FIELD OF INVENTION
The present invention relates to a composition comprising a botanical extract of oil palm
vegetation liquor, and more particularly this invention relates to use of said composition
for improving vascular health including treating and preventing vascular diseases and
diseases related thereto.
BACKGROUND OF THE INVENTION
The ripe oil palm fruit bunch (Elaeis guineensis) used in the extraction of palm oil boasts
a rich mix of deep colours (maroon, red, orange and yellow) which are indicative of an
abundance of phenolics, carotenes and related phytochemicals. Indeed, palm carotenes
(rich in a-and β-carotenes) and palm vitamin E enriched in tocotrienols reside in the
mesocarp and are recovered from extracted crude palm oil. These products are currently
available commercially.
The extraction of oil from the palm fruit results in a large aqueous stream of vegetation
liquor which is currently under-utilized. This aqueous stream accounts for twice the
tonnage of crude palm oil production.
In contrast to lipid-soluble compounds present in the oil phase, the aqueous stream of
vegetation liquor that originates in the milling process and extraction of oil has been
identified as a rich source of water soluble phenolic compounds. It has been found that
the water soluble phytochemicals rich in phenolics, are extracted into the sterilizer
condensate and other aqueous streams of the milling process. The use of high
temperatures for the sterilisation of palm fruit bunch also inactivates the polyphenol-
oxidases and preserves the phenolics.

FIGS. 5a-5b provide a panel of two graphs for the pooled data (all test samples resulted
in similar outcome) of protective effects of OPP against ischemia induced VT and VF;
FIGS 6a-6b provide a panel of two graphs for cardioprotective actions of OPP following
12 week dietary administration;
FIG. 7 provides a panel of three graphs for effect of dietary OPP supplementation on
cardiac arrhythmia parameters in rats treated with L-NAME,
DETAILED DESCRWTION
In line with the above summary, the disclosed description and examples relates to a
composition, use and method thereof for promoting cardio-vascular health and treatment
and/or prophylaxis of a cardiovascular disease (CVD) using a water soluble extract from
the vegetation liquor of the palm oil milling process.
In addition to direct use of an extract, it is also possible to use different purified fractions
of the oil palm phenolic extract
The biologically active extracts of palm vegetation liquor useful in this invention are
those obtained from the vegetation liquor of the palm oil milling process according but
not confined to the methods disclosed in US Patent No. 7,387,802.
Although the extract contains a variety of compounds including phenolic compounds,
fruit acids, fruit sugars and glycerol, for purposes of standardization the concentrations of
the extracts used were measured in terms of phenolic content i.e gallic acid equivalent
Content of phenolic compounds in oil palm phenolic (OPP) extract
100 μl of OPP extract was mixed with 0.2 ml Folin-Ciocalteau reagent (Sigma), 2ml of
H20, and one ml of 15 %Na2CO3 and the absorbance was measured at 765 nm after 2h

incubation at room temperature. A standard curve was plotted with different
concentrations of gallic acid and the total phenolic acid content expressed as gallic acid
equivalent (GAE).
Accordingly, two strengths of OPP were used in the vascular experimental studies herein:
1) OPP (liquid) at 1,050 parts per million (ppm)
2) OPP concentrate prepared in the form of freeze dried concentrate of OPP
- 30,000 ppm, GAE
Vascular Relaxation
Generally, the elevation of total peripheral vascular resistance or also known as systemic
resistance in hypertension is primarily due to changes in structural properties of the blood
vesels (vascular remodeling and/or hypertrophy) and the functional state; increased
vascular tone arising from an unbalanced or impairment of the vasoconstrictor and
vasodilator systems. It is also known that the changes in the balance between sympathetic
vasoconstriction and endothelium-dependant vascular relaxation, mediated primarily via
nitric oxide (NO), can influence the regulation and maintenance of normal blood pressure
(BP). Such changes also form the basis for elevation in BP in several forms of
experimental hypertension.
A number of studies have reported that dietary polyphenols may influence vascular
function and influence blood pressure in several experimental models of hypertension.
Methods for studying the functional properties of larger (conductance) vessels for the
purpose of the present invention include the aortic ring preparation, which is known in
the art as a common and reliable method for in vitro experimental technique. This
method was used for the assessment of the potential vasodilatory effects, which include
the assessment of the role of vascular endothelium and the possible mode of actions
thereof Another significant aim of using the method was to investigate and thus
determine the association between modifications in vascular reactivity or compliance in
larger vessels (stiffness/elasticity) and vascular flow characteristics.

Methods for studying the blood pressure regulation and in hypertension for the purpose
of the present invention include the study on mesenteric vascular bed. The imperative
indications relative to measuring or analyzing blood pressure regulation and hypertension
can be observed via the systemic vascular resistance. The vascular resistance is defined
in the art as the resistance to flow that needs or must be overcome in order to provide
blood within the organ system. As the mesenteric vascular bed is known in the art as a
significant contributor to systemic vascular resistance and compared to larger vessels
such as aorta, contractions of smaller arteries are more relevant to blood pressure
analysis. It is known that both an increased cardiac output and increased vascular
resistance in peripheral circulation can lead to hypertension.
Comprehendingly, in order to determine the effect of OPP in vascular resistance, tissue
preparations which measure vascular function in resistance vessels is therefore selected,
hence the perfused mesenteric vascular bed study. In the perfused mesenteric preparation
for the present invention, the changes in intra-luminal pressure development due to the
constriction of resistance arteries (peripheral circulation) are measured.
It is further known that agents that influence total peripheral resistance via vasorelaxation
are considered beneficial, as functional studies have demonstrated that anti-hypertensive
therapy increase blood flow, decrease vascular resistance and modify structural vascular
alterations both in experimental animal models and in humans.
The effect of OPP of the present invention in vascular resistance were observed and
results show that OPP exerts strong endodielium-dependant vascular relaxation in
isolated vascular preparations, as depicted in FIG la -lb, and FIG 2a-2b
The present invention will now be described by way of example only, the invention is not
to be limited to the discussion of the examples below:
Materials and Methods:

Oil Palm PhenolICS (OPP) obtained through a patented process (US Patent No.
7,387,802) were used in these studies. The concentration of OPP in the extract was
determined using the Folin-Ciocalteu assay . Folin Ciocalteu reagent was mixed with
serial dilutions of test extracts and gallic acid standards, and incubated for 1S mins.
Sodium carbonate (1.9 mol/L) was added, mixture vortexed and incubated for 60 mins.
The absorbance (76S nm) was read against gallic acid standards. Results were expressed
as gallic acid equivalents (GAE).
EXAMPLE 1
Aortic Ring Preparation
Wista-Kyoto (WKY) rats were used for to determine the effect of OPP on vascular
functions.
Isolated segments (3 mm) of thoracic aorta from normotensive Wistar-Kyoto (WKY) rats
(12-14 weeks old) were mounted under isometric conditions in organ bath chambers. In
some rings the endothelium was removed by careful rubbing of the intima with a
moistened cotton swab. The tissues equilibrated for at least 60 minutes before contracting
with KG (20 mmol/L) to test tissue viability. The change in tension was monitored by a
computer based data acquisition system. The rings were pre-contracted with half-
maximal (EC50) dose of noradrenalin (NA) or isoprostaneE2 (8-iso-prostaglandinE2). Test
extracts were dissolved and diluted serially with buffer and added in cumulatively,
directly to the bath The change in tension recorded and the extent of relaxation
calculated.
Results
FIGS, la - FIG lb plot the response following accumulative addition of OPP to
endothelium intact aortic rings from normotensive WKY rats pre-contracted with

noradrenalin (NA). It is observed from the graphs that OPP caused dose-dependant
relaxation in aortic rings pre-contracted with either noradrenalin or 8-iso-PGE2
Results obtained were accordingly recorded and plotted as seen in FIG 1 a -FIG 1 b for
OPP liquid and freeze-dried OPP preparation respectively.
As shown in FIG la and FIG lb OPP liquid showed lower potency compared to that of
the freeze-dried OPP preparation . Subject to the extent of concentration of the active
ingredient, it is observed that the freeze-dried OPP preparation caused substantial
vasorelaxation activity that was therefore considered dose-dependant It is eminent from
the graph that the freeze-dried OPP preparation caused approximately over 75%
relaxation at the highest dose tested. It would be appreciated by a person skilled in the art
that from the results it is further observed that the relaxation profile and the maximum
level exerted by freeze-dried OPP preparation were similar to those observed with the
pharmacological agent acetylcholine, an established vascular relaxant.
FIG 2 plots the response following the cumulative addition of OPP to endothelium
denuded aortic rings from normotensive rats pre-contracted with noradrenalin. It is
observed from the said graph that there was lack of relaxation activity of OPP in the
absence of vascular endothelium denuded ring, therefore suggesting OPP promotes
vasodilation via endothelium derived nitric oxide related mechanism.
EXAMPLE 2
Mesenteric Vascular Bed Test
Blood vessel preparations are from spontaneously hypertensive rats (SHR), which have
impaired vasorelaxation and do not respond effectively to several pharmacological agents
including acetylcholine.

The superior mesentery artery was cannulated and flushed with heparin-saline. The entire
mesenteric bed, including the intestinal tract was removed, and the gut content was
removed by flushing with saline. The preparation was mounted in a 50 ml organ bath
chamber and continuously perfused with oxygenated Krebs-Henseleit medium. After 30
min. of equilibration, tissue viability was assessed by cumulative intra-luminal injection
of various agonists (KC1, NA). The pressure was raised by the addition of NA (EC50) in
the bath perfusate.
Vasorelaxation responses to the pharmacological agent acetylcholine (ACh) and OPP
were determined by measuring the extent of pressure reduction following intra luminal
administration. The pressure changes were monitored using MLT844 Physiological
Pressure Transducer connected to a pressure amplifier (DA100C, BIOPAC Systems Inc.)
and a computer based data acquisition system (MP100WSW High performance data
acquisition unit, BIOPAC Systems Inc.)
Results
Results obtained were accordingly recorded and plotted as seen in FIG 3a -3b.
FIGS. 3a - 3b demonstrate the results for the mesenteric vascular bed study, whereby
there is provided the responses following intra luminal administration of OPP to perfused
mesenteric vascular bed from normotensive (normal blood pressure) rats pre-contracted
with noradrenalin (EC50) and responses following intra luminal administration of OPP to
prefused mesenteric vascular bed from spontaneously hypertensive (high blood pressure)
rats pre-contracted with noradrenalin (EC50) respectively. The graphs indicate induced
relaxation in the resistance blood vessels (mesenteric vascular bed) for both animals,
whereby it is observed that intra luminal administration of OPP caused relaxation in
vessels of the normotensive and hypertensive animals. Accordingly, OPP has shown
efficacy in causing over 70% relaxation at the highest dose tested.

Blood Pressure Lowering
In regulating blood pressure (BP), nitric oxide (NO) serves the main function of inducing
vasodilation by way of diffusing across the endothelium into the adjacent smooth muscle,
and thus causing the said smooth muscle to dilate. It is produced from L-arginine, upon
stimulation, with the assistance of a catalyst, the endothelial nitric oxide synthase
(eNOS). Understandably, reduced amount of NO will result to hypertension.
In order to determine the effect of OPP in blood pressure reduction, a method utilizing a
nitric oxide (NO) deficient model of hypertension was used. This animal model involves
the inhibition of endogenous NO production with L-NAME (N°-nitro-L-arginine methyl
ester) that leads to elevation in BP.
The present invention provides positive results in blood pressure lowering study which
will be described and thus understood by reference to the examples herein.
EXAMPLE 3
Blood Pressure Study
Dietary Feeding
12 weeks old Sprague-Dawley rats were fed a standard laboratory rat diet, formulated to
contain low n-3 polyunsaturated fatty acids (Glen Forrest Stock feeders, Glen Forrest,
W.A., Australia). The OPP was provided as a drink at a rate of 30ml/rat/day. OPP were
provided at two different strengths - as liquid (1500 GAE) and in a concentrated liquid
form at 3000 GAE (prepared using the freeze-dried preparation at 30,000 GAE). Rats
were pre-treated for four weeks prior to challenge with L-NAME. To minimise any
adverse effect due to sudden exposure to L-NAME, in the first two weeks rats were
maintained at a 10 mg/Kg before increasing the dose to ISmg/Kg and maintained at this
level for the next 6 weeks along with OPP. The four experimental groups included (I)
control (2) L-NAME (3) L-NAME+OPP liquid and (4) L-NAME+OPP liquid
concentrate. The intakes were adjusted every two days to allow for gain in bodyweight.

Blood Pressure Monitoring
BP was monitored fortnightly by standard photoelectric tail-cuff procedure (IITC, Life
Sciences, Woodlands Hills, CA, USA) as described in detail previously Briefly, rats
(pre-trained) were placed in restraining tubes (HTC Life Sciences model 805 and model
815) and the tails were occluded with an appropriate sized cuff coupled to a pneumatic
pulse transducer and electrophysiograph (IITC model 65-12). The pulse was detected as
the cuff pressure was reduced and the pressure at which the first pulse detected was taken
as the systolic blood pressure and computed automatically (IITC software package). The
ambient temperature was carefully controlled (30 °C) and the average of 3-4 readings was
taken as the final reading.
Results
Results obtained were accordingly recorded and plotted as seen in FIG 4a -4b
It is noted that in this study, rats were pre-treated for 4 weeks with OPP before treatment
with Af-nitro L-arginine methylester (L-NAME), an antagonist of L-arginine. The main
objective of the said pre-treatment with L-NAME is to elevate the blood pressure of the
rats.
FIG 4a indicates that the introduction of L-NAME (lOmg/Kg) resulted in a sharp rise in
BP (18 mmHg) in the L-NAME control group. However, such initial rise was not
observed (p<0.05) in the rats that received OPP. The increase in L-NAME dosage to
15mg/Kg as shown in the graph continued to result in a gradual increase in BP during the
course of the treatment period. This observation is in contrast to the untreated control
group; whereby the L-NAME treated (15 mg/Kg) rats displayed significantly higher
blood pressure values throughout the duration of the experiment.

It is evident from the plotted graphs that the rats supplemented with OPP showed
retardation in blood pressure development when compared to the L-NAME treated group.
This was more clearly apparent in the OPP concentrate fed animals which received a
higher amount or dose of extract. Referring to FIG 4b, the animals in this group
demonstrated more stable BP values throughout the experiment The final BP values
(mmHg) at the conclusion of the experiment were - untreated control 139±1.6; L-NAME
(control) 176±2.5, OPP liquid 160±2.1** OPP liquid concentrate 156±4.4*** (**p<0.01,
***p<0.001, n=12 per group).
The results have therefore indicated that OPP has significant effect on the blood pressure
system and thus reducing elevated blood pressure in. In addition, oral administration of
OPP lowered the blood pressure in a NO-deficient model of hypertension in dose-related
manner. In can be further highlighted that in view of OPP providing antioxidant
properties, the blood pressure may be lowered by way of scavenging the elevated amount
of ROS and thus reducing or inhibiting oxidant stress which in most cases, may lead to
hypertension or atherosclerosis.
Cardioprotection
In another embodiment of the present invention, the OPP of the present invention were
found to demonstrate protective actions, whereby the said protective actions including
protection against the oxidation of low density lipoprotein (LDL) and thus the
development of atherosclerosis, protection against ischemia induced ventricular
tachycardia (VT), ventricular fibrillation (VF) and sudden cardiac death (SCD).
Generally, the VT incidence and VF data are often associated with the occurrence of
sudden cardiac death; therefore it is highly essential that these data are quantified within a
region.

The following study was designed to evaluate the cardioprotective actions of OPP as
mentioned in the preceding paragraph, whereby the obtained results will be described
shortly herein.
EXAMPLE 4
Protective effects of OPP following acute (intravenous) administration
Twelve-week old male normotensive (WKY) rats were fed a standard laboratory rat diet
formulated to contain low n-3 polyunsaturated fatty acids (Glen Forrest Stockfeeders,
Glen Forrest, Western Australia). The total fat (w/w) content was increased to 10%
(w/w) using lard. The base diet contained no fish meal as a protein source, thereby
minimising the possible presence of long chain n-3PUFAs. Previous experience in this
laboratory has demonstrated that pro-arrhythmic effects of lard may be counteracted by
the presence of fish meal in the base diet. The animals were maintained on this diet for a
period of three months before experimentation. It has previously been demonstrated that
both the age and the type of fat in the diet are key determinants of arrhythmic
vulnerability
EXAMPLES
Protective effects of OPP following dietary feeding
Male WKY rats aged 12 weeks of age (Animal Resource Centre, Western Australia) were
housed in groups of 6 and randomly allocated to different experimental groups.
Bodyweights were recorded fortnightly throughout the experiment. Animals were
provided with ad libitum access to food and water and were maintained under a 12/12
light/dark cycle. They were fed a diet as described above (study 1) and the OPP was
provided as a drink at a rate of 30 ml/day, and replenished thrice weekly. The OPP was
kept frozen in individual 1 litre bottles and thawed prior to use. Two dose levels -
undiluted (100% OPP i.e 1050 ppm GAE), and after 1:1 (v/v) dilution with Milli Q water
(50% OPP) - were used. Supplementary water was provided as required to individual
cages when the test compounds had been consumed. The duration of intervention was 3
months. Bodyweights were recorded fortnightly, and animals were monitored routinely
for overall health and well being.

EXAMPLE 6
Protective effects following OPPfeeding in a nitric oxide deficient model)
12-week old WKY rats were treated with L-NAME (A/G-nitro-L-arginine methyl ester,
40mg/kg bodyweight) for 5 weeks in the absence (water control) or presence of OPP. The
intakes were adjusted every two days to allow for gain in bodyweights Undiluted OPP
liquid (1500 GAE) was provided (30ml/rat/day) throughout the experiment. After the
withdrawal of L-NAME at 5 weeks, the animals continued on their dietary OPP treatment
for further 5 weeks before subjecting to coronary artery ligation as described below.
EXAMPLE 7
Coronary artery ligation
Myocardial ischemia was induced by a temporary occlusion of the LAD (left anterior
descending coronary artery) as described in detail previously. In brief, rats were
anaesthetised (sodium pentobarbitone 60mg/ml stock; 1.0 ml/kg, ip) and intubated. After
tracheotomy, animals were ventilated with room air using a rodent ventilator (model
7025, Ugo Basile, Comerio Varese, Italy) with a stroke volume of approximately 18
ml/kg body weight and at a rate of 60 strokes per min.
The femoral artery and vein were cannulated to monitor blood pressure and for the
administration of saline/drugs/test compounds as required. After left thoracotomy the
heart was exteriorised from the thoracic cavity, and using an atraumatic needle (6/0
braided silk), a ligature was placed just beneath and around the LAD coronary artery. The
heart was returned to the chest cavity and the animals were allowed to stabilise (5 min)
prior to infusion of test compounds. For study 1, the test compounds were made in saline
at a stock concentration of 100 mg/ml The test extracts were infused at a volume of

0.1 ml/1 OOg body weight (a dose level of O.lg/kg body weight) via the femoral vein at a
rate of 0.13 ml/min using an automated syringe pump (model 34IB, Sage Instruments,
Boston, MA) 30 min. prior to coronary occlusion. Regional ischemia (occlusion phase)
was induced by tightening the ligature. The LAD coronary artery occlusion was
maintained for period 30 min. to assess ischemia-induced arrhythmia. At the completion
of the experiment, rats were exsanguinated, hearts excised and perfused with dye (Evans
blue, 0.1 ml) to determine the zone at-risk.
Blood pressure (BP), heart rate (HR) and electrocardiogram (ECG) changes were
monitored throughout the experimental period and recorded using a computer based data
acquisition system (BioPak-MPlOO). Ventricular ectopic activity was assessed according
to the criteria advocated on the Lambeth convention. The parameters computed include
the number of ventricular ectopic beats (VEB), and the incidence and duration of
ventricular tachyarrhythmia, including ventricular tachycardia (VT) and ventricular
fibrillation (VF).
Statistical Analysis
It is noted that data were expressed as mean±SEM The incidences of VT and VF were
compared using the Chi-square test. Differences in the number of VEB, the duration of
VT and VF between the control and individual treatments were analysed using the Mann-
Whitney rank-sum test The pooled data from concentrated samples were subjected to
one way ANOVA followed by Bonferroni's post hoc test A p value of <0.05 was
considered significant. The statistical analyses were performed using the Graph Pad-/n
Stat (version 4.0) computer software program.
In order to determine the cardioprotective effects of the present invention in the
aforementioned animal models, OPP of the present invention was prepared in the
following concentrations and referred herein as Test Samples.
Test Sample 1 : OPP liquid (1,050 Gallic Acid Equivalent, GAE)

Test Sample 2 : 2.5 fold concentrate of the OPP liquid (2,625 GAE)
Test Sample 3 : 3 fold concentrate of the OPP liquid (3,150 GAE)
Test Samples 4: 12 5 fold concentrate of the OPP liquid (13,125 GAE)
Test Samples 5: Freeze dried preparation of OPP (30,000 GAE)
OPP was assessed following intravenous administration, directly into the blood stream.
Results
Acute Administration Study
The results obtained for all studies related to cardioprotection effects of OPP will be
described shortly herein.
Table 1 below provides the values as accorded for the test on the effects of different OPP
concentrations on ventricular tachycardia (VT) durations.
Table 1
Effects of OPP on ischemia induced ventricular tachycardia (VT) in the anaesthetized rat
Ventricular tachycardia (VT)
Compound N Incidence (%) Duration (sec)
Control (saline) (11) 100 27 0 ±8.0
Test Sample lfTSl) (5) 80 18.5 ±12.8
Test Sample 2 (TS2) (9) 33* 1.0 ±0.7*
Test Sample 3 (TS3) (5) 60 2.5 ±1.1*
Test Sample 4 (TS4) (6) 83 2.8 ±1.3*
Test Sample 5 (TS5) (3) 67 13 ±0.8*

Values reported are the mean ± SEM of the duration and the incidence of VT during a 30min.
occlusion period. N refers to the number of animals per group. Test compounds were infused 30
min. prior to the induction of ischemia by ligation of the left anterior descending coronary artery.
Statistical significance (*) ot the level of 5% or better.
As shown in Table 1 the VT incidence has reduced approximately 20% with the
administration of Test Sample 1 when compared to that of the control model. A
substantia! reduction of VT incidence is observed when models were administered with
Test Sample 2.
FIG 5a -5b plot the pooled data (all test samples resulted in similar outcome) of
protective effects of OPP against ischemia induced VT and VF, whereby as seen in the
said graphs there are significant difference between the control and OPP administered
animals in the VT and VF incidence.
The incidence of ventricular fibrillation (VF) in the control group was 36% (4/11). OPP
Test Sample 1 (OPP liquor) was without effect against VF and this group showed similar
VF incidence (40%). In contrast, none of the animals in the concentrated OPP samples
(Test Samples 2-5) developed VF.
From the values tabulated in Table 1 and as summarized in FIG 5a-5b, it is evident that
OPP of the present invention demonstrates significant protection or inhibition against the
% incidence and the duration of ventricular tachycardia (VT) by way of reducing the
incidence and duration of VT following acute administration (intravenous) directly into
the circulation. Based on these results, it is shown that OPP may provide anti-arrhythmic
effect for the in vivo animal model of cardiac arrhythmia used for the purpose of the
present invention.
Dietary Feeding Study
The main objective of this study is to analyse the effect of OPP on % incidence and
duration of VF and mortality from VF during the 30 minutes ischemic period of rats fed
OPP liquor (50% and 100% strength's) for a duration of three months.

As shown in Table 2 appended below, as opposed to the control (water) group whereby
there were 3/12 deaths, treated animals showed no deaths. This therefore indicates that
the OPP feeding facilitates in reducing the VF (% incidence and duration).

Values reported are the mean ± SEMofthe duration and the incidence and mean duration ofVF
during a 30min. occlusion period. N refers to the number of animals per group. * p<0.05 (vs.
control), Chi-square test. * denotes significance difference from OPP treated groups (ANOVA;
Turkey s test; P<0.05 or better)
Table 3 appended below tabulates the pooled data recorded for effects of OPP long term
dietary feeding (3 months) to animals, whereby the positive results demonstrated are
summarized as FIG 6a-6b Results presented in FIG 6a-6b are the pooled data (N=25)
from animals fed (30ml/day) diluted (50%) and undiluted (100%) liquid.
Table 3
%Incidence of VT and VF in rats fed a pro-arrhythmic diet supplemented with OPP
(pooled data)


It is therefore observed that treating OPP to the animals via long term dietary feeding
provided a significant effect on the reduction of VF and VT (incidence and duration)
which are the similar effects observed following an intravenous administration to the rats.
Dietary Feeding (Nitric Oxide Deficient Model)
It is noted that in order to determine the effect of dietary administration of OPP on the
vulnerability to cardiac arrhythmia, the rats were treated with L-NAME. The rationale for
this was based on the premise that OPP may be able to exert cardioprotection by
promoting the availability of nitric oxide (NO) in this NO-deficient model. It has been
reported that impaired production/availability of NO is pivotal to the cardiovascular
abnormalities observed in this model.
At the end of this study, the administration of OPP demonstrated cardioprotection effect
similar to that observed with untreated animals fed a pro-arrhythmic diet and as plotted in
FIG 6a-6b
Therefore, Figure 7 shows that OPP feeding resulted in considerable cardioprotection in
the NO-deficient model. For instance, it is evident from the results that the total number
of VEB was reduced (p<0.05) in the OPP supplemented group compared to the control

rats and was also reflected in the extent of VT incidences since compared to 100%
incidence (13/13 animals) in the control group, OPP group displayed only 55% (5 out of
9). This protection by OPP against %VT (p<0.05, Chi-square test) was also apparent
when the duration of VT activity was compared In addition, the OPP feeding led to a
significantly shortened duration (seconds) of VT (control 35.7 ± 7.8; OPP 2.3 ± 1.0*,
p<0.05).
Conclusively, based on the results obtained it is evident that OPP provides considerable
protection against ischemia induced cardiac arrhythmias (VEB, VT), ventricular
fibrillation and sudden cardiac death. Accordingly, the results obtained from the said
studies verifies the observations made with the concentrated OPP test samples in an
acute setting following administration directly into the blood stream as seen in Table 1
and FIG 5.
Atherosclerosis
In numerous plant extract studies, several results obtained have shown atherogenesis
prevention, whereby it was found that the compounds were able to decrease the
susceptibility of low density lipoprotein (LDL) to lipid oxidation and retard the
development of atherosclresosis in animal model such as rabbits fed pro-atherogenic diet.
A study was conducted to determine another embodiment of the present invention, the
protective effects of OPP against in vito oxidation of human LDL and the development of
athrematous lesions in the rabbit model of atherosclerosis. Results from this study were
accordingly recorded and plotted in Tables 4-6.
Example 8
Inhibition of LDL oxidation by OPP
Materials and Methods

Oil Palm Phenolics (OPP) obtained through a patented process (US Patent No 7,387,802) were
used in these studies. The concentration of OPP in the extract was determined using the Folin-
Clocalteu assay . Folin Ciocalteu reagent was mixed with serial dilutions of test extracts and
gallic acid standards, and incubated for 15 mins. Sodium carbonate (1.9 mol/L) was added,
mixture vortexed and incubated for 60 mins. The absorbance (765 nm) was read against gallic
acid standards. Results were expressed as gallic acid equivalents (GAE).
Plasma LDL isolation
Blood from six healthy normocholesterolemic adult volunteers was collected into
vacutainer tubes containing EDTA and centrifuged (1000 x g for 15 min) to isolate
plasma. LDL was isolated by density gradient centrifugation in a Beckman
Ultracentrifuge using a 50.2 Ti rotor, based on die method of Sundram et at. (2). The
LDL layer following sequential isolation at density 1.063 kg/L was removed and dialyzed
against 4 L of PBS, pH 7.4 and purged with nitrogen for 12-18 h. After dialysis, the
cholesterol concentration in the pooled LDL sample was determined using an automated
Cholesterol assay. The LDL fraction was used immediately or immediately stored under
nitrogen at 4°C and analyzed for LDL oxidation within 48h.
Copper-mediated LDL oxidation
To evaluate the ability of OPP to inhibit copper (Cu) mediated LDL oxidation,
conjugated dienes (CD) were continually monitored at 5-min intervals at 37°C by UV
absorption at 234 nm LDL oxidation was initiated by addition of copper sulfate at a
final concentration of 6 umol/L to 90 ug of LDL cholesterol in a final volume of 1 mL.
The purified test compounds (catechin obtained from Sigma Chemical) and OPP extracts
were added immediately before the addition of the oxidant The extracts were added at a
final concentration range of 0.25-1.0 umol/L GAE and the purified compounds at a final
concentration of 0.25 or 0.5 umol/L. All LDL oxidations were performed in triplicate.
The lag time in the presence or absence of the test compounds was determined as the
intercept of the slopes for the lag and propagation phases. This was compared with the
control oxidized LDL to determine the percentage of LDL oxidation inhibition.

Oxidation of LDL initiated by Cu ions and conjugated diene (CD) formation was
measured at 234 nm. Extracts at concentrations of 0.25, 0.5 and 1.0 umol/L GAE
inhibited Cu mediated LDL oxidation in a dose-dependent manner since LDL lag phase
was lengthened up to 10%, 53% and 81%, respectively, relative to the control LDL.
Purified catechin was used as control phenolic compounds and compared with control
LDL. The highest concentration of catechin (0.5 umol/L) resulted in 55% increases in
lag time with Cu-mediated oxidation system.

EXAMPLE 9
Rabbit Arthrosclerosis Study
Twenty-four New Zealand White rabbits were divided into three groups of 8 and fed an
atherogenic diet for 100 days. The diet contained 35% energy from fat, with added
cholesterol 0.15% (w/w). The fat comprised 67.1% saturated fatty acids, primarily as
02:0+04:0 (Table 5). Group 1; the Control group (CTR) was fed the atherogenic diet,
and was provided distilled water as their drinking water. Two concentrations of OPP

were tested in two groups of 8 animals each and these were fed the atherogenic diet and
palm phenolics (at a concentration of 500 mg gallic acid equivalents/L (mg GAE/L)) or
1000 mg GAE/L was provided as drinking fluid; Groups 2 and 3 respectively..

Animals were fed diets ad libitum and had free access to drinking fluids. Fasting blood
was obtained prior to sacrifice and plasma total cholesterol (TC), high density
lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C) and
triglycerides (TG) were analyzed. At the end of the 100 days supplementation, the rabbits
were killed by a lethal injection of Nembutal and the entire aorta was traced, dissected
and fixed in 10% formalin. The said aortas were then stained with Oil Red-O which
resulted to the lipid deposits in the aorta to be stained red thus delineation of
atheromatous deposits in the intima and lesions were quantified using a digital image

analysis system. It is noted that the intensity of staining was directly proportional to the
amount of the lipid deposits. Aortic lesions were scored both visibly and on the basis of
the intensity of lipid stains recorded by the said digital image analysis system. Means
were calculated and a final score assigned to the control and experimental groups. These
observations were further verified by plasma and aortic lipid analyses.
Table 6 shows the results for the effect of OPP on atherosclerosis. It is observed that the
lipid profile analysis in all the experimental groups did not show any significant
differences Fibrous plaques (% of total aorta area) were detected in the CTR group
(7.96±4.82) but were significantly reduced in OPP supplemented groups (OPP-500 mg
GAE/L 1.41±4.45 units???? ) groups. Similar outcomes were observed for fatty plaques.
The results obtained indicate that compared to the control group, the provision of OPP
provided significant protective effect in a dose-dependent against the development of
atherosclerotic lesions in this animal model. It is noteworthy that this protection was
acheived against a pro-atherogenic high-fat background diet.



Values are % of total aorta area (mm); n = 9 animals per group and presented as means
t SD. Means with common superscript are significantly different (p<0.O5). Total plasma
cholesterol was > 15 mmol/dL in all groups. LDL and HDL levels were not significant
among groups in this cholesterol driven atherosclerosis model.
CTR - Control group provided drinking water without any antioxidants added, water
soluble OPP (oil palm phytochemicals) at 500 or 1000 mgGAE/L and provided mixed in
the drinking water of these animals.
Fibrous Plaque - Raised nodular lesions, continuous, intense red, white hard
and visible to naked eyes
Fatty Plaque - Raised distinct lesions, intensely stained red
Fatty streak - Lipid accumulation, stained light red
Lesion Free - Healthy intima
Dosage and Formulation
Generally, the composition comprising OPP may be prepared in various suitable forms
for direct or oral administration for improving vascular heath, treating and preventing
vascular diseases and diseases related thereto
The compositions of the invention include those suitable for oral, rectal, optical, buccal
(for example, sublingual), parenteral (for example, subcutaneous, intramuscular,
intradermal and intravenous) and transdermal administration. The most suitable route in
any given case will depend on the nature and severity of the condition being treated and
the state of the patient,
Compositions suitable for oral administration may be presented in discrete units, such as
capsules, sachets, lozenges, or tablets, each containing a pre-determined amount of the
extract: as a powder or granules; as a solution or a suspension in an aqueous or non-
aqueous liquid. Such compositions may be prepared by any suitable method of pharmacy

which includes the step of bringing into association the extract of the present invention
and one or more suitable carriers (which may contain one or more accessory ingredients
as noted below). In general, the compositions of the invention are prepared by uniformly
and intimately admixing the extract with a liquid or finely divided solid carrier, or both,
and then, if necessary, shaping the resulting mixture. For example, a tablet may be
prepared by compressing or moulding a powder or granules containing the extract,
optionally with one or more accessory ingredients.
Compressed tablets may be prepared by compressing in a suitable machine, the extracts
in the form of a powder or granules, optionally mixed with a binder, lubricant, inert
diluents, and/or surface active/dispersing agent(s). Moulded tablets may be made by
moulding, in a suitable machine, the powdered compound moistened with an inert liquid
binder.
Suitable carriers may be fillers, such as sugars, for example lactose, saccharose, mannitol
or sorbitol, cellulose preparations and/or calcium phosphates, for example, tricalcium
phosphate or calcium hydrogen phosphate, and also binders, such as starch pastes using
for example, corn, wheat, rice, or potato starch, gelatin, tragacanth, methylcellulose
and/or polyvinylpyrrolidone and, if desired, disintegrators, such as the above-mentioned
starches, also carboxymelhyl starch, cross linked polyvinylpyrrolidone, agar or algin acid
or a salt thereof, such as sodium alginate.
Excipients may be flow conditioners and lubricants, for example silicic acid, talc, stearic
acid or salts thereof, such as magnesium or calcium stearate, and/or polyethylene glycol.
Dragee cores are provided with suitable, optionally enteric coatings, there being used,
inter alia, concentrated sugar solutions which may comprise gum Arabic, talc,
polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, or coating solutions in
suitable organic solvents or solvent mixtures, or for the preparation of enteric coatings,
solutions of suitable cellulose preparations, such as acetylcellulose phthalate or
hydroxypropylmethylcellulose phthalate. Dyes or pigments may be added to the tablets or
dragee coatings, for example, for identification purposes or to indicate different doses of
active ingredients.

Formulations suitable for buccal (sublingual) administration include lozenges comprising
the extracts in a flavoured base, usually sucrose and acacia or tragacanth; and pastilles
comprising the extract in an inert base such as gelatine and glycerin or sucrose and
acacia.
Compositions of the present invention suitable for parenteral administration conveniently
comprise sterile aqueous preparations of the extracts, which preparations are preferably
isotonic with the blood of the intended recipient. These preparations are preferably
administered intravenously, although administration may also be effected by means of
subcutaneous, intramuscular, or intradermal injection. Suitable compositions include
water soluble extracts and also suspensions of the active ingredient, such as
corresponding oily injection suspensions, there being used suitable lipophilic solvents or
vehicles, such as fatty oils, for example sesame oil, or synthetic fatty acid esters, for
example ethyl oleate or triglycerides, or aqueous injection suspensions comprising
viscosity-increasing substances, for example sodium carboxymethylcellulose, sorbitol
and/or dextran, and, where appropriate, also stabilizers. As an example, compositions
may conveniently be prepared by admixing the extracts with water or a glycine buffer
and rendering the resulting solution sterile and isotonic with the blood. Injectable
formulations according to the invention may contain from 0.1% to 60% of the extract and
may, for example, be administered at a rate of 0.1 ml/minute/kg.
Formulations suitable for rectal administration are preferably presented as unit dose
suppositories These may be prepared by admixing Ihe extracts with one or more
conventional solid carriers, for example cocoa butter, then reshaping the resulting
mixture.
Compositions suitable for transdermal administration may be presented as discrete
patches adapted to remain intact with the epidermis of the recipient for a prolonged
period of time. Such patches may contain the extracts in an optionally buffered aqueous
solution.

Compositions suitable for transdermal administration may also be delivered by
iontophoresis (see, for example, Pharmaceutical Research 3 (6): 318 (1986) and typically
take the form of an optionally buffered aqueous solution of the extracts. Such
compositions may, for example, contain citrate or bis/tris buffer (pH6) or ethanol/water,
with for example 0.05% to 30% w/w extract.
Compositions may be prepared in a manner, and in a form/amount as is conveniently
practiced. See, for example, Goodman & Gillman, The Pharmacological Basis of
Therapeutics (7th Edition, 1985) and Remington's Pharmaceutical Science (Mack
Publishing Company, 10th Edition), both of which are incorporated herein by reference.
Compositions may contain, for example, from 0.1 mg to 2g OPP such as 0.1 mg to 200mg,
more particularly 15mg to 50mg OPP.
The compositions of the invention may also be administered to a human in a dietary
supplement form. Dietary supplements incorporating the active composition can be
prepared by adding the composition to a food in the process of preparing the food. Any
food may be used including, but not limited thereto, meats such as ground meats,
emulsified meats and marinated meats; beverages such as nutritional beverages, sports
beverages, protein fortified beverages, juices, milk, milk alternatives, and weight loss
beverages; cheeses such as hard and soft cheeses, cream cheese, and cottage cheese;
frozen desserts such as ice cream, ice milk, low fat frozen deserts, and non dairy frozen
deserts; yoghurts; soaps; puddings; bakery products; salad dressings; and dips and
spreads such as mayonnaise, butter, butter substitute, and other fat containing spreads.
The composition is added to the food in an amount selected to deliver a desired dose of
the composition to the consumer of the food.
An effective amount of the compositions of the present invention is administered to a
human subject The actual dosage levels will depend upon the a number of factors, such
as specific mode of administration, the condition being treated, the condition of the
patient and the judgement of the health care giver. Examples of dosages of the

composition of the present invention are about 0.1 mg to about 200mg per day, such as in
the order of 1 5mg/kg (body weight)/day. A convenient dosage form contains about 25mg
to 50mg OPP as described herein
The composition comprising OPP of the present invention may be prepared for use in a
pharmaceutically effective or nutraceutically effective amount, solely on its own or in
combination with other agents or compounds deemed appropriate by a person skilled in
the art.
It is noted that the term 'pharmaceutically effective' and 'nutraceutically effective*
amount includes a quantification that is acceptable for improving vascular health, treating
and preventing vascular diseases and diseases related thereto.
In one embodiment the composition comprising OPP may be administered in form of
doses, within a predetermined period of time, whereby it may be administered for
example but not limiting to daily, weekly or monthly.
In another embodiment the composition comprising OPP may be provided in
conventional treatment forms, pharmaceutical formulations or as nutritional supplement.
In one embodiment the composition of the present invention may be provided in a
nutraceutical form.
The compositions according to the present invention may include one or more
pharmaceutically acceptable or nutraceutically acceptable carriers. Carriers are selected
so as to be acceptable in the sense of being ingredients in the composition and must not
be deleterious to the patient The carriers may be solid or a liquid, or both, and may be
formulated with an extract containing the OPP at the desired ratios as a unit does, for
example a tablet, which may contain from 0.5% to 80% by weight of the extract or up to
100% by weight of extract

Compositions may be prepared by any of the well known techniques of pharmacy, for
example admixing the extract, optionally including excipients, diluents (for example
water) and auxiliaries as are well known in the pharmaceutical field. The compositions
may include one or more agents, such as vitamins (for example, Vitamin A, Vitamin B
group, Vitamin C, Vitamin D, Vitamin E and Vitamin K), and minerals (for example
magnesium, iron, zinc, calcium and manganese in the form of pharmaceutically or
nutraceutically acceptable salts).
It is noted from the results of the studies referred herein as exemplifications evidently
show that the composition comprising OPP of the present invention have the following
properties: promoting vascular health, improving vascular integrity thus limiting
adherence of cells and build up of fatty plaques, inducing vasodilatation, reducing overall
oxidative burden, limiting and thus inhibiting the oxidation of LDL cholesterol, inhibiting
atheroma formation, lowering blood pressure, reducing cardiac and vascular hypertrophy,
improving renal function, providing cardioprotective effect, preventing atherogenesis,
and maintaining heart rhythm at normal rate in event that the blood flow is impaired or
disrupted.
It is understood by a person skilled in the art that the methods for experiments and studies
are described as exemplifications herein and thus the results 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, agents or starting materials which must be utilized
exclusively in order to practice the present invention. It is therefore understood mat the
invention may be practiced, within the scope of the appended claims, with equivalent
methods for the experiments than as specifically described and stated in claims.

We claim:
1. Use of a composition in promoting cardio vascular health wherein the composition
comprising water soluble extracts having components with molecular weights below 41,000
Daltons, obtained from vegetation liquor of a palm oil milling process.
2. Use of a composition in the treatment and/or prophylaxis of cardio vascular diseases,
wherein said composition comprising water soluble extract having components with
molecular weights below 41,000 Daltons, obtained from vegetation liquor of a palm oil
milling process.
3. The use as claimed in anyone of Claim 1 or 2, wherein the extract vegetation liquor is
from an oil palm fruit bunch which includes the fruit, spikelet and stalk.
4. The use as claimed in anyone of Claim 1 or 2, wherein the extract vegetation liquor is
from an oil palm frond.
5. The use as claimed in Claim 2, wherein the cardio vascular disease includes
hypertension and associated maladies, atherosclerosis and cardiac arrhythmias.
6 The use as claimed in anyone of Claim 1 or 2, in at least one of the following:
promoting vascular health, improving vascular integrity thus limiting adherence of cells
and build up of fatty plaques, inducing vasodilatation, reducing overall oxidative
burden, limiting and thus inhibiting the oxidation of LDL cholesterol, inhibiting atheroma
formation, lowering blood pressure, reducing cardiac and vascular hypertrophy,
improving renal function, providing cardio protective effect, preventing atherogenesis
and maintaining heart rhythm at normal rate in event that the blood flow is impaired or
disrupted.
7. The use as claimed in Claim 1 having at least one of the following properties:
increasing or promoting availability of NO and including vaso relaxation.

8. The use as claimed in Claim 1 wherein the composition is provided in dose dependent
manner.
9. The use as claimed in Claim 1 wherein the composition is provided in solid dosage
form.
10. The use as claimed in Claim 1 wherein the composition is provided in liquid dosage
form.
11. The use as claimed in Claim 1 wherein the composition is provided in solution form.
12. The use as claimed in Claim 1 wherein the composition is provided in a
pharmaceutically effective form.
13. The use as claimed in Claiml wherein the composition is provided in nutraceutical
form.
14. The use as claimed in Claim 1 wherein the composition is in nutritional supplementary
form.
15. The use as claimed in Claim 1 wherein the composition is in suppository form.
16. The use as claimed in Claim 1 wherein the composition is provided in a form suitable
for oral administration.
17. The use as claimed in Claim 1 wherein the composition is provided in a form suitable
for intravenous administration.

18. A method for preparing a food ingredient for promoting cardio vascular health and
protecting against cardio vascular disease, characterized in that, said food ingredient
consists of a water soluble extract having components with molecular weights below
41,000 daltons obtained from vegetation liquor of a palm oil milling process.
19. A method of promoting cardio vascular health in a mammal, comprising administering
a composition comprising water soluble extract obtained from vegetation liquor of
palm oil milling process, characterized in that the extract comprises components with
molecular weights below 41,000 Daltons.
20. The method for treating and/or prophylaxis of cardio-vascular disease in humans and
animals by administering an effective dose of a water soluble extract having
components with molecular weights below 41,000 daltons obtained from vegetation
liquor of a palm oil milling process.
21. Use of a water soluble extract having components with molecular weights below
41,000 daltons obtained from vegetation liquor of a palm oil milling process in the
manufacture of a medicament for promoting vascular health in a human and animal.

ABSTRACT

The present invention relates to a composition for the use in improving vascular health,
including treating and preventing vascular diseases and diseases related thereto, using a
botanical extract of oil palm vegetation liquor from the palm oil milling process.