FIELD OF INVENTION
The present invention relates to the use of a composition for the prevention and
treatment of cardiovascular disease, in particular the invention relates to a
composition obtained based on extracts obtained from palm oil effluent, comprising
vitamin E, phenolic flavanoids and combinations thereof, for the preparation of a
means for the prevention and treatment of cardiovascular disease, particularly
atherosclerosis.
BACKGROUND OF INVENTION
Atherosclerosis is an exemplary of a cardiovascular disease (CVD) that requires life
long management in both prevention and treatment. It is recently considered the
leading cause of death in South East Asia which accounts for approximately 4 million
fatal cases each year. This particular disease is characterized by the hardening and
thickening the inner lining of an artery with deposits that consist of fatty substances,
cholesterol, cellular waste products, calcium and fibrin. Consequently, if not treated,
the buildup known as plaque or atheroma, which embedded the walls of arteries will
eventually block the arteries, and is the main contributory factor for heart attack,
chest pain or stroke.
The main carrier in relation to the occurrence of cholesterol and its main source of
damaging accumulation and blockage as mentioned above in the arteries is low-
density lipoproteins (LDL). Generally, LDL carries cholesterol to peripheral tissues
and accordingly passes through the endothelium that causes further development of
plaques and therefore forms arterial wall cholesterol. Conclusively, patients with high
amount of LDL have significantly high risk of atherosclerosis.

From the above, and recognizing the fact that by reducing or lowering the amount of
LDL in patients may significantly assist to reduce the occurrence of atherosclerosis,
the development of scientifically validated medicaments and treatments have been
primed over the years based on this vital factor.
Ongoing scientific advancements include several chemically developed drugs that
inhibit or lower the production of cholesterol; such drugs for instance include statins;
and pharmaceutical compositions. Alternative approaches at present also include the
consumption of juices or substances containing high level of antioxidants, for instance
the pomegranate juice. The presence of antioxidants can assist to neutralize free
radical damage. Free radicals are highly reactive chemical substances that can
damage cellular materials, and therefore causes major degenerative illnesses
including cardiovascular disease and cancer.
Additionally, botanical or plant based compositions have also played a major role in
providing effectual remedies in relation to cardiovascular disease.
The major constituents in plant extract having beneficial properties comprise of
antioxidants which have been widely known for treatment and prevention of a range
of cholesterol related disease, wherein examples of antioxidants in plants are
phenolics acids and flavanoids. Other plant based components or minerals with
astronomical medicinal benefits which may be extracted from plants include
potassium, calcium and magnesium, wherein the said minerals are well recognized for
preventing and managing disorders such as hypertension, cardiovascular disease, and
diabetes.
Following the above, the use of natural plant extracts having antioxidant properties
instead of synthetic antioxidants for treatment of cardiovascular disease is now
gaining momentum.

consumption and weight gain. The consumption of the palm phenolics of the AO and
AOE group was measured over a 14-day continuous duration and was found to be
approximately 150 mg GAE/day/animal.
Table 2 : Content of major fatty acids (%) in the experimental diets.

At the end of the feeding trial, all animals were sacrificed. Prior to sacrifice the
animals were fasted overnight, anesthetized with a mixture of katemin and zoletil (0.1
mL/kg body weight) and 30 mL blood was drawn by heart puncture. Plasma was
prepared by centrifugation at 3000 g for 20 minutes and stored at -80 °C until
analyses. The animals were then overdosed with sodium pentobarbital before autopsy
to remove various organs of interest such as liver, heart, lung and kidneys. The aorta
system was carefully traced, dissected and cleaned of adherent adventitial tissue. The
aorta was then cut open and preserved in 10% formalin solution before staining with
oil Red-0 for quantification of atherosclerotic lesions.
2. Biochemical analyses and measurements of atherogenic indices.

All laboratory analyses listed below are current and standard in most relevant
laboratories.
Plasma lipid analysis
Plasma lipids (TC, triglycerides (TG), HDL-C), were analyzed using enzymatic assay
kits (Roche Diagnostics GmbH, Mannheim, Germany) as per manufacturer's protocol
on the clinical chemistry autoanalyzer, Roche/Hitachi 902.
Plasma antioxidant status
Plasma antioxidant status was measured by two methods; the 2,2' - azinobis (3-
ethylbenzothiazoline) 6-sulfonic acid radical cation (ABTS·+) decolorization assay and
ferric reducing ability of plasma (FRAP).
The ability of rabbit plasma to scavenge the ABTS·+ was measured using the method
of Re et al. (1999) as adapted by Balasundram (2006). Essentially this assay
measures the ability of antioxidants in the plasma to scavenge preformed ABTS·+
produced by the oxidation of ABTS by potassium persulfate. The intensely coloured
ABTS·+ is relatively stable, but in the presence of an antioxidant, it is readily reduced
to the colourless ABTS·2-. The loss of absorbance at 734 nm after 6 min is taken as a
measure of the ABTS·+ scavenging activity. Standard ABTS·+-scavenging curves were
constructed using 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox)
and the ABTS·+ scavenging capacity of the plasma is reported in terms of mg Trolox
equivalents/mL (mg TE /mL).
The ferric reducing antioxidant power of plasma was determined using the method of
Firuzi et al. (2005) as adapted by Balasundram (2006). The original FRAP assay was
developed by Benzie & Strain (1996) as a test to measure the ferric reducing ability of

plasma, i.e. the ability to reduce a ferric-2,4,6-tripyridyl-s-triazine (Fe3+-TPTZ) to
ferrous-2,4,6-tripyridyl-s-triazine (Fe2+-TPTZ) via an electron transfer mechanism.
This reduction resulted in the production of an intensely blue coloured reduced
complex, with maximum absorption at 593 nm (Benzie & Strain, 1996).
Histopathological examination
The aorta was stained with oil Red-O and quantification of the atherosclerotic lesions
was undertaken by using an imaging analysis software (IDT-Solution). Lesions were
categorized as shown in Table 3 below :
Table 3 : Lesions characterization.

Statistical analyses
All data were analyzed using the analysis of variance (ANOVA) and the post hoc
Tukey HSD to test differences between dietary groups/treatments. Differences were
considered statistically significant at p< 0.05.

RESULTS
Mean body weights of the rabbits, prior to and at the end of the study, were not
significantly different between all dietary groups after 100 days feeding (Table 4).
This indicates that the experimental diet did not have any adverse outcomes on the
normal growth curves of the experimental animals.
Table 4 : Animal body weights after 100 days treatments.
n=8 animals/group

At sacrifice, the weight of the liver, heart, lung and kidneys showed no significant
difference between the treatment groups (VIT E, AO, AOE) and control group (Table
5).
Table 5: Effect of antioxidants on various organs of rabbits at autopsy


The plasma lipid profiles of the rabbits in the four treatment groups are shown in
Figure 1A-D. These results do not show any significant difference between groups. It
must be noted that these plasma samples were highly lipaemic. Nanji (1984) has
reported that measurement of lipid profile of lipaemic samples is problematic as the
lipaemia interferes with a variety of clinical chemistry analyses. Hence, further
pretreatment of the samples was required to obtain more accurate results. Results
were plotted as FIG 1, whereby the effect of the treatments on plasma lipid profiles in
rabbits can be observed. (A: Total cholesterol, B:LDL-cholesterol, C: HDL-cholesterol,
D:Triglyceride). Data are presented as means ± sd from 8 animals/group.
All rabbits in this experiment were observed to have developed atherosclerotic lesions
after feeding on the atherogenic diet for 100 days (Fig. 2A-D). The CTR group had
significantly higher fibrous plaque (Fig. 2A) score compared to the other three groups
(VIT E, AO and AOE). In the AOE group, all animals did not show occurrence of
fibrous plaques and this was significantly different (lower) compared to the CTR, the

VIT E and AO groups. Fatty plaques (Fig. 2B) were significantly higher in both the CTR
and VIT E groups compared to the AO and AOE treatments. In addition, the lowest
fatty plaque score was apparent with the AOE treatment. Fatty streaks were highest
in the VIT E treated animals followed by the CTR group (Fig. 2C). AO and AOE groups
had significantly lower occurrence of fatty streaks compared to CTR and VIT E groups.
Lesion free area (Fig. 2D), denoting the total area that was not altered
morphologically (i.e. free of fatty streaks, fatty and fibrous plaques), was significantly
higher following the AOE and AO treatments compared to the VIT E and CTR diets.
This suggests protective effects due to AO and AOE treatments compared to the CTR
and VIT E treatments, against occurrence of atherosclerosis.
FIG 2 shows the development of atherosclerotic lesions (A: Fibrous Plaque, B: Fatty
Plaque, C: Fatty Streaks, D: Lesion Free) after 100 days treatments. Values are
means ± SD ( n=8 rats in each group) Means with common superscripts are
significantly different (p<0.05).
To measure the antioxidant capacity of the rabbits plasma, two methods were used
i.e the ABTS1+ decolorization assay and FRAP assay. Though the FRAP results for AO
(12.32 ± 5.69 mg TE/mL ) and the AOE group (10.53 ± 4.84 mg TE/mL) were
comparatively higher than that of the Control group (7.18 ± 2.66 mg TE/mL), the
differences were not statistically significant (Table 6). Similarly, there were no
significant differences in the plasma ABTS·+ scavenging capacity of all the four
groups. The use of two methods for measurement of antioxidant activity was based
on the suggestion by Frankel & Meyer (2000) and Verhagen et al. (2003) on the need
to evaluate antioxidant activity by using different methods. These two methods were
chosen as they are amongst those most commonly used to measure the antioxidant
capacity due to their rapidity of analysis, ease of use and high sensitivity
(Maisuthisakul et al., 2007).

Table 6: Effect of the treatments on the antioxidant capacity in the rabbit plasma

In this study, the AO and AOE groups given palm phenolics as their drinking fluid did
not exhibit significant changes in their plasma lipid profile (TC,TG,LDL-C and HDL-
C),though the HDL-C level was observed to be slightly higher (statistically not
significant) than the CTR and VIT E groups. However, these animals (AO and AOE
groups) had significantly reduced incidence of atherosclerosis (i.e less fatty streaks
and plaques) than the CTR and VIT E groups. Similar observation was also reported
by Wang et al. (2005) that no significant changes were seen in plasma lipid
parameters between the control and any of the experimental groups at the end of the
12 week feeding duration. These authors found that dealcoholized red wine containing
known amounts of resveratrol suppressed atherosclerosis in hypercholesterolemic
rabbits and concluded that the phythochemicals in red wine can suppress
atherosclerosis without affecting plasma lipid levels. In another rabbit study, Chen et
al. (2005) fed a high-cholesterol diet for 10 weeks to rabbits, supplemented with or
without mulberry fruit extract at two different concentrations (i.e. either 0.5% or
1%), and showed a significant decreased in plasma TC, LDL and TG levels.
The effect of palm phenolics in this study in inhibiting the formation of fibrous plaque
was obvious in both the AO and AOE groups compared to the CTR and VIT E group,
suggesting that palm phenolics may potentially inhibit atherosclerosis. This finding is
in agreement with the findings of Fuhrman et al. (2005), who reported that fresh

grape powder attenuated atherosclerotic development by reducing macrophages
uptake of oxidized LDL and reduced macrophages cholesterol accumulation in
apolipoprotein E-deficient (E°) mice. These authors found no changes in the lipid
profiles of the E° mice. Similarly, although no changes were seen in plasma LDL-C or
HDL-C upon feeding of red wine to E° mice, reduced progression of lesions was
reported by Hayek et al. (1997). Other recent animals studies also reported that
consumption of pomegranate juice by mice (Kaplan et al., 2001), grape extract by
hamsters (Auger et al., 2004). Similarly, consumption red wine, dealcoholized wine
and wine polyphenols was found to reduce atherosclerotic lesions in E° mice, hamsters
and rabbits (Manach et al., 2005). These results therefore show that the phenolics
from palm could be similarly protective against atherosclerosis tendencies.
The compound of this invention may be prepared independently, in dosage form as
described above, and can also be prepared combined together as combination
product.
The dosage form as used herein above includes any suitable vehicle for the
administration of medications known in the pharmaceutical art, including, by way of
example, capsules, tablets, syrups, elixirs and solutions for parenteral injection with
specified ranges of drug concentrations.
These examples are 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 used in the prevention and treatment of atherogenic diseases, comprising
extracts from oil palm oil mill effluents, wherein the composition exhibits inhibitory effects in the
formation of fatty and fibrous plaque and atherosclerotic lesions in a mammal.
2. A composition as claimed in Claim 1 wherein the composition further comprises Vitamin E.
3. A composition as claimed in any one of Claims 1 to 2 wherein the composition comprises
phenolics and flavanoids extracted form oil palm oil mill effluents.
4. The composition as claimed in Claim 1, wherein the composition further provided with
pharmaceutically acceptable carriers

5. Use of a therapeutically effective amount of a composition comprising extracts from palm oil mill
effluents in the prevention and treatment of atherogenic diseases in a mammal in need thereof,
characterized in that the composition reduces and inhibits the formation of fatty and fibrous plaque
and inhibits the development of atherosclerotic lesions in said mammal.
6. A method for inhibiting the formation of fatty and fibrous plaques and atherosclerotic lesions in the
treatment of atherogenic diseases in a mammal comprising administering a composition comprising
extracts from palm oil mill effluents.

ABSTRACT

The invention provides a composition the prevention and treatment of cardiovascular
disease, wherein said composition compounds obtained from palm oil mill effluents, in
particular from vegetative liquor from the milling of palm oil fruit.