FIELD OF INVENTION
The present invention relates to the use of a composition for the prevention and
inhibition of haemolysis; in particular the invention relates to a composition obtained
from the aqueous stream of palm oil milling (palm oil vegetation liquor) for the
prevention and inhibition of oxidative stress and haemolysis in human red blood cells
(RBC).
BACKGROUND OF INVENTION
Haemolysis can be considered as the final intracellular event in regards to impairment of
a human erythrocyte affected by oxidative distress. In this condition, the human
erythrocyte or red blood cells (RBC), is break open and consequently hemoglobin is
released into the plasma.
The intracellular events of human erythrocytes or red blood cells upon induced by any
oxidative agents may include cell morphological transformations or alteration, enzyme
modifications, depletion of intracellular glutathione (GSH) and haemolysis.
Comprehendingly, the prevention of oxidative stress facilitates to inhibit potential red
blood cells impairment.
Invariable medical research and investigations conducted over several years on the
deformability or impairment of human red blood cells (RBC) has perceived that, apart
from the deficiency on glucose-6-phosphate dehydrogenase (G6PD), extraordinary
amount of reactive oxygen species, including free radicals production and increased level
of radical-generating substance are primary causative factors in relation to haemolysis of
the human red blood cells (RBC).
The indirect surplus of free radicals and oxidants is induced by environmental causes or
disrupted physiological processes, one of the most common physiological processes
complications include the failure of human biological system's ability to detoxify reactive
intermediates or damages. Such condition will gradually result to oxidative distress of
the blood cells.
Critical level of oxidative distress in human blood cell may result to tissue injury related
diseases, including cardiovascular disease or neurodegenerative disease, while
haemolysis leads to haemolytic anemia, whereby in this condition, the bone marrow
activity cannot counteract the high amount of red blood cells disruption.
Haemolysis can be reduced by prevention or protection of the blood cells from oxidative
distress. One of the most effective amelioration methods in this regard is the presence of
free radical defense.
Free radical defense may include the supplementation of antioxidants which play a major
role in alleviating the destructive effects of excessive oxidants and free radicals, either in
cellular or supplemented forms. Examples of cellular antioxidants are enzymes
superoxide dismutase (SOD), catalase and gluththione peroxidase, wherein these
enzymes function as free radical scavengers and chain reaction terminators. Chemical
antioxidants however act by donating electron to a free radical and thus converting it
into a non-radical form.
Another widely known medical application involving antioxidants or free radicals
scavengers is from a plant based source. It is evident nowadays that the primary
constituents based on plant extracts having astronomical medicinal properties comprise
antioxidants which are in the form of phenolics and flavanoids. These antioxidants are
preferred compared to chemical antioxidants as they have lower risks of releasing or
forming their own free radical upon scavenging a free radical. It follows that the use of
compositions containing extracts based on plant having antioxidant properties instead of

synthetic or chemical antioxidants for treatment of various diseases are now gaining
momentum. An exemplary of an excellent source of two major phytochemicals namely
vitamin E (tocopherols and tocotrienols) and carotenoids, both of which are fat soluble,
is the oil palm fruit. Palm vitamin E has been reported to act as a potent biological
antioxidant, protecting against oxidative stress and the atherosclerotic process.
Palm fruit (Elaeis guineensis) extracts have been reported to exhibit scavenging activity
in relation to reactive oxygen species, via hydrogen - and electron-donating mechanisms.
It is the primary objective of the present invention to provide a composition for
preparing a medicament for the prevention of oxidative stress and haemolysis to human
red blood cells (RBC), based on a plant extract.
It is further an objective of the present invention to provide a composition for preparing
a safe medicament for the prevention of impairment of human red blood cell due to
oxidative stress and haemolysis, said composition containing palm oil phenolics.
SUMMARY OF INVENTION
The present invention relates to a composition comprising phenolics and flavanoids
obtained from the aqueous stream of palm oil milling, for the prevention and inhibition of
oxidative stress and haemolysis in human red blood cell.
The invention further relates to a use of therapeutically effective amount of a
composition containing the said palm oil extracts in the preparation of a medicament for
prevention and inhibition oxidative stress and haemolysis in human red blood cell in an
individual by administering to an individual in need thereof.

DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to the identification of excellent remedial effects for the
prevention of oxidative stress and haemolysis in human red blood cells (RBC) based on
palm phenolics and vitamin E, and thus the production of a composition on the same.
Erythrocytes are suitable models for studies on oxidative stress and human erythrocytes
have been used as model systems to investigate the antioxidant activity of natural
extracts from black tea, and turmeric, as well as polyphenol -rich fractions from tea,
virgin olive oil and red wine.
The following experiments was conducted to show that Palm fruit (Elaeis gunieensis)
exhibits antioxidant activity, acting as scavengers of reactive oxygen species via
hydrogen-and electron- donating mechanisms and to evaluate the antioxidant properties
of palm fruit crude extract referred herein as CE and ethanolic extract referred as (EE).
For the purpose of this invention, the said experiments was conducted in an ex vivo
model system using human red blood cells (RBC). Blood sample was obtained from
healthy volunteers and collected in heparinized tubes.
The methods and results obtained based on respective assays to determine the
effectiveness of the said compounds in palm oil vegetation liquid will be described herein.
The preferred embodiments of the present invention will now be illustrated by way of a
working example.

BEST MODE FOR CARRYING OUT THE INVENTION
Working Example
MATERIALS
2, 2'-azobis - (2-amidinopropoane) dihydrochloride (AAPH) was obtained from Wako
Pure Chemical Co., Ltd. All other reagents used were of analytical grade.
The biologically active extracts of palm vegetation liquor useful in this invention can be
prepared by any means capable of extracting phenolic compounds from the vegetation
liquor using standard extraction techniques or techniques as described in US Patent
Application No. 20030031740 (Sambanthamurthi, Tan and Sundram 2004) .Such
extractions include but are not limited to ethanol, methanol, acetone, ethyl acetate and
butanol.
The extract as described in the above patent (referred herein as crude extract CE) and
ethanolic extracts (EE) were prepared, wherein the extracts were successfully recovered
from the vegetation liquor generated from the milling of oil palm fruits.
Fresh blood was obtained from healthy volunteers by venipuncture, and collected in
heparinized test tubes.
METHODS AND RESULTS
Methods
I. Haemolysis of Erythrocytes

Blood was centrifuged at 3000 RPM for 20 minutes. Subsequently, the plasma and buffy
coat were removed by aspiration. The RBCs were washed three times with 10mM
phosphate buffered saline (PBS) pH 7.4 and resuspended in the same buffer to yield a
haematocrit (Hct) of 10% when incubated at 37°C with 50nM 2,2'-azobis - (2-
amidinopropoane) dihydrochloride (AAPH), without extracts - referred herein as Control,
or with CE or EE at a final concentration .
Accordingly, palm fruit extracts in PBS were added to final concentration of 0.025 to 0.10
mM GAE before addition of 50 mM in AAPH in PBS. For control samples, an equivalent
volume of PBS was added before the addition of AAPH. Samples were then incubated in a
shaking water bath 37°C, and aliquots were taken at timed intervals for determination of
degree of haemolysis, GSH concentration and MetHB content. The degree of haemolysis
was determined according to the conventional method of Zou et al. The concentration of
GSH was determined by reaction with 5, 5'- dithiobis ( 2- nitrobenzoic acid (DTNB)
according to the method of Buetler. MetHb was measured by spectrophotometric
II. Methaemoglobin Formation in Haemolysates
Haemolysates were prepared by suspending 40 µL packed RBCs in 10 mL of phosphate
buffer (15mM, pH 7.2). The suspension was vortexed and then centrifuged at 4000 RPM
for 10 min. The haemoglobin (Hb) content of the supernatant was adjusted to 100mg
Hb/dL with phosphate buffer (PB). A total amount of 100 µL of extract solution in PB was
added to 0.8 mL of haemolysate in a microcuvette. Oxidation was initiated by addition of
100 µL l8mM NaNO2 and the absorbance at 630nm was continuously monitored. For the
control, 100 µL of buffer was used in place of the extract solution.

RESULTS

The above result (Figure 1) depicts the effects of extracts of the present invention on
haemolysis.
AAPH
In the first series of experiments, red blood cell (RBC) suspensions (10% Hct) were
incubated with AAPH (50mM) alone or in the presence of 0.1mM CE and EE. The graph
plotted the progression of AAPH-induced haemolysis of human RBCs, which increases
over time. It is shown that the degree of haemolysis is reduced in the presence of CE
and EE of the present invention, while there is a long lag time before the onset of
haemolysis. This indicates that both extracts effectively protect RBCs against ROS-
induced cytotoxicity.
Referring to the above plotted graph, (Figure 2) depicts that the protective effect of CE
and EE of the present invention on erythrocytes was found to be dose-dependent and a
significant protective effect was seen even at a concentration as low as 25 uM GAE of
extract. Hydrophillic radical scavenging antioxidants have been found to be effective in
preventing AAPH-induced oxidation, as shown in the plotted graph.
In accordance to a preferred embodiment of the present invention, the protective effect
of the extracts of the invention may be due to their ability to scavenge peroxyl radicals
produced in the aqueous phase before these radicals cause oxidative damage to the RBC
membrane.
NaNO2
The second series of experiments for the purpose of the present invention involved the
evaluation of the effectiveness of the extracts in inhibiting nitrite-induced oxidation of
haemoglobin (Hb) to methaemoglobin (MetHb). Nitrite is a well known oxidizing and
MetHb-forming agent.
In this experiment, the haemolysates (100mg/dL) were incubated with:
A. For Crude Extract (CE)
a) Added with NaNO2 (1.8 mM) without extracts (Control);
b) Added with CE 0.05 to 0.10 mM GAE
B. For Ethanol Extract (EE)
a) Added with NaNO2 (1.8mM) without extracts (Control)
b) Added with EE 0.05 to 0.10 mM
It is shown that the extracts in both forms inhibited the formation of MetHb in
haemolysates, and the effect was seen to be dose-dependent as shown in Table 1 below.

For the first series of the experiment and as shown in Figure 2, the degree of haemolysis
in theControl was 64.3 ± 14.6 % compared with 25.9± 11.7% and 24.38 ± 12.9 %for
RBC treated with CE and EE respectively. No consistent effects were observed in GSH
and MetHb concentrations over 6 hours.
Based on the plotted graphs (Figure 3 and Figure 4), for the second series of the
experiment in accordance to the present invention, the changes in the amount of MetHb
formed in relation to time at different extract concentrations. The oxidation of Hb to
MetHb by nitrite comprise a biphasic reaction, the first phase was an initial lag phase
followed by a rapid autocatalytic phase. During the lag phase the MetHb formed may
have reacted with H2O2 to generate the ferrylhaemoglobin radical and thus accomplished
the autocatalytic phase.
The autocatalytic process is however slowed down in the presence of the extracts of the
present invention.
It is therefore concluded that the extracts of the present invention can protect the RBCs
from oxidative distress and thus haemolysis by way of inhibiting the rapid progression of
MetHb formation, due to the scavenging of H2O2 generated, as evident during the lag
phase. In addition, the results also suggest that palm fruit extracts delayed AAPH-
induced haemolysis in human red blood cells.
The working example is not intended, however, to limit or restrict the scope of the
invention in any way and should not be construed as providing conditions, parameters,
reagents or starting materials which must be utilized exclusively in order to practice the
present invention.

WE CLAIM:
1. A composition comprising phenolic acids and flavanoids obtained from the
aqueous stream of palm oil milling (palm oil vegetation liquor), for the prevention and
inhibition of oxidative stress and haemolysis in human red blood cell.
2. The composition as claimed in Claim 1, for the prevention of oxidative impairment
in human red blood cell, due to oxidative stress and haemolysis.
3. The composition as claimed in Claim 1 wherein the composition prevents and
inhibits oxidative stress by way of inhibiting the rapid progression of MetHb formation,
due to the scavenging of H2O2 generated, said scavenging performed by phenolic acids
and flavanoids contained therein.
4. The composition as claimed in Claim 1 wherein the composition is able to delay
AAPH induced haemolysis in human red blood cells (RBCs).
5. Use of therapeutically effective amount of a composition as claimed in claim 1, in
the preparation of a medicament for prevention of oxidative stress and haemolysis in
human red blood cell in an individual in need thereof.

The invention provides a composition for the prevention and inhibition of oxidative
stress and haemolysis in human red blood cell wherein said composition compounds
obtained from the aqueous stream of palm oil milling ( palm oil vegetation liquor) , in
particular from vegetative liquor from the milling of palm oil fruit.