SUPPLEMENTARY MATERIAL
Antigiardial Activity of glycoproteins and glycopeptides from Ziziphus Honey
Seif Eldin A. Mohammeda, Ahmed S. Kabashib, Waleed S. Kokob, and M. Kamran Azimc*
a
Environment and Natural Resources Research Institute, National Centre for Research, P.O. Box
6096, Khartoum, Sudan,
b
Medicinal and Aromatic Plants Research Institute, National Centre for Research, P.O. Box
2404 Khartoum, Sudan,
c
H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological
Sciences, University of Karachi-75270, Karachi, Pakistan
*Corresponding author. Email: kamran.azim@iccs.edu; mkamranazim@yahoo.co.uk
ABSTRACT
Natural honey contains an array of glycoproteins, proteoglycons and glycopeptides. Size
exclusion chromatography fractionated Ziziphus honey proteins into five peaks with molecular
masses in the range of 10->200 kDa. The fractionated proteins exhibited in vitro antigiardial
activities against Giardia lamblia with IC50 values ≤25 µg/ml. Results indicated that honey
proteins were more active as antiprotozoal agents than Metronidazole. Present study indicated
the potential of honey proteins and peptides as novel antiprotozoal agents.
Keywords: Giardia lamblia; Honey proteins; antiprotozoal
EXPERIMENMTAL
Processing of honey samples
Twenty grams of Ziziphus honey was dialyzed at 4 °C (Spectra/Por membrane MWCO 2000,
Spectrum laboratories, Inc., Rancho Dominguez, CA, USA) against distilled water followed by
lyophilization. The lyophilized sample was reconstituted in 10 ml deionized water and
centrifuged at 5000 g at 4 °C for 10 minutes. Supernatants were precipitated with 80%
(NH4)2SO4 (Merck, Damstadt, Germany) and centrifuged at 5000 g at 4 °C for 10 minutes.
Protein precipitates were collected and dissolved in buffer A (20 mM Tris HCl, 100 mM NaCl, 1
mM EDTA pH 7.2).
Separation of honey proteins and peptides
The protein was separated by size exclusion chromatography using Superdex 75 prep grade gel
matrix (GE Healthcare Biosciences, Sweden) packed in a column with dimensions 0.8 x 60 cm.
Protein samples (150 µl) were applied on the column and elution was carried with buffer A at
flow rate of 0.5 ml/minute. Constant flow rate was maintained by peristaltic pump (Miniplus 3,
Gilson Inc., Middleton, WI, USA) and 1 ml fractions were collected using SuperFrac fraction
collector (GE Healthcare Biosciences, Sweden). The eluted peaks were detected by UV
absorbance at 280 nm. All chromatographic procedures were carried out at 4 °C. The collected
fractions were lyophilized and stored at -20 °C until used for the in vitro parasite susceptibility
assays.
Antiparasitic assays
Giardia lamblia used in the experiments were isolated from clinical specimen of Giardiasis
patients. All positive Giardia lamblia samples were examined by wet mount preparation and
trophozoites were maintained in RPMI 1640 medium containing 5% bovine serum at 37 °C and
were employed in the log phase of growth. In vitro susceptibility assays were carried out in 96
well plates using sub-culture method of Valdez et al. (2002) with slight modification. Eighty
microliters of culture medium containing trophozoites were added to wells. Twenty microliters
of protein fractions with final concentrations of 25, 50, 100 and 200 µg/ml were added in the
wells in triplicates. The final volume of assay mixture was 100 µl. Metronidazole (a
trichomonocide) [(1-(2-hydroxyethl)-2-methyl-5 nitroimidazole] was used as positive control in
concentration 312.5 µg/ml, whereas untreated cells were used as a negative controls (culture
medium plus trophozoites). For counting, the samples were mixed with Trypan blue in equal
volume. The parasite count was determined with haemocytometer in triplicate readings after 24
hours. The mortality (%) of trophozoites for each treatment was carried out according to the
following formula:
Mortality (%) = (control negative – test sample) × 100/control negative
Supplementary Figure S1: Size exclusion chromatogram of Ziziphus honey proteins. Five
chromatophic peaks are labeled as SZ3A to SZ3E.
REFERENCE
Valdez J, Cedillo R, Hernández-Campos A, Yépez L, Hernández-Luis F, Navarrete-Vázquez G,
Tapia A, Cortés R, Hernández M, Castillo R. 2002. Synthesis and antiparasitic activity
of 1H-benzimidazole derivatives. Bioorg Med Chem Lett. 12(16):2221-2224.