1100, quaternary pump G 1311A, vacuum degasser G 1322A, column oven G 1316A, photodiode array detector G 1315A, column C₁₈ silica 10 µm particle size, Lichrocart, Water Ireland).

Plant material

Fresh leaves of L. leucocephala were collected from a mature tree growing in Hadayek El-Ahram, Cairo, Egypt, during May 2009. Identification of the plant was confirmed by Dr. Tearse Labib, Department of Flora and Taxonomy, Orman Garden, Cairo, Egypt. Voucher specimen (Reg. no. L-1) was kept in the herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Helwan University, Cairo, Egypt.

Chemicals

DPPH (1,1-diphenyl-2-picrylhydrazyl) was purchased from Sigma-Aldrich Co. (St Louis, MO). Sodium phosphate, ammonium molybdate, Folin-Ciocalteu’s reagent, ascorbic acid, gallic acid were purchased from Merck Chemical Co (Darmstadt, Germany). All other chemicals, solvents and reagents used in chromatography were of analytical grade. Authentic reference phenolic compounds were obtained from Phytochemistry Laboratory, Department of Molecular and cell Biology, University of Texas at Austin (Austin, TX).

Cell line and culture medium

HepG₂ cells were maintained in RPMI-1640 medium containing 10% (v/v) heat inactivated fetal bovine serum supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C under 5% CO₂ in air.

Extraction and isolation

Powdered, air-dried leaves of L. leucocephala (1 kg) were exhaustively extracted with hot 80% MeOH (4 × 3 L), under reflux. The dry residue obtained (140 g) was extracted with chloroform (3 × 1 L). The aqueous residue (100 g) was fractionated on a polyamide column (Ø 5.5 × 120 cm) and was eluted with water followed by water/methanol mixtures of decreasing polarities to afford five collective fractions (I–V). While the chloroformic soluble portion concentrated under vacuum (30 g) and fractionated on Sephadex LH-20 column (Ø 3.5 × 100 cm) using BIW/MeOH for elution. Eleven compounds were isolated and purified as shown in the flow chart (Figure 1).

Figure 1.  Flow chart of fractionation, extraction and purification of polyphenolic compounds isolated from the leaves of L. leucocephala.

DPPH radical scavenging activity

The ability of 80% MeOH, EtOAc, n-BuOH extracts of L. leucocephala as well as the isolated pure compounds (4–8) to scavenge DPPH radicals was evaluated according to the procedure described by Mensor et al. (2001). Each sample (3 ml) at a concentration of 100 µg/ml was mixed with 1 ml of 0.1 mM DPPH in methanol. The mixture was then shaken and left for 30 min at room temperature in the dark. The absorbance was measured at 517 nm using a spectrophotometer. Ascorbic acid was used as a reference standard. All experiments were carried out in triplicate. The activity of each extract was expressed as percentage DPPH radical scavenging relative to the control using the following equation:

The scavenging effect (antioxidant activity) of each extract was expressed as SC₅₀ which is the concentration of the extract required for 50% scavenging of DPPH radicals compared with that of the standard ascorbic acid.

Determination of total antioxidant capacity

The antioxidant activity of 80% MeOH, EtOAc, n-BuOH extracts of L. leucocephala as well as the isolated pure compounds (4–8) was determined according to phosphomolybdenum method (Prieto et al., 1999) using ascorbic acid as standard. In this method, 0.3 ml of each sample (100 µg/ml) in methanol was combined in dried vials with 3 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The vials containing the reaction mixture were capped and incubated in a thermal block at 95°C for 90 min. After the samples had cooled at room temperature, the absorbance was measured at 695 nm against a blank. The blank consisted of all reagents and solvents without the sample, and it was incubated under the same conditions. All experiments were carried out in triplicates. The antioxidant activity of the extracts was expressed as the number of equivalents of ascorbic acid.

Determination of total phenolic content

The total phenolic content of 80% MeOH, EtOAc, n-BuOH extracts of L. leucocephala was determined using Folin-Ciocalteu reagent according to the method described by Kumar et al. (2008). Gallic acid was used as standard. In this method, (100 μl) of each extract in concentrate of (100 µl/ml) was combined with 500 μl of the Folin-Ciocalteu reagent and 1.5 ml of sodium carbonate (20%). The mixture was shaken and made up to 10 ml using distilled water, allowed to stand for 2 h. Then the absorbance was measured at 765 nm. All determinations were carried out in triplicate. The total phenolic content was expressed as mg gallic acid equivalent (GAE)/g extract.

Determination of total flavonoid content

The total flavonoid content of 80% MeOH, EtOAc, n-BuOH extracts of L. leucocephala was determined using the procedure described by Kumaran and Karunakaran (2006) using rutin as a standard. Plant extract (100 µl) in methanol (100 mg/ml) was mixed with 100 µl of aluminum trichloride in methanol (20 mg/ml) and then diluted with methanol to 500 µl. The absorption at 415 nm was read after 40 min against the blank. All determinations were carried out in triplicate. The total flavonoid in each plant extract was determined as mg rutin equivalents (REs)/g extract.

Reducing power assay

Reducing power of 80% MeOH, EtOAc, n-BuOH extracts of L. leucocephala as well as the isolated pure compounds (4–8) was determined according to the method of Oyaizu (1986). Each sample (250 µl) in methanol was mixed with 250 µl of sodium phosphate buffer (0.2 M, pH 6.6) and 250 µl of 1% K₃Fe(CN)₆ incubate at 50°C for 20 min. After adding 250 µl of trichloroacetic acid, the mixture was centrifuged at 3000 rpm for 10 min. The supernatant solution (100 µl) was then taken out and immediately mixed with 100 µl of methanol and 25 µl of 0.1% ferric chloride. After incubation for 10 min, the absorbance against blank was determined at 7000 nm. Three replicates were made for each tested sample. Increased absorbance of the reaction mixture indicated increased reduction power. Ascorbic acid standard was used for comparison.

Cytotoxic assay

The cytotoxic activity of 80% MeOH, EtOAc, n-BuOH extracts of L. leucocephala as well as the isolated pure compounds 7 and 8 was carried out according to the method described by Skehan et al. (1990). This colorimetric assay (Sulforhodamine-B “SRB”) estimates cell number indirectly by staining total cellular protein with the dye SRB. Hep G₂ cells were seeded in a 96-well plate at a concentration 5 × 10⁴ cells/well in a fresh medium and left to attach to the well-plate for 24 h at 37°C in a humidified atmosphere of 5% CO₂. Then the tested sample was applied to the wells at different concentrations (0, 1, 2.5, 5 and 10 µg/ml) and the plate was incubated for 24, 48 and 72 h under the same condition. Control cells were left without any treatment. The cells were fixed with 50 µl cold 50% trichloroacetic acid for 1 h at 4°C then the wells were washed 5 times with distilled water and left to dry in air, SRB stain (50 µl: 0.4% in 1% acetic acids) was added to each well and allowed to be in contract with the cell for 30 min. Subsequently, to remove excess dyes, the plate was washed with 1% acetic acid, rinsed 4 times until only dye adhering to the cells was left. The plate was dried and 100 µM tri-base (tri-hydroxy-methyl-amino-methane), pH 10.5 was added to each well to solubilize the dye. The plate was shaken gently for 20 min on a shaker (Orbital shaker O5-20, Boeco, Germany) at 1600 rpm. The optical density (OD) of each well was measured spectrophotometrically at 564 nm with an ELIZA micro-plate reader. The mean background absorbance was automatically subtracted and the mean value of sample concentrations was calculated. The experiment was repeated three times for each cell line. The percentage of cell survival was calculated according to the following equation:

The IC₅₀ values were calculated from the prism program obtained by plotting the percentage of surviving cells vs. the concentration interpolated by cubic spine. According to the National Cancer Institute guideline, an extract and/or a compound with IC₅₀ values < 20 µg/ml is considered active (Boyed, 1997).

Statistical analysis

All experimental results were expressed as means ± SD. Analysis of variance was performed by ANOVA procedures. Correlation coefficient (R²) was used to determine two variables. SPSS software was used for statistical calculations. The results with P < 0.05 were regarded to be statistically significant.

Results

A new natural product

Myricetin 3-O-(2′,3′,4′-tri-O-galloyl)-α-l-rhamnopyranoside (7)

Dark yellow amorphous powder (40 mg)

R_f values: 0.40 (S₁) and 0.71 (S₂); deep purple fluorescent spot under UV light turns to orange–red with Naturstoff, green color with FeCl₃ and indigo–red color with KIO₃ spray reagents, respectively. UV λ_max (nm), MeOH: 215, 265, 360; (+ NaOMe): 217, 274, 410; (+ NaOAc): 275, 290, 365; (+ NaOAc + H₃BO₃): 230, 263, 375; (+ AlCl₃): 230, 274, 420; (+ AlCl₃ + HCl): 229, 275, 422. Negative ESI-MS: m/z. 919 (M-H), 766.9 (M-galloyl)⁻, 461 (M-3-galloyl)⁻ and 169 (gallic acid -H)⁻. ¹H NMR: (300 MHz, DMSO-d₆): δ ppm 6.92, 6.90, 6.89 (each 2H, s, 3 × H-2′′′/6′′′ galloyl), 6.87 (2H, s, H-2′/6′), 6.39 (1H, d, J = 1.8 Hz, H-8), 6.21 (1H, d, J = 1.8 Hz, H-6), 5.82 (1H, dd, J = 3.40, 1.5 Hz, H-2′), 5.35 (1H, d, J = 1.5 Hz, H-1’), 5.32 (1H, dd, J = 9.8, 3.40 Hz, H-3′), 5.28 (1H, dd, J = 9.8, 6.0 Hz, H-4′) 3.63 (1H, m, H-5’), 1.00 (3H, d, J = 6.0 Hz, CH₃-6’). ¹³C NMR: (75 MHz, DMSO-d₆): δ ppm 177.34 (C-4), 165.29, 164.33, 164.19 (3 × C-7′′′), 161.20 (C-5), 161.19 (C-7), 157.73 (C-9), 156.43 (C-2), 145.77, 145.46, 145.26 (3 × C-3′′′/5′′′), 145.27 (C-3′/5′), 138.77, 138.41, 138.42 (3 × C-4′′′), 136.47 (C-4′), 134.24 (C-3), 120.71, 120.66, 120.63 (3 × C-1′′′), 120.54 (C-1′), 108.80 (3 × C-2′′′/6′′′), 108.02 (C-2′/6′), 104.02 (C-10), 99.23 (C-1’), 99.24 (C-6), 93.56 (C-8), 71.66 (C-4′), 71.65 (C-2′), 70.88 (C-3′), 69.29 (C-5’), 17.42 (C-6’).

Isolated compounds from L. leucocephala for the first time

Apigenin 7-O-β-d-glucuronopyranoside methyl ester (5)

Reddish brown amorphous powder (25 mg)

R_f values: 0.44 (S₁) and 0.22 (S₂); deep purple fluorescent spot under long UV light turns to greenish yellow with Naturstoff and green color with FeCl₃ spray reagents. UV λ_max (nm), MeOH: 222, 265, 333. Negative ESI-MS: m/z. 459 (M-H)⁻. ¹H NMR: (300 MHz, DMSO-d₆): δ ppm 7.89 (2H, d, J = 8.3 Hz, H-2′/6′), 6.92 (2H, d, J = 8.3 Hz, H-3′/5′), 6.82 (1H, s, H-3), 6.74 (1H, d, J = 1.8 Hz, H-8), 6.43 (1H, d, J = 1.8 Hz, H-6), 5.22 (1H, d, J = 6.6 Hz, H-1’), 3.64 (1H, d, J = 9.0 Hz, H-5’), 3.26 (3H, s, CH₃-ester). ¹³C NMR: (75 MHz, DMSO-d₆): δ ppm 182.65 (C-4), 169.87, (C-6’), 162.94 (C-2), 161.83 (C-7), 161.82 (C-4′), 161.63 (C-5), 157.60, (C-9), 129.35 (C-2′/6′), 121.65 (C-1′), 116.78 (C-3′/5′), 106.13 (C-3), 106.14 (C-10), 99.79 (C-6), 99.71 (C-1’), 95.41 (C-8), 75.69 (C-3′,C-5’), 73.26 (C-2′), 71.90 (C-4′), 52.89 (C-methyl ester).

Luteolin 7-O-β-d-glucuronopyranoside methyl ester (6)

Reddish brown amorphous powder (20 mg)

R_f values: 0.41 (S₁) and 0.51 (S₂); deep purple fluorescent spot under long UV light turns to orange fluorescent with Naturstoff and green color with FeCl₃ spray reagents. UV λ_max (nm), MeOH: 221, 267, 345. Negative ESI-MS: m/z. 475 (M-H)⁻. ¹H NMR: (300 MHz, DMSO-d₆): δ ppm 7.42 (2H, m, H-2′/6′), 6.92 (1H, d, J = 8.0 Hz, H-5′), 6.70 (1H, s, H-3), 6.60 (1H, d, J = 1.6 Hz, H-8), 6.42 (1H, d, J = 1.6 Hz, H-6), 5.12 (1H, d, J = 6.8 Hz, H-1’), 3.38 (1H, d, J = 6.0 Hz, H-5’), 3.12 (3H, s, CH₃-ester). ¹³C NMR: (75 MHz, DMSO-d₆): δ ppm 182.64 (C-4), 171.25, (C-6’), 165.37 (C-2), 163.19 (C-7), 161.61 (C-5), 158.05 (C-9), 150.51, (C-4′), 146.28 (C-3′), 121.73 (C-1′), 120.07 (C-6′), 116.84 (C-5′), 114.05 (C-2′), 106.14 (C-10), 103.74 (C-3), 100.21 (C-1’), 99.89 (C-6), 95.49 (C-8), 76.07 (C-5’), 75.81 (C-3′), 73.34 (C-2′), 71.94 (C-4′), 49.36 (C-methyl ester).

1,3,6-Tri-O-galloyl-β-d-glucopyranose (8)

Off-white amorphous powder (30 mg)

R_f values: 0.42 (S₁) and 0.37 (S₂); shine violet fluorescent spot under short UV light turns to deep blue color with FeCl₃ and red-pink color with KIO₃ spray reagents, UV λ_max (nm), MeOH: 220, 275. Negative ESI-MS: m/z. 635.13 (M-H)⁻, 483 (M-galloyl)⁻, 331 (M-2 galloyl)⁻ and 169 (gallic acid -H)⁻. ¹H NMR: (300 MHz, DMSO-d₆): δ ppm 7.03, 6.97, 6.92, (each 2H, s, 3 × H-2′/6′, 2′/6’, 2′′′/6′′′ galloyl), sugar moiety 5.73 (1H, d, J = 7.5 Hz, H-1), 5.17 (1H, t, J = 8.0 Hz, H-3), 4.45 (1H, m, H-6a), 4.36 (1H, brd, J = 13 Hz H-6b), 3.84 (1H, m, H-5), 3.74–3.64 (2H, m, H-2/4). ¹³C NMR: (75 MHz, DMSO-d₆): δ ppm galloyl moieties: 165.85, 165.57, 164.59 (C-7′/7’/7′′′), 145.72, 145.68, 145.58 (3 × C-3′/5′), 139.31, 138.73, 138.43 (3 × C-4′), 120.80, 120.20, 119.57 (3 × C-1′), 109.46, 109.20, 108.97 (3 × C-2′/6′), sugar moiety: 94.49 (C-1), 77.55 (C-3), 74.82 (C-5), 71.03 (C-2), 67.98 (C-4), 63.09 (C-6).

Discussion

The dried residue of 80% MeOH extract, which was extracted with chloroform for defatting and extracting of aglycones (Marzouk et al., 2009) was chromatographed on a polyamide column followed by successive separation on Sephadex LH-20 and cellulose columns affording eleven pure compounds, among which was compound 7, that exhibited chromatographic characteristics and UV spectral maxima in MeOH closely similar to those reported for myricetin 3-O-glycoside (Hyoung et al., 1998; Kyung, 2000; Ahn et al., 2002). On complete acid hydrolysis (2N HCl, 3 h, 100°C), yielded gallic acid, myricetin in the organic layer and rhamnose in the aqueous layer (comparative paper chromatography, CoPC), suggesting that compound 7 may be myricetin-O-galloyl rhamnoside. The negative ESI-MS spectrum showed molecular ion peak at m/z 919 for (M-H)⁻ together with three fragment ion peaks at 766.9 (loss of galloyl), 461 (loss of 3 galloyl) and 169 of gallate ion. These peaks giving evidence for the molecular formula C₄₂ H₃₂ O₂₄ together with molecular weight 920 m/z, the structure was expected to be myricetin 3-O-galloyl rhamnoside. The positions of substitutions of galloyl moieties were determined by ¹H NMR analysis, as the aglycone myricetin was identified by its three proton resonance of H-2′/6′, H-8 and H-6 at 6.87, 6.39 and 6.21 δ ppm, respectively. The presence of gallic acid moieties were conducted by the three singlets each integrated for two protons at δ 6.92, 6.90 and 6.89 ppm of 2′′′/6′′′. The location of gallic acid moieties was detected by the down-field shifts of H-2′ (δ 5.82), H-3′ (δ 5.32) and H-4′ (δ 5.28). The conformation of the rhamnosyl moiety was identified as α-l-rhamnopyranoside according to δ and J values of all other signals of H-1’, H-5’ and CH₃-6’. Accordingly, the compound was identified as myricetin 3-O-(2′,3′,4′-tri-O-galloyl)-α-l-rhamnopyranoside, and was confirmed by ¹³C NMR spectrum that showed the methyl carbon resonance (C-6’) at δ 17.42 ppm of the rhamnose, the characteristic three signals at δ 165.29, 164.33 and 164.19 ppm for the three carbonyl carbons of the three galloyl groups whose positions were detected by the down-field shift of carbons C-2′, 3′ and 4′. This complete assignment confirmed the structure of compound 7 to be myricetin 3-O-(2′,3′,4′-tri-O-galloyl)-α-l-rhamnopyranoside which has not been reported previously in nature, (Figure 2).

Figure 2.  Structures of compounds isolated from the leaves of L. leucocephala.

Compounds 5, 6 and 8 were isolated from L. leucocephala for the first time. Compound 5 gave chromatographic properties of a flavonoid data (Mabry et al., 1970) of the characteristic band I at λ_max 333 nm for apigenin nucleus. The absence of bathochromic shift in band II upon the addition of NaOAc reagent indicated a 7-O-substituted apigenin compound, this evidence supported by complete acid hydrolysis yielding apigenin in the organic layer and d-glucuronic acid in the aqueous layer detected by CoPC, suggesting that compound 5 expected to be apigenin 7-O-glucouronic acid. ¹H NMR spectrum showing an AX system exhibiting two ortho doublets each integrated for two protons of H-2′/6′, and of H-3′/5′ indicating 1′,4′-disubstituted B-ring. The down-field shift of both H-6 and H-8 to 6.43 and 6.74 meta doublet and the anomeric proton signal at δ 5.22 ppm gave evidence for the presence of β-glycosidic moiety at 7-position (Harborne & Mabry, 1982). The presence of a clear doublet at δ 3.64 ppm and a singlet (3 protons) at δ 3.26 ppm assigned to H-5’ and methyl ester, respectively, led to characterize the sugar moiety as β-methyl-glucuronate. ¹³C NMR spectrum showed 13 carbon signals characteristic for apigenin nucleus, glycosidation at 7-OH was indicated by slight up-field shift of C-7 and the down-field shift of C-6 and C-8 (Agrawal, 1989), the glucuronic acid ester was confirmed by carbon signal at δ 169.87 ppm for C-6’ (carbonyl carbon) and the methyl ester carbon at 52.89 ppm. According to the obtained data and reported data of structural related compounds (Agrawal, 1989; Harborne, 1994), compound 5 was identified as apigenin 7-O-β-d-glucuronopyranoside methyl ester (Figure 2).

Compound 6 gave characteristic chromatographic properties of a flavonoid data (Mabry et al., 1970) of the characteristic band I at λ_max 354 nm for luteolin nucleus. The absence of batho-chromic shift in band II upon the addition of NaOAc reagent indicated a 7-O-substituted luteolin compound, this evidence supported by complete acid hydrolysis yielding luteolin in the organic layer and d-glucuronic acid in the aqueous layer detected by CoPC, suggesting that compound 6 expected to be luteolin 7-O-glucouronic acid. ¹H NMR spectrum shows an ABX system exhibiting an ortho doublets signal for H-5′ and a multiplet signal for H-2′/6′ indicating 3′,4′-disubstituted B-ring. The down-field shift of both H-6 and H-8 to 6.40 and 6.60, respectively, meta doublets as well as the β-anomeric proton signal at δ 5.12 ppm gave evidence for the presence of β-glycosidic moiety at 7-position (Harborne & Mabry, 1982). The presence of doublet at δ 3.38 ppm and a singlet (3 protons) at δ 3.12 ppm assigned to H-5’ and methyl ester, respectively, led to characterize the sugar moiety as β-methyl-glucuronate. ¹³C NMR spectrum showed 15 carbon signals characteristic for lutleoin nucleus, glycosidation at 7-OH was indicated by slight up-field shift of C-7 and the down-field shift of C-6 and C-8 (Agrawal, 1989), the glucuronic acid ester was confirmed by carbon signal at δ 171.25 ppm for C-6’ (carbonyl carbon) and the methyl ester carbon at 49.36 ppm. According to the obtained data and reported data of structural related compounds (Agrawal, 1989; Harborne, 1994), compound 6 was identified as luteolin 7-O-β-d-glucuronopyranoside methyl ester (Figure 2).

Compound 8 gave characteristic chromatographic properties of a gallotanin data (Marzouk et al., 2004), this evidence was supported by complete acid hydrolysis (gallic acid and d-glucose) and negative ESI-MS analysis [molecular ion peak at m/z. 635.13 (M-H)⁻, 483 (M-galloyl)⁻, 331 (M-2 galloyl)⁻ and 169 (gallic acid -H)⁻], suggested a tri-O-galloyl- glucose compound. Confirmation was obtained through ¹H NMR spectrum that showed three singlet signals at δ 7.03, 6.97 and 6.92, each integrated to two protons of the three gallic acid moieties. Galloylation positions were detected at C-1, C-3 and C-6 due to down-field shift of H-1 at 5.73, H-3 at 5.17 and H-6 at 4.45 ppm. ¹³C NMR spectrum showed characteristic three signals at δ 165.85, 165.57 and 164.59 for three carbonyl carbon resonances of the three galloyl groups. The structure was also confirmed with comparison with published data (Nawwar et al., 1982; Haddock et al., 1982; Marzouk et al., 2004) as 1,3,6-tri-O-galloyl-β-d-glucopyranose (Figure 2).

Compounds 1, 2, 3, 4, 9, 10 and 11, which were isolated and identified as gallic acid, methyl gallate, ellagic acid, ellagic acid 3-O-methyl ester, luteolin, apigenin and myricetin, were isolated before from L. leucocephala (Harborne & Mabry, 1982; Markham, 1982; Lowry et al., 1984; Wheeler et al., 1994; Harzallah-Skhiri & Ben-Jannet, 2005).

Antioxidant activity

The antioxidants are able to reduce the stable radical DPPH to yellow colored diphenyl picrylhydrazine. The method is based on the reduction of DPPH in alcoholic solution in the presence of a hydrogen-donating antioxidant due to the formation of non-radical from DPPH-H in the reaction (Tuba & Gulicn, 2008). Accordingly, the DPPH radical scavenging activity (SC₅₀) of 80% methanol, ethyl acetate and butanol extracts of L. leucocephala leaves were determined where the lower SC₅₀ value corresponds to the higher scavenging and higher antioxidant activity. Results in Table 1 revealed that all three tested extracts have antioxidant activity. When compared to the standard ascorbic acid, the 80% methanol extract showed significantly (P < 0.05) higher activity. The antioxidant activity (SC₅₀) of the tested extracts is ranked as follows, 80% MeOH extract (3.94 µg/ml), EtOAc extract (4.41 µg/ml) and BuOH extract (7.16 µg/ml). The activity of the extracts exhibited positive correlation with their phenolic contents (Figure 3). The correlation coefficient (R²) was equal to 0.98. Therefore, it can be noted that the strong antioxidant properties may be attributed to the phenolic components in the extracts. From the results in Table 1, it was appeared that the 80% MeOH extract contains high phenolic components (220.7 mg GAE/g plant extract). The ethyl acetate extract has the highest of flavonoid content (101.33 mg RE/g extract). These results are in full agreement with the reported data as the DPPH radical scavenging activity of a plant extract is associated with its phenolic and flavonoid contents (Wu et al., 2008; Tung et al., 2009). DPPH radical scavenging activity of the three tested extracts of L. leucocephala was supported by measuring their total antioxidant capacity using phosphomolybdenum method which was based on the reduction of Mo (IV) to MO (V) by the tested extract and the subsequent formation of green phosphate/MO (V) compound with maximum absorption at 695 nm (Prieto et al., 1999). Table 1 also shows that the three tested extracts have high total antioxidant capacity and that 80% MeOH extract is the most active (677.98 mg equivalent to ascorbic acid/g extract). Also, the antioxidant capacity of the three extracts exhibited positive correlation with their phenolic contents as shown in Figure 4, (correlated coefficient R² = 0.99).

Table 1.  DPPH radical scavenging activity, TPC, TF and MeOH, EtOAc and n-BuOH extracts of leaves of L. leucopholea.

Extract	DPPH SC50 (µg/ml)	TPC (mg GAE/g ext)	TF (mg RE/g)	Total antioxidant (mg AAE/g ext.)
80% MeOH	*3.94 ± 0.34	220.7 ± 3.1	63.15 ± 2.1	677.98 ± 3.70
EtOAc	*4.41 ± 0.27	200.49 ± 2.87	101.33 ± 10.06	600.34 ± 4.72
n-BuOH	*7.16 ± 0.31	145.35 ± 2.17	53.33 ± 8.32	550.66 ± 7.64
Ascorbic acid	7.90 ± 0.20	—	—	—

Results are means ± SD (n = 3).

GAE, gallic acid equivalent; RE, rutin equivalent; AAE, ascorbic acid equivalent; TPC, total phenolic content; TF, total flavonoid content.

*Significant differences vs. standard P < 0.05.

Owing to the high antioxidant activity of 80% MeOH extract, it was subjected to chromatographic fractionation and the major isolated compounds (4–8) were evaluated as antioxidant agents. The results in Table 2 show that compound 7 has highly significant activity (P < 0.05) when compared to the antioxidant reference (SC₅₀ = 2.49 µg/ml). Also it exhibited high antioxidant capacity (665.62 mg equivalent to ascorbic acid/g compound).

Table 2.  DPPH radical scavenging activity and total antioxidant capacity of compounds (4–8) isolated from 80% MeOH leaf extract of L. leucopholea.

Compound	DPPH SC50 (µg/ml)	Total antioxidant capacity (mg AAE/g compound)
7	2.49 ± 0.57*	665.62 ± 5.10
8	5.08 ± 0.23*	491.13 ± 3.63
4	7.16 ± 0.31*	470.86 ± 4.85
5	14.03 ± 0.37	227.6 ± 5.55
6	68.75 ± 1.5	71.3 ± 4.8
Ascorbic acid	7.90 ± 0.20	—

Results are means ± SD (n = 3).

AAE, ascorbic acid equivalent.

*Significant differences vs. standard P < 0.05.

Figure 3.  Correlation of DPPH radical scavenging activity of 80% methanol, ethyl acetate and n-butanol extracts of leaves of L. leucocephala to their phenolic contents.

Figure 4.  Correlation of antioxidant activity of 80% methanol, ethyl acetate and n-butanol extracts of the leaves of L. leucocephala to their phenolic contents.

Reducing power

Many reports have revealed the direct correlation between antioxidant activity and the reducing power of certain plant extract (Yildirim et al., 2000; Mohamed et al., 2008; Singh et al., 2009b). In this assay, the ability of 80% MeOH, EtOAc and n-BuOH extracts of the leaves of L. leucocephala to reduce Fe³⁺ to Fe²⁺ was determined. The ability of the extracts as antioxidant agents in reduction of Fe³⁺/ferric cyanide complex to the ferrous complex is measured by absorbance at 700 nm. From Figure 5, it is evident that the tested extracts have reducing power (high absorbance at 700 nm corresponds to high reducing power). When compared the reducing power of the extracts to standard reference (ascorbic acid), it was appeared that the 80% MeOH extract has the highest reducing power. This also attributed to that the 80% MeOH extract contains the largest amount of phenolic components. Results in Figure 6 showed that the reducing power of compound 7 is the highest followed by compounds 8, 4, 5 and 6, respectively, when compared to ascorbic acid as standard. Based on the obtained data, of the methanol extract of L. leucocephala showing the strongest antioxidant and free radical scavenging activity and the most reducing power. It was used for the cytotoxic assay and subjected to the chromatographic isolation and identification of its phenolic content.

Figure 5.  Reducing power of 80% methanol, ethyl acetate and n-butanol extracts of the leaves of L. leucocephala relative to ascorbic acid (n = 3).

Figure 6.  Reducing power of the isolated compounds (4–8) from the methanol leaf extract of L. leucocephala relative to ascorbic acid (n = 3).

Cytotoxic activity

Human hepatoma cell line (Hep G₂) represents one of the most widely experimental models for in vitro studies of human HCC. Therefore, in this study, the effect of 80% methanol extract of L. leucocephala and both compounds 7 and 8 were investigated against Hep G₂ using the SRB method. The assay estimates cell number indirectly by staining total cellular protein with the dye SRB (Skehan et al., 1990). The results in Table 3, which are also presented in Figure 7, show that the survival fractions of the cells treated with 80% methanol extract (in different concentrations) are less than those of compounds 7 and 8 throughout 24-, 48- and 72-h treatment periods. The National Cancer Institute (NCI) indicated that the cytotoxicity of a plant extract is considered effective with the IC₅₀ below 20 µg/ml (Boyd, 1997). The results in Table 4 exhibited that 80% methanol extract of L. leucocephala and both compounds 7 and 8 have cytotoxic activity against Hep G₂ with IC₅₀ values 1.41, 2.14 and 2.81 µg/ml, respectively. Accordingly, the methanol extract is the most active followed by the new isolated natural compound 7 and finally compound 8. The results were in accordance with the data reported for different Leucenia species as a rich source of naturally occurring antioxidant cytotoxic phenolic compounds (Kaur et al., 2002; Palewski et al., 2002; Wu et al., 2005).

Table 3.  Cytotoxic activity of 80% methanol extract and compounds 7 and 8 of L. leucocephala against Hep G2 cell line.

Concentration (µg/ml)	Survival fraction
	80% MeOH extract	Compound 7	Compound 8
0.00	1.00 ± 0.00	1.00 ± 0.00	1.00 ± 0.00
1.00	0.556 ± 0.05	0.623 ± 0.06	0.634 ± 0.06
2.50	0.446 ± 0.04	0.474 ± 0.04	0.521 ± 0.05
5.00	0.373 ± 0.03	0.381 ± 0.04	0.390 ± 0.05
10.00	0.241 ± 0.02	0.304 ± 0.03	0.376 ± 0.04

Results are means ± SD of 24, 48 and 72 treatment periods (n = 3).

Table 4.  IC₅₀ of 80% methanol extract, compounds 7 and 8 of L. leucocephala against Hep G2 cell line.

Sample	IC50 µg/ml
80% MeOH extract	1.41 ± 0.17
Compound 7	2.14 ± 0.26
Compound 8	2.81 ± 0.35

Results are means ± SD (n = 3).

Figure 7.  Cytotoxic activity of 80% methanol extract, compound 7 and compound 8 of L. leucocephala against Hep G2 cell line (n = 3).

Conclusions

In conclusion, the methanol extract of the leaves of L. leucopholea contains a considerable amount of polyphenolic compounds that have high antioxidant properties and thus have great potential as a source for natural health products.

Acknowledgment

The authors would like to thank the Department of Tumor Biology, National Cancer Institute, Cairo University for hosting the cytotoxicity activity in the department.

Declaration of interest

The authors report no conflicts of interest.