Int J Pharm Pharm Sci, Vol 6, Issue 8, 108-111Original Article

CHROMATOGRAPHIC ISOLATION OF ALLIUM CEPA (SSP. RED ONION) AND ITS CYTOTOXIC ACTIVITY AGAINST HUMAN LIVER CARCINOMA CELL LINES (HEPG2)

MAHFOUZ M ABDEL-GAWAD1, MAHER A EL-HASHASH2, MORTADA M EL-SAYED1, EMAN A EL-WAKIL1, EZZAT E ABDEL-LATEEF*1

1Theodor Bilharz Research Institute, Laboratory of Medicinal Chemistry, Korniash El-Nile, 12661Warrak El-Hadar, Giza, Egypt, 2Ain-Shams University, Faculty of Science, Department of Chemistry, El-Khalifa El-Mamoun, 11566 Abasia, Cairo, Egypt.
Email: ezzat_ea@yahoo.com

Received: 18 Jun 2014 Revised and Accepted: 20 Jul 2014

ABSTRACT

Objective: The present study aimed to find out cytotoxic agent from Allium cepa L. (ssp. red onion) family Alliaceae as well as chromatographic isolation of its active constituents.

Methods: The defatted methanol extract of Allium cepa (ssp. red onion) was fractionated with chloroform, ethyl acetate and n-butanol. Each of ethyl acetate and butanolic fraction was separately submitted to chromatographic isolation using different chromatographic techniques such as column chromatography (CC), thin layer chromatography (TLC) and paper chromatography (PC). The structures of the isolated compounds were elucidated through spectroscopic analysis.

Results: Four compounds were isolated from ethyl acetate fraction and identified as kaempferol (1), quercetin (2) isorhamnetin (3) and isorhamentin-4`-O-β- D-glucopyranoside (4) whereas two compounds were isolated and identified from the butanolic fraction as α -D-glucopyranose (5), β-D-glucopyranosyl- α- D-glucopyranoside (6). The cytotoxic activity of defatted methanolic extract of A. cepa (ssp red onion), ethyl acetate and butanolic fractions against human liver carcinoma cell line (HepG2) was IC50 = 10.9, 6.08 and 9.95 µg/ ml respectively. Also, compounds 4 isolated from ethyl acetate fraction and compound 6 isolated from butanolic fraction showed cytotoxic activity (IC50 = 11.90 and 22.0 µg/ ml) respectively.

Conclusion: It was appeared that the ethyl acetate fraction was the most active due to it contain high amount of phenolic compounds.

Keywords: Allium cepa, Cytotoxic, Human liver carcinoma cell lines HepG2, Chromatographic isolation.

INTRODUCTION

Cancer is a complex disease involving numerous tempospatial changes in cell physiology, which ultimately lead to malignant tumors. Abnormal cell growth is the biological endpoint of the disease. Tumor cell invasion of surrounding tissues and distant organs is the primary cause of morbidity and mortality for most cancer patients [1]. Hepatocellular cancer (HCC) is the fifth most common cause of cancer and the third leading cause of cancer-related deaths worldwide [2]. The main risk factors for HCC, chronic infections of hepatitis B virus (HBV), hepatitis C virus (HCV), aflatoxin, alcohol, smoking as well as other dietary agents are the most important in humans [3].

The plant kingdom represents an enormous reservoir of biologically active phytochemicals with various chemical structures and protective properties. These phytochemicals are often secondary metabolites in the plants and including alkaloids, steroids, flavonoids, terpenoids, tannins, and other groups. Many of these groups have scavenging activity for free radicals therefore; they can be reducing the tissue injury [4-6]. Now several drugs used in medicine are of plant origin, therefore much current research devoted to phytochemical of the plants [7].

The Allium (family Alliaceae) has over 700 members each of them has special tastes, forms and colors. They have several pharmacological and biological properties such as anti-bacterial, anti-fungal and anti-inflammatory activities [8]. Many of these biological effects are related to the volatile sulfur compounds which are responsible of their characteristic pungent aroma and taste. However, these compounds are unstable and give rise to transformation products. For this reason, recent attention has been focused on polar compounds that are more stable to cooking and to the storage. Among these compounds, sapogenins, saponins, and flavonoids are the main classes found in Allium family [9]. In this study the defatted methanolic extract of A. cepa (ssp. red anion) was fractionated using different organic solvent as CHCl3, EtOAc and n-BuOH. Each of the ethyl acetate and n-butanol fractions was submitted to chromatographic isolation and evaluated as cytotoxic agent against Hep-G2 cell lines.

MATERIALS AND METHODS

General experimental procedures

Melting points were determined on an electrothermal apparatus (Electrothermal 9200). The nuclear magnetic resonance as 1H-NMR and 13CNMR spectra were recorded on a Brucker Avance- 500 and JEOL GX-spectrometer (500 MHz for 1H and 125 MHz for 13C). The chemical shifts were expressed in δ (ppm) with TMS as reference and coupling constant (J) in Hertz. UV spectra (λmax) of the isolated compounds were measured in methanol before and after addition of different reagents on Camspec M550 UV/Vis spectrophotometer (UK). Silica gel 60 (70-230 mesh, Merck) and Sephadex LH-20 (25-100 μm, Sigma) were used for column chromatography. Analytical and preparative thin layer chromatographies (TLC) were performed on silica gel GF254 pre-coated plates (Merck). Paper chromatography (PC) was carried out on Whatmann No. 1 and No. 3 paper sheets. Spots were visualized by absorption of UV radiation and spraying with ethanolic AlCl3 (2%), FeCl3 (1%) and 20% H2SO4 followed by heating for flavonoids compounds and sugars.

Chemicals

Aluminum chloride, Sodium acetate, Boric acid and Sodium methoxide were purchased from Merck (Darmstadt, Germany), Quercetin, kaempferol and isorhamnetin, were purchased from Sigma Aldrich (Milan, Italy) and all solvents are analytical grade.

Plant Materials

The bulbs of Allium cepa (ssp. red onion) were collected from El-Sharkia governorate, Egypt in March 2012. The collected plant was kindly identified by Prof. Dr. Waffa Amer, Professor of plant taxonomy, Faculty of Science, Cairo University. Voucher specimen of the plant was deposited at laboratory of medicinal chemistry, Theodor Bilharz Research Institute, Giza, Egypt. Then the plant materials were submitted to extraction process.

Extraction and Fractionation

Fresh bulbs of Allium cepa (ssp. red onion) (4 Kg) were cut to small pieces then grinded with electric mixer to be ready for extraction process. The pieces of plant bulbs were extracted with pure methanol on cold several times till exhaustion. The solvent was distilled using rotatory evaporator till dryness to give dried extract 290 g. The dried methanolic extract (250 g) was defatted with petroleum ether. The defatted methanolic extract was successively extracted with aqueous chloroform, ethyl acetate and n-butanol. The obtained different fractions were evaporated under reduced pressure to dryness using rotatory evaporator. The ethyl acetate and butanolic extracts were kept for chromatographic separation process.

Chromatographic Separation of EtOAc Extract

Ethyl acetate extract of A. cepa (ssp. red onion) (5.50 g) was subjected to column chromatography (3×60 cm) packed with silica gel 60 (70-230 mesh, Merck) as stationary phase. Elution started with petroleum ether, petroleum ether/ CHCl3 mixture, CHCl3 and then CHCl3/ MeOH gradient and ending with pure methanol. Fractions 250 ml each were collected, concentrated and examined by TLC silica gel using solvent systems; Benzene: MeOH (9:1 and 8:2), CHCl3: MeOH (9:1 and 8:2) and CHCl3: MeOH: H2O (7: 3: 0.5) and on PC using solvent system 15% AcOH and 4: 1: 5 BuOH: AcOH: H2O (BAW). Chromatograms were visualized under UV before and after spraying with AlCl3 (2%), FeCl3 (1%) and 20% H2SO4. Four major fractions (I-IV) were obtained. Fraction (I) eluted with pure CHCl3 was concentrated and purified on sephadex LH-20 column (2×50 cm) using H2O/ MeOH as eluent and the resulted fractions were examined by paper chromatography Whatmman No (1) using solvent system 15% AcOH to give compound 1. Fraction II eluted with CHCl3 - MeOH (98: 2) and re-chromatographed over sephadex LH-20 column using H2O/ MeOH as eluent and then purified on preparative paper chromatography Whatmman No (3) using solvent system 15% AcOH to give compound 2. Fraction III eluted by 95% CHCl3 was subjected to sub- column sephadex LH-20 and H2O/ MeOH as eluent to give compound No 3 and 4.

Compound 1 was obtained as yellow powder, its m.p. 273-278 ˚C, Rf values= 0.036 (15% Acetic acid, PC), 0.68 (n-butanol, acetic acid, water, 4:1:5, PC). UV λmax (MeOH) 255, 268sh and 368; (MeOH+NaOMe) 275, 318,420; (MeOH + AlCl3) 265, 303 sh, 352 sh 423; (MeOH + AlCl3 + HCl) 266, 303sh, 354 sh, 424; (MeOH + NaOAc) 268, 301 sh, 386; (MeOH + NaOAc + H3BO3)262, 298 sh, 373.

Compound 2 was obtained as yellow powder, m.p. 315-317 ˚C, Rf values= 0.051 (15% AcOH, and 0.60 (n-BuOH: AcOH: H2O; 4: 1: 5, PC). UV λmax (MeOH) 256, 268 sh, 372; (MeOH+NaOMe) 273, 330,415; (MeOH + AlCl3) 275, 303 sh, 455; (MeOH + AlCl3 + HCl) 269, 354 sh, 427; (MeOH + NaOAc) 275, 320, 392; (MeOH + NaOAc + H3BO3) 261, 303 sh, 388.

Compound 3 was obtained as yellowish brown powder, m.p. 306-308 ˚C, Rf values= 0.10 (15% AcOH, and 0.56 (n-BuOH: AcOH: H2O; 4: 1: 5, PC). UV λmax (MeOH) 254, 268 sh, 303 sh, 369; (MeOH+NaOMe) 270, 327, 430; (MeOH + AlCl3) 268, 304 sh, 428; (MeOH + AlCl3 + HCl) 266, 302 sh, 427; (MeOH + NaOAc) 274, 325 sh, 390; (MeOH + NaOAc + H3BO3) 257, 275 sh, 377.

Compound 4 was isolated as yellow powder, m.p. 242-244 °C, Rf value= 0.155 (15% AcOH, PC), 0.50 (n-BuOH: AcOH: H2O; 4:1:5, PC) and 0.48 (n-BuOH: MeOH: H2O; 4: 1: 1, TLC). UV λmax (MeOH) 254, 303 sh, 365; (MeOH+NaOMe) 264, 402; (MeOH + AlCl3) 263, 305 sh, 423; (MeOH + AlCl3 + HCl) 260, 302 sh, 420; (MeOH + NaOAc) 272, 316 sh, 386; (MeOH + NaOAc + H3BO3) 257, 275 sh, 368. The 1H-NMR δ; 12.39 (1H, s, 5-OH), 9.52 (1H, s, 3-OH), 8.99 for (1H, s, OH), 7.66 (1H, d, J= 2.0 Hz, H-2'), 7.59 (1H, dd, J= 8.6 and 2.1 Hz, H-6'), 7.21 (1H, d, J= 8.6 Hz, H-5'), 6.41 (1H, d, J= 2.0 Hz, H-8), 6.16 (1H, d, J= 2.0 Hz, H-6), 5.15 (1H, d, J= 6.7 Hz, H-1'', Glc) and 3.70 (3H, s, OCH3). 13C-NMR δ: 176.57 (C-4), 164.61 (C-7), 161.22 (C-5), 156.74 (C-9), 147.29 (C-2), 146.84 (C-3`), 146.40(C-4`), 136.95 (C-3), 125.61 (C-1`), 120.04 (C-6`), 116.27 (C-5`),115.64 (C-2`),103.59 (C-10), 101.82(C-1``), 98.78 (C-6), 98.04 (C-8), 77.79(C-3``), 76.44 (C-5``), 73.77 (C-2``),70.40 (C-4``), 69.72 (C-6``), 61.19 (3`-OCH3).

Chromatographic Separation of n-BuOH Extract

Butanolic extract of Allium cepa (ssp. red onion) (30 g) was subjected to column chromatography (6×120 cm) packed with silica gel 60 (70-230 mesh, Merck) as stationary phase. Elution started with pure CHCl3 and then CHCl3/ MeOH gradient and ending with pure methanol. Fractions 250 ml were collected, concentrated and examined by TLC silica gel using solvent systems; Benzene: MeOH (9:1 and 8:2), CHCl3: MeOH (7: 3) and CHCl3: MeOH: H2O (7: 3: 0.5). The TLC of each fraction was visualized under UV before and after spraying with 20% H2SO4. Also each fraction was examined by paper chromatography Whatmann No 1 using different solvent systems, 15% AcOH and 4: 1: 5 BuOH: AcOH: H2O (BAW). The paper chromatography (PC) was visualized under UV before and after spraying with FeCl3 (1%) and AlCl3 (2%). Three major fractions were obtained. Fraction I eluted with 90% CHCl3/MeOH were concentrated and purified with preparative TLC using solvent system CHCl3: MeOH (7: 3) to give compound 5. Fraction III eluted by 75 % CHCl3/MeOH were collected and submitted to further purification on sub column silica gel (2× 50 cm) with gradient mobile phase CHCl3 the CHCl3/ MeOH to give compound 6.

Compound 5 was obtained as a white amorphous powder, its m.p. 160-162 ˚C, Rf values= 0.45 (CHCl3: MeOH: H2O; 65: 35: 5, TLC), 0.74 (n-BuOH: MeOH: H2O, 4:1:0.5, TLC). 1H-NMR δ: 6.18(1H, d, J= 3.8 Hz, H1), 4.85 (1H, d, H6), 4.75 (1 H, dd, H5), 4.63 (1H, d, H3), 4.75 (1 H, dd, H5), 4.36 (1 H, d, H4). 13C-NMR δ: 92.70 (C-1), 73.55(C-3), 72.82 (C-2), 72.44 (C-5), 71.01 (C-4), 61.67 (C-6).

Compound 6 was obtained as a white crystals, its m.p. 196-198 ˚C, Rf values= 0.50 (CHCl3: MeOH; 7: 3, TLC), 0.66 (CHCl3: MeOH: H2O; 70: 30: 5, TLC) and 0.68 (n-BuOH: MeOH: H2O, 4: 1: 0.5, TLC). 1H-NMR δ: 5.18 (1H, d, J= 3.6 Hz, H1), 4.803 (1H, d, J= 8 Hz, H1`), 4.50 (1H, d, H6`), 4.41 (1 H, d, H3), 4.37 (1H, dd, H5), 3.83 (1H, d, H6), 3.62 (1 H, d, H2), 3.49 (1 H, d, H3`), 3.44 (1 H, d, H4), 3.43 (1 H, dd, H5`), 3.40 (1 H, d, H4`), 3.35 (1 H, d, H2`). 13C NMR δ: 104.48 (C-1`), 92.22 (C-1), 83.0 (C-2), 77.44 (C-5`), 74.70 (C-3`), 73.30 (C-2`), 72.09 (C-3), 70.27 (C-5), 62.60 (C-4), 62.51 (C-4`), 60.94 (C-6), 60.94 (C-6`).

Cytotoxic Assay

The defatted methanolic extract of A. cepa (ssp. red onion) and their fractions (EtOAc and n-BuOH) were investigated toward cytotoxic assay of human liver carcinoma cell line (HepG2). This part was carried out at National Cancer Institute, Cairo, Egypt, according to protocol of Skehan et al., 1990 [10]. This is a colorimetric assay that estimates cell number indirectly by staining total cellular protein with the dye sulphorhodamine-B (SRB). Cells were seeded in 96-well microtiter plates at a concentration of 5 × 104-105 cell/well in a fresh medium and left to attach to the plates for 24 h. For each sample, different concentrations (0, 5, 12.5, 25, and 50 𝜇g/mL) were added to 100 𝜇L wells. Wells were completed to total of 200 𝜇L volume/ well using fresh medium and incubated for 48 h at 37∘C in 5% CO2. Following 48h treatment, the cells were fixed with 50 𝜇L cold 50% trichloroacetic acid for 1 h at 4∘C. Wells were washed 5 times with distilled water and stained for 30min at room temperature with 50𝜇L 0.4% SRB dissolved in 1% acetic acid. The plates were air-dried, and the dye was solubilized with 100 𝜇L/well of 10mM tris base (pH 10.5) for 5min on a shaker and optical density (O.D.) of each well was measured spectrophotometrically at 564 nm with an ELIZA microplate reader (Meter tech. Σ 960, USA). The experiment was repeated 3 times. The percentage of cell survival was calculated according to the following equation:

Survival fraction (%) = [O.D. of treated cell/O.D. of control cells] × 100.

Statistical analysis

All experimental were carried out in triplicate, and statistical analysis were performed using SPSS (13) software and Microsoft Excel program.

RESULTS AND DISSCUSION

Compound 1 was obtained as yellow powder, its m.p 273-278 ˚C, Rf values 0.036 (15% Acetic acid, PC), 0.68 (n-butanol, acetic acid, water, 4:1:5, PC). UV spectrum of methanolic solution of the compound 1 showed characteristic bands of flavonol structure at 255, 268sh and 368 nm. Bathochromic shift of band I (52 nm) was observed with NaOMe indicating the presence of free OH at 4`. Also, bathochromic shift of band II (20 nm) was observed indicating the presence of free 5 and 7 OH. Bathochromic shift of band II with AlCl3 (10 nm) reflected the presence of free OH at position 5. Also, bathochromic shift of band II (13 nm) after addition of NaOAc indicated the presence of OH group in position 7. Addition of NaOAc+H3BO3 led to small shift of band I (5 nm) indicating the absence of OH group in position 3`. Compound 1 showed no depression in m.p. when mixed with authentic kaempferol. Its UV data was in good agreement with that of kaempferol [11, 12]. By comparing its m.p., Co-PC behavior with authentic sample of kaempferol. Thus compound 1 was identified as kaempferol.

Compound 2 was obtained as yellow powder, m.p. 315-317 ˚C, Rf values 0.051 (15% AcOH, and 0.60 (n-BuOH: AcOH: H2O; 4: 1: 5, PC). UV spectrum of the compound in methanol showed the presence of major absorption bands at 256, 268 sh, 372 which confirmed the flavonol structure. Observation of bathochromic shift in both bands I and band II with NaOMe indicated the presence of free 7-OH, 5-OH, 3`OH and 4`OH groups. There are strong bathochromic shift in band I with AlCl3 (73 nm) reflected the presence of O-dihydroxy group in B-ring at 3` and 4` positions bathochromic shift of band II with (19 nm) indicated of free 5-OH group Hypsochromic shift in band I with AlCl3/ HCl (28 nm) indicated the presence of 3`, 4`OH groups. Bathochromic shift (16 nm) in band I with NaOAc/H3BO3 also indicated the presence of O-dihydroxy groups in B-ring at 3` and 4` positions [13, 14]. By comparing its m.p., Co-PC behavior with authentic sample of quercetin. Thus compound 2 was identified as quercetin.

Compound 3 was obtained as yellowish brown powder, m.p. 306-308 ˚C, Rf values 0.10 (15% AcOH, and 0.56 (n-BuOH: AcOH: H2O; 4: 1: 5, PC). UV spectrum of compound 3 in methanol showed the presence of major absorption bands at 254, 268 sh, 303 sh, 369nm which confirmed the flavonol structure. Observation of bathochromic shift in band II with NaOMe indicated the presence of free 7-OH and 5-OH group. Bathochromic shift in band I with AlCl3 (61 nm) reflected the presence of free OH group in B-ring at 3` or 4` positions and band II with (14 nm) indicated of free 5-OH group. A very small hypsochromic shift in band I with AlCl3/HCl (1 nm) indicated the presence only free OH at 3`or 4`position. There is a limited Bathochromic shift (8 nm) in band I with NaOAc/H3BO3 also indicated the absence of O-dihydroxy groups in B-ring at 3` and 4` positions [13, 15]. By comparing their m.p., Co-PC behavior with authentic sample of isorhamnetin. Thus compound 3 was identified as isorhamnetin.

Compound 4 was isolated as yellow powder, UV spectrum exhibited characteristic absorption bands of flavonoid structure at 254, 303 sh and 365 nm [13, 16]. Bathochromic shift in band II (10 nm) was clear by addition NaOMe. This reflected the presence of free OH at C-7. Moderate bathochromic shift (58 nm) in band I by addition of AlCl3 reflected the absence of free OH groups at position 3` and 4`. Also no hypsochromic shift with AlCl3/ HCl was observed which means that 3` and 4` positions are substituted. Bathochromatic shift was obtained by band I (21nm) with NaOAc indicating the presence of substituted groups at position 3` and 4` [13]. The 1H-NMR spectrum showed sharp singlet was appeared at δ 12.39 for (1H, s, 5-OH), 9.52 for (1H, s, OH) and 8.99 for (1H, s, OH). Other protons of the aglycone moiety was appeared at δ 7.66 (1H, d, J= 2 Hz), 7.59 (1 H, d, J=8.6 Hz ), 7.21 (1H, d, J= 8.6 Hz), 6.41 (1H, d, J=2.1 Hz) and 6.16 (1 H, d, J=2 Hz) corresponding to H-2`, H-6`, H-5` and H-8 and H-6 respectively. Also the anomric proton signal was appeared at δ 5.15 (1 H, d, J= 6.7 Hz, H-1`` Glc) and methoxy group signal at δ 3.70 (s, 3H) [13, 17, 18]. 13C-NMR showed characteristic peak of (C-4) at δ 176.57, 101.82 for (C-1``) of anomric carbon of glucose unit and 61.19 for 3`-OCH3 group. This confirmed that compound 4 was identified as isorhamnetin - 4`-O-β-D- glucopyranoside.

Compound 5 was obtained as a white amorphous powder. The 1H-NMR spectrum showed the presence of six peaks in region δ 4.3- 6.18 this area of sugar moiety. There are a sharp doublet peak was appeared at δ 6.18 for (1H, d, J= 3.8 Hz, H1 anomric proton). Other protons were appeared at δ 4.85 (1H, d, H6), 4.75 (1 H, dd, H5), 4.63 (1H, d, H3), 4.44 (1H, d, H2) and 4.36 (1 H, d, H4) these signals of D- glucose unit. In the carbon spectra of gluco-oligosaccharides three chemical shift regions for the signals are usually seen resulting from the anomeric (90–105 ppm), methane (69–83 ppm), and methylene carbons (61–69 ppm) [19, 20]. 13C-NMR of compound 5 showed the presence a single peak of anomric carbon (C1) at δ 92.70 of D-Glucose and other five carbon atoms at δ 73.55, 72.82, 72.44, 71.01 and 61.67 for C3, C2, C5, C4 and C6 respectively. The conformational space of α and β -D- glucose has been widely studied with experimental and theoretical methods [19]. In vacuo the α - anomer is more stable than the β-anomer because of the anomric effect, but in aqueous solution the α / β ratio is reversed due to solvent effects and hydrogen bonding [21]. Therefore from 1H, 13C NMR data and co-TLC compound 5 was identified as α -D - glucopyranos. This is the first time to isolate this free glucose from A. cepa (ssp. red onion).

Compound 6 was obtained as white crystals. The 1H-NMR spectrum showed the presence of peaks in region δ 3.344- 5.185 this area of sugar moiety. There are a doublet peak was appeared at δ 5.185 for (1H, d, J= 3.6 Hz, H1 anomric proton of α-D-glucose unit) and another peak appeared at δ 4.803 for (1H, d, J= 8 Hz, H1` anomric proton of another β-D-glucose unit). The signals of the anomeric protons in the non-reducing unit of the α - or β -disaccharides were close to each other whereas those of the reducing unit at α - and β -equilibrium were well separated [19]. The 1HNMR signals indicate the presence of two D- glucose units. The13C-NMR data of compound 6 showed the presence of peak at δ 104.48 of anomric carbon (C1) of β-D-Glucose, carbon atom at δ 92.22 of anomric carbon (C1) of α -D-Glucose. It was observed that the α -linked anomeric carbon of the non-reducing end resonates in the region 97.0-104.6 ppm, while the β –linked non-reducing anomeric carbon resonates at 98.4-103.5 ppm [19, 20]. From reported data, 1H and 13C NMR data compound 6 was identified as β -D- glucopyranosyl (1→2) - α -D – glucopyranoside. This is the first time to isolate this dimer of glucose from A. cepa (ssp. red onion).

Cytotoxic Assay

Many investigations indicate that phenolic compounds are of great value in preventing the onset and progression of cancer. They are toxic to cancer cells, but are not toxic or are less toxic to normal cells. So it plays an important role in the prevention of cancer [22, 23]. In present study, ethyl acetate fraction which derived from the defatted methanolic extracts of A. cepa (ssp. red onion) is the most active extract (IC50 = 6.08 µg/ ml) against human liver carcinoma cell lines HepG2 as shown in figure (1) and its activity is nearby doxorubicin (IC50 = 4.0 µg/ ml) which used as cytotoxic standard. The butanolic fraction and defatted methanolic extract have IC50 = 9.95 and 10.9 µg/ ml respectively lower than ethyl acetate fraction. Compound 4 (isorhamnetin-4`-O-β-D-gloucopyranoside) which isolated from EtOAc fraction has IC50 = 11.9 µg/ ml and compound 6 (β-D-glucopyranosyl- α- D-glucopyranoside) which isolated from n-BuOH fraction has IC50 = 22.0 µg/ ml. According to the National Cancer Institute guideline, an extract and/or a compound with IC50 values < 20 𝜇g/mL is considered active on hepatocellular carcinoma cell line [24, 25]. These results indicated and confirmed that phenolic compounds especially flavonoids have high cytotoxic activity and it depend on the number of hydroxylation and glycosylation of phenolic compounds as reported by Kathrin et al., 2007 [26] who stated that there is inverse correlation between the cytotoxicity and the number of hydroxyl phenolic groups of flavonoids.

Fig. 1: Cytotoxic activity of defatted methanolic extract of A. cepa (ssp. red onion), derived fractions and its pure isolated compounds against Hep G2 cell line.

CONCLUSION

The present study showed the cytotoxic activity of defatted methanolic extract of Allium cepa (ssp. red onion) and is derived fractions (EtOAc and n-BuOH) as well as chromatographic isolation of their active chemical constituents. The results indicated that the two fractions contain phenolic and free sugars which have cytotoxic properties against Hep G2 cell line.

CONFLICT OF INTERESTS

Declared None

ACKNOWLEDGMENT

The authors are thankful and grateful to Science and Technology Development Fund (STDF), Ministry of Scientific Research, Cairo, Egypt for financial support.

REFRENCES

  1. Seyfried NT, Laura MS. Cancer as a metabolic disease. J Nutr Metabol 2010;7 (7):1-22.
  2. Parikh S, David H. Hepatocellular Cancer: A Guide for the Internist. Am J Med 2007;120:194-202.
  3. Gao J, Li X, Wan-Shui Y, Wei Z, Shan G, Jing W, Yong-Bing X. Risk Factors of Hepatocellular Carcinoma-Current Status and Perspectives. Asian Pac J Cancer Prev 2012;13:743-52.
  4. Kaneria M, Baravalia Y, Vaghasiya Y, Chanda S. Determination of antibacterial and antioxidant potential of some medicinal plants from Saurashtra region, India. Indian J Pharma Sci 2009;71-406.
  5. Dai J and Russell JM. Plant Phenolics:Extraction, Analysis and Their Antioxidant and Anticancer Properties. J Molecules 2010;15:7313-52.
  6. Saxena M, Jyoti S, Rajeev N, Dharmendra S, Abhishek G. Phytochemistry of Medicinal Plants. J Pharm Phytochem 2013;1 (6):168-82.
  7. Peteros PN, Mylene MU. Antioxidant and cytotoxic activities and phytochemical screening of four Philippine medicinal plants. J Med Plants Res 2010;4(5):407-14.
  8. Benkeblia N. Free-radical scavenging capacity and antioxidant properties of some selected onions (Allium cepa L.) and garlic (Allium sativum L.) extracts. J Brazilian Arch Bio Tech 2005;48:753-9.
  9. Lanzotti V. The analysis of onion and garlic. J Chromatogr A 2006;1112:3-22.
  10. Skehan PR, Storeng D, Scudiero. New colorimetric cytotoxicity assay for anticancer drug screening. J Nat Cancer Inst 1990;82 (13):1107-12.
  11. Mabry TJ, Markham KR, Thomas, MB. The systematic identification of flavonoids. Springer-Verlag. New York: Heidlberg Berlin;1970,p. 354.
  12. Abdel-Moaty HI. Evaluation of the primary and secondary products of Nepeta septemcrenata Erenb. J Nat Prod 2009;2:81-8.
  13. Mohamed GA, Ibrahim SRM and Sayed HM. Phenolic constituents of Cucurbita pepo L. cv Eskandrani` (Summer Squash) Flowers. J Bull Pharm Sci Assiut Univ 2009;32(2):311-9.
  14. El-Sayed MM, Maher MA, Mahfouz MA, El-Sayed SA, Wafaa SA, Ezzat EA. Chemical constituents and cytotoxic activity of Cassia glauca Linn. leaves. Life Sci J 2013;10(3):1617-25.
  15. Rajvaidhya S, Gajendra KS, Badri PN, Saroj KP. Extraction, isolation and chemical structure elucidation of isorhamnetin from leaves and stems of Argemone mexicana Linn of Bhopal, Madhya Pradesh, India. Indian J Appl Res 2014;4(2):10-13.
  16. Sikorska M and Irena M. Kaempferol and isorhamnetin and their glycosides in the flowers of Asclepias syriaca L. J Acta Pol Pharm Drug Res 2001;58(4):269-72.
  17. Park YK and Chang YL. Identification of isorhamnetin 4`-glucoside in onions. J Agric Food Chem 1996;44:34-6.
  18. Guvenalp ZL, Omur D. Flavonol glycosides from Asperula arvensis L. Turk J Chem 2005;29:163-9.
  19. Roslund, MU, Petri T, Matthias N, Rainer S. Complete assignments of the 1H and 13C chemical shifts and JH,H coupling constants in NMR spectra of D-glucopyranose and all D-glucopyranosyl-D-glucopyranosides. J Carbohydrate Res 2008;343:101-12.
  20. Leeuwen SS, Bas RL, Gerrit JG, Johannis PK. Development of a 1H-NMR structural-reporter-group concept for the primary structural characterization of α-D-glucans. J Carbohydrate Res 2008;343:1114-19.
  21. Corchado JC, Sa´nchez ML, Aguilar MA. Theoretical study of the relative stability of rotational conformers of alpha and beta-D-glucopyranose in gas phase and aqueous solution. J Am Chem Soc 2004;126, 7311-19.
  22. Nijveldt JR, Els N, Danny ECH, Petra GB, Klaske N, Paul AML. Flavonoids:a review of probable mechanisms of action and potential applications. Am J Clin Nutr 2001;74:418-25.
  23. Bulbul IJ, Laizuman N and Mahmuda H. Antibacterial, cytotoxic and antioxidant activity of chloroform, n-hexane and ethyl acetate extract of plant Coccinia cordifolia. Agric Bio J North Am 2001;2(4):713-9.
  24. Boik J. Natural compounds in cancer therapy. Oregon Medical Press, Princeton, Minn, USA 2001.
  25. Abdel-Hameed ES, Salih AB, Mahmood SS. Characterization of the phytochemical constituents of Taif rose and its antioxidant and anticancer activities. J Biomed Res Int 2013;1:1-13.
  26. Kathrin P, Gabriele K, Eleni K, Elke R, Peter R, Axel R, Peter S, Carsten S. Structure-activity relationships of flavonoid-induced cytotoxicity on human leukemia cells. J Arch Biochem Biophys 2007; 460:1-9.