International Research Journal of Public and Environmental Health Vol.7 (2),pp. 37-44, March-April 2020 Available online at https://www.journalissues.org/IRJPEH/ https://doi.org/10.15739/irjpeh.20.006 Copyright © 2020 Author(s) retain the copyright of this article ISSN 2360-8803

Original Research Article

Characterization and cytotoxic activity of amygdalin extracted from apricot kernels growing in Egypt

Received 15 January, 2019, Revised 3 March, 2020 Accepted 10 March, 2020 Published 21 March, 2020

Sherif EA Badr1*, Vitamin B17 (Amygdalin) is D-mandelonitrile-bi-glucoside naturally found in Ola Aly Wahdan2 fruit seeds. Amygdalin was extracted and purified from the kernels of apricots cultivated in Egypt. Identification of the extract was performed by and High-performance liquid chromatography (HPLC), where a major amygdalin Mohamed Saleh peak was obtained at retention time 3.5. Chemical structure of amygdalin AbdelFattah3 was verified using Fourier-transform infrared spectroscopy (FTIR) and Nuclear Magnetic Resonance (1H-NMR) spectroscopy. Human hepatic (Huh- 1N.M.R. Lab., Regional Center for Food & Feed “RCFF”, Agriculture 7) and colorectal (HT-29) cancer cell lines were treated as indicator for Research Center “ARC”, Giza, Egypt. cytotoxicity of extracted amygdalin. The IC50 valued >100 µM and 30 µM, 2Regional Center for Food & Feed respectively. “RCFF”, Agriculture Research Center “ARC”, Giza, Egypt. Keywords: Apricot kernels, amygdalin, HPLC, FTIR, NMR, liver and colorectal 3Natural Products Research Unit, cancer cell lines, cytotoxicity. Faculty of Science, Helwan University, Cairo, Egypt.

*Corresponding Author Email: [email protected]

INTRODUCTION

Apricot (Prunus armeniaca L.) is a member of Rosaceae, The level of amygdalin in apricot kernels ranged between subfamily Prunoideae and can be consumed fresh, dried or 0.08-0.4 mg/g in apricots with sweet seeds up to 1.4-4.44 processed. Apricot kernels are used in the production of mg/g in apricots with bitter seeds Karsavuran et al. (2015). oils, cosmetics, active carbon, and aroma perfume Yıldız Kernels contained 50-150µMol/g cyanogenic glycocides, (1994). Physico-chemical properties of apricot kernels mainly amygdalin and prunasin Tuncel and Brimer (1998). were studied by Gezer et al.(2011) who found that at 2.46% The mean lethal dose (LD50) of amygdalin in rats was found moisture level,94% of kernels were between 0.45g and to be 880 mg/kg body weight (BW) by oral administration 0.68g in weight,96% between 16.5mm and 19.5mm in (Adewusi and Oke, 1985). Human can present systemic length,98% between 8.77mm and 11mm in width, and 98% toxicity after oral administration of amygdalin 4 g per day, between 4.5mm and 6mm in thickness. lasted for half a month or intravenous injection of a month Amygdalin is identified according to International Union (Bromley et al., 2005 and O'Brien et al., 2005). of Pure and Applied chemistry (IUPAC) as [(6-O-β-D- GC-MS analysis of raw apricot kernel and roasted kernel glucopyranosyl-β-D-glucopyranosyl)oxy](phenyl) revealed the existence of amygdalin at 2.47 and 0.83%, acetonitrile with formula: C20H27NO11; Molar mass: respectively. Whereas, soaked kernels and roasted soaked 457.431 g/mol, containing two molecules of glucose, kernels were free from amygdalin as reported by (Badr et hydrogen cyanide and benzaldehyde Figure 1 with 10 % al., 2017). solubility in hot water. Papyri from pharaonic time mentioned the use of Int. Res. J. Public Environ. Health 38

Figure 1: Chemical Structure of amygdalin

amigdalorum for the treatment of some skin tumor for HPLC analysis. For FTIR and 1H-NMR measurements, (Contreras, 1980). Diet rich in amygdalin reduced cancer amygdalin was purified by crystallization. The finely rate especially in tavajo Indians, Eskimos and the ground kernels was washed three times with chloroform Karakorum (Enculescu, 2009). Naturally occurring and allowed to dry at room temperature for 20 min. This amygdalin manifested its role against cancer by induction material was immersed in 30 mL of diethyl ether at 40°C of apoptosis and cell cycle arrest in tumor cells (Saleem et and cooled slowly for 24 h to about 2°C. After evaporation al., 2018). of the solvent, amygdalin appeared as white pure crystals. El-Desouky et al. (2020) studied the anticancer effect of amygdalin on hepatocellular carcinoma cell lines [HepG2] HPLC separation of amygdalin in the presence and absence of zinc. A highly significant apoptotic of hepatocellular carcinoma cell lines [HepG2] Amygdalin crude extract (1.5 ml) was centrifuged for 5 was noticed upon treatment with amygdalin and 800 µmol minutes at 10,000 rpm using a micro centrifuge and filtered zinc. Amygdalin induced cell cycle arrest at G2/M and with PTFE filters. HPLC analysis of Amygdalin was increased the level of P53, Bax, cytochrome c and caspase-3 performed using HPLC-PDA (SYKM S500 series, Germany) significantly, while it decreased the level of anti-apoptotic equipped with column (C18 Column, 4.6×250 mm). The Bcl2. mobile phase was acetonitrile/water (CH3CN/H2O) at flow In the current study, amygdalin was extract from apricot rate of 1.0 ml/min. Amygdalin was identified by photodiode kernels cultivated in Egypt, followed by chromatographic array detector at 214 nm. UV-vis spectra of purified separation using HPLC-PDA and characterization by FTIR amygdalin was recorded after chromatographic separation and NMR spectroscopy. Anti-tumor activity of extract on the HPLC equipment. against liver cancer (Huh-7) and colorectal (HT-29) cell lines was determined to assess its effectiveness as newly Characterization of the chemical structure of prepared cytotoxic agent. amygdalin

Chemical structure of amygdalin was verified using FTIR MATERIALS AND METHODS (JASCO, 6100 FTIR-6100) and 1H- NMR (Bruker 400 MHz) spectroscopy. For NMR experiment, dimethyl sulfoxide Extraction of amygdalin from apricot kernels (DMSO-d6) was used as a solvent.

Ten grams of grounded apricot kernels were extracted with Cell cultures 300ml of ethanol under sonication for 15 minutes at 30°C. The extract was filtered through Whatman filter paper No. Liver cancer cell line (Huh-7) and colorectal cancer cell line 1 and the solvent was completely evaporated under (HT-29) were obtained from Nawah Scientific Inc., Egypt. vacuum. At room temperature, 50 ml of diethyl ether was Huh-7 Cells were maintained in DMEM media added to the dried sample and vortex to precipitate supplemented with 100 mg/ml streptomycin, 100 units/ml amygdalin. Diethyl ether was removed by decantation and penicillin and 10% heat-inactivated fetal bovine at 37°C. the solid residue was dried at 50°C overnight. The The HT-29 cells were maintained in RPMI media precipitate of amygdalin was dissolved in water (2 mg/ml) supplemented with 100 mg/ml streptomycin, 100 units/ml Badr et al. 39

Figure 2a: 96–well plate contained amygdalin and liver cancer cell line (Huh-7)

Figure 2b: 96–well plate contained amygdalin and colorectal cancer cell line (HT-29)

penicillin and 10% heat-inactivated fetal bovine at 37°C. RESULTS AND DISCUSSION

Cytotoxicity assay Chromatogram of amygdalin obtained from the crude extract of apricot kernels is presented in Figure 3. The Cell viability was assessed by Sulforhodamine B assay (SRB chromatogram is characterized by a major amygdalin peak assay).Aliquots of 100µl cell suspension(5×103 cells) were at retention time of 3.5, indicating a high extraction placed in 96–well plates (Figures 2a, 2b and incubated in specificity of the used method and good separation of complete media for 24 hours. The cells were treated with amygdalin by HPLC method. Viorica-Mirela et al. (2006) 100µl amygdaline extract at various concentrations ranging and Chunhua et al. (2007) reported that methanol/water from 0.01, 0.1, 1, 10 and 100µg/ml. After 72 hours, the cells mixtures were used for the separation of amygdalin from were fixed with 150 µl of 10% TCA and incubated at 4°C for natural sources. 1 hour. TCA solution was removed, and the cells were Hyun et al.(2019) extracted amygdalin from apricot washed with distilled water. Aliquots of 70 µl SRB solution kernels and verified its quality by HPLC method, where (0.4% W/V) were added and incubated in a dark place at concentration of amygdalin was 129.34±0.99 mg/g. room temperature for 10 min. The plates were washed 3 times with 1% acetic acid and allowed to air-dry overnight. Spectroscopic characterization of amygdalin Then, 150 µl of Tris (10 mM) was added to dissolve protein bound SRB stain. The absorbance was measured at 540 nm UV-vis spectrum, from HPLC-PDA, of amygdalin sample using a BMG LABTECH-FLUOSTAR omega micro plate showing maxima around 190 and 250 nm (Figure 4). These reader (Ortenberg, Germany) according to Skehan et absorption maxima were found to be matched to those al.(1990) and Allam et al. (2018) methods. reported by Amaya-Salcedo et al. (2018) attributed to Int. Res. J. Public Environ. Health 40

Figure 3: HPLC chromatogram of the crude extract containing amygdalin

Figure 4: UV-vis spectrum of amygdalin from HPLC-PDA.

aromatic p→p* transitions. The bands at 1744 and 1635 cm-1 are the result of C=C The FTIR spectrum of amygdalin (Figure 5) illustrates a stretching vibrations from the benzene ring. The stretching broad band centered at 3327 cm-1 and was assigned to the vibrations for C-O bonds, from ether and hydroxyl groups OH vibrations of the glucose moiety. The absorptions of are observed at 1274 and 1040 cm-1, respectively. Also, the lower intensity at 2924 and 2655 cm-1 were ascribed to intense absorption band at 698 cm-1 was assigned to out-of- aromatic and aliphatic C-H stretching modes respectively. plane C-H bending vibrations. No band was observed Badr et al. 41

Figure 5: FTIR spectrum of amygdalin

Figure 6:1H-NMR (400 MHz) spectrum of amygdalin in DMSO-d6

around 2200 cm-1 for CN group given its very low expected Anticancer activity of apricot amygdalin intensity. 1 The H-NMR spectroscopy in DMSO-d6 characterized the Figures 7 and 8 illustrated the cytotoxicity of extracted structure of the isolated amygdalin more precisely (Figure amygdalin against liver (Huh-7) and colorectal (HT-29) 6). The peaks are in good agreement and very similar to cancer cell lines. Syrigos et al. (1998) mentioned that those reported previously for the compound Wei et al. amygdalin killed cancer cells selectively at the tumor site (2009), thus confirming its identity as cyanogen glycoside without systemic toxicity. Krebs (1940) discovered that amygdalin. Beta-glucosidase splits vitamin B17 into toxic cyanide ions Int. Res. J. Public Environ. Health 42

120 AMG 100

80

60

% Viability

40 IC50: >100 uM R fraction: N/A 20

0 0.001 0.01 0.1 1 10 100 1000

Conc (uM)

Figure 7: Effect of amygdalin on survival percent of Huh-7

120 AMG 100

80

60

% Viability

40 IC50: 30 uM R fraction: N/A 20

0 0.001 0.01 0.1 1 10 100 1000

Conc (uM)

Figure 8: Effect of amygdalin on survival percent of HCT-29

and benzaldehyde that tend to kill tumor cells in a sort of (2001) that is responsible for anticancer activity of pseudo-apoptosis. In human body, enzyme Rhodanese is amygdalin. present only in normal tissues with maximum Results ensured that amygdalin extracted from apricot concentration in the liver. This enzyme breaks hydrogen kernels had appreciable anticancer activity towards cyanide and benzaldehyde into thiocyanates and benzoic colorectal (HT-29) cancer cell line with IC50 valued 30 µM. acid necessarily in nourishment of healthy cells and Our results were in accordance with Moon et al. (2015) production of vitamin B12. Ernest Krebs (1940) described who concluded that amygdalin inhibited cell growth and the mode of action of amygdalin by characterizing the down-regulation of telomerase activity by increasing ß- existence of enzyme Rhodanese in whole body except glucosidase activity. Amygdalin had antitumor by inducing tumor cells. Contrarily, the enzyme beta-glycocidase was apoptosis and regulating immune function Shi et al. (2019). found only in cancerous cells. Amygdalin is decomposed by In-vivo studies are needed to determine the safe dose of the action of enzyme ß-D-glucosidase found in cancerous amygdalin necessary to exert its cytotoxicity effect. cells not healthy cells to yield hydrocyanic acid Isozaki et al. Our results are closely related to Waseem and Nijoud Badr et al. 43

(2019) who evaluated the effect of amygdalin treatment on Adriano A, Federico A, Arben M (2010). Gold Nanoparticle breast cancer patients. Carcinoma 15.3 (CA 15.3) was Based ELISA for a Breast Cancer. Biomarker. Anal. Chem. measured as a tumor biomarker. A significant elevation in 82:1151–1156. CA 15.3 (85.1 U/mL) for breast cancer patients compared Allam RM, Al-Abd AM, Khedr A, Sharaf OA, Nofal SM, Khalifa to healthy control (5.1 U/mL). Treatment with amygdalin AE, Mosli HA Abdel-Naim AB (2018). Fingolimod led to significant decrease in CA 15.3 (3.4 U/mL) compared interrupts the cross talk between estrogen metabolism with serum of breast cancer patients. CA 15.3 determined and sphingolipid metabolism within prostate cancer cells. the extent of patient's respond to treatment Adriano et al. Toxicology Letters Apr 11; 291:77-85. (2010). Amaya-Salcedo JC, Cárdenas-González OE, Gómez-Castaño Cassiem and de Kock (2019) cited that amygdalin of JA (2018). Solid-to-liquid extraction and HPLC/UV various apricot and peach kernels reduced HT-29 colon determination of amygdalin of seeds of apple cancer cell growth. The inhibition of proliferative activity (Maluspumila Mill): Comparison between traditional- through modulation of cell cycle progression and the solvent and microwave methodologies. Acta Agron. 67: 3. induction of a temporary S-phase block. Badr SEA, Ramis ES, Wahdan OA, Sakr DM, Elghandour HMA (2017). Evaluation of protein quality, phytochemical characterization and the effect of soaking Conclusion and roasting processes on raw apricot kernels. The first international conference of Nutrition, Hurghada, the Amygdalin was extracted from apricot kernels cultivated in Egyptian Nutrition Society, 253- 287. Egypt. HPLC chromatogram indicated separation of Bromley J, Hughes BG, Leong DC, Buckley NA (2005). Life- amygdalin as a major sharp peak at retention time 3.5. FTIR threatening interaction between complementary spectrum of amygdalin ensured that no band was observed medicines: Cyanide toxicity following ingestion of around 2200 cm-1 for CN group given its very low expected amygdalin and vitamin C. Ann Pharmacother;39:1566- intensity. 1H-NMR of amygdalin confirmed its identity as 1569. glycoside amygdalin. Amygdalin exerted cytotoxicity Cassiem W, de Koc M (2019). The anti-proliferative effect of against Liver (Huh-7) and colorectal (HT-29) cancer cell apricot and peach kernel extracts on human coloncancer lines with recorded IC50 valued >100 µM and 30 µM, cells in vitro. Complementary and Alternative Medicine, respectively. Further studies are needed to assess safe dose 19:32. level of amygdalin as new anticancer agent extracted from Chunhua Z, Kunsong C, Chongde S, Qingjun C, Wangshu Z, natural botanical source. and Xian L (2007). 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