1. A COMPARATIVE EVALUATION OF BUFFERS FOR THE EXTRACTION OF NIGELLA PROTEINS
OMORO OGHALE* BIRTHE NIELSEN
oo355@greenwich.ac.uk
University of Greenwich, School of Science, Central Avenue, Chatham Maritime, Kent ME4 4TB
MATERIALS & METHODSMATERIALS & METHODS
MATERIALS & METHODSMATERIALS & METHODSINTRODUCTIONINTRODUCTION
LITERATURE CITEDLITERATURE CITED
.
Nigella seeds crushed to powder in
cold N2 with a Mortar and Pestle
Defatting (diethyl ether)
Protein extraction in: 0.6M NaCl & 0.1% HCl Buffer (pH 7.8),
50 mM Phosphate Buffer (pH 7.8), 50 mM Tris-HCl buffer, (pH
8), 50mM Tris HCl & 0.5M NaCl, (pH 7.8), 10% TCA Acid
(pH 8.5) and 50mM H2SO4 (pH 5.0)
Collection of supernatant
Partial fractionation with Ammonium
Sulphate (NH4)2SO4 (30% -95%
saturation)
Reconstitution of pellet in
extraction buffer
Dialysis against dH2O
Anionic ion Exchange column
Chromatography of dialysed
samples
Separation and
collection of
fractions
Freezing drying of all
fractions
SDS-PAGE
Total protein
content
determination
(Bradford Assay)
SDS-PAGE
RESULTS AND
DISCUSSION
RESULTS AND
DISCUSSIONQuantitative estimation using the method of Bradford
have shown that protein were successfully extracted
from the (NH4)2SO4 precipitated samples (Figure 1).
PBS with a yield of (20.2 mg/g) gave the best
performance
Figure (1) Total yield Extracted from N.sativa by various
buffers used
SDS-PAGE profile (Figure 2) revealed the polypeptide chain
resolved and gave an indication of the performance of various
buffers used.
Lanes: (M) MW Marker (1) 50mM H2SO4 (2) 0.6M NaCl (3) 50
mM PBS (4) 10% TCA (5) 50Mm Tris-HCl pH 8 (6) 50 mM pH
7.8
SDS-PAGE revealed a significant difference in the banding
patterns of the extracted proteins depending on the buffer. This has
been confirmed by previous studies (Sheoran et al., 2008)
CONCLUSIONSCONCLUSIONS
Table (1) further highlights the extracted yield in relation to
the number of polypeptide resolved.
Dialysed sample from Tris-HCl pH 8 was further purified
(Fig 3) using Ion exchange chromatography and resolved
on SDS-PAGE
Results showed partial separation indicating that
fraction 45-52 is enriched in ~33-42kD while 107-
111 was enriched in (~29 -38 kD)
This research has not only successfully highlighted the fact
that N.sativa accumulate extractable proteins in quantities that
is economically useful, but has further demonstrated an
inexpensive extraction technique to enable the pharmacist or
formulation scientist to adequately quantify their standardized
drugs with the right milligram (mg) of proteins at the time of
formulation and production of drugs.
Natural products in the form of plant extract
either as pure compounds or standardized
extracts, have given rise to limitless opportunities
to facilitate the discovery of new drugs chiefly as
a result of their unmatched availability.
Medicinal plants research is therefore directed at
the extraction, purification and identification of
naturally occurring compounds and as such it is
viewed as an effective means of discovering
novel chemical compounds with immense
potentials that can function as drug leads.
Nigella sativa belonging to the Ranunculaceae is
a natural medicinal remedy composed of (16-
19%) protein constituent(Paarakh, 2010).
SCOPE OF RESEARCHSCOPE OF RESEARCH
 Exploration of six Solvent
Extraction buffers
 Detailed analysis with:
1.SDS-PAGE
2. Total Protein content estimation
3. Ion exchange Chromatography
1. Paarakh, Padmaa M. "Nigella sativa Linn.–A comprehensive
review." Indian J Nat Prod Resour 1 (2010): 409-29
2. Sheoran, I. S., Ross, A. R., Olson, D. J. and Sawhney, V. K.
(2009) 'Compatibility of plant protein extraction methods with
mass spectrometry for proteome analysis', Plant science, 176(1),
99-104.