Field of Invention
The present invention relates to a composition enriched but not
confined to phenolic antioxidants obtained from a botanical
extract of oil palm vegetation liquor obtained from the palm oil
milling process, and more particularly this invention relates to
a composition for promoting immunity in general and most
essentially against Human Immunodeficiency Virus (HIV) related
infections.
Background of Invention
Over the past decade, scientific research has provided increasing
evidence that crude extracts of plants may provide lead molecules
in relation to drugs for a variety of critical ailments.
Accordingly, substantial progress has been made in this regard in
order to further determine and thus provide a natural origin
solution to illnesses.
In plants, phenolics and flavonoids are the most abundant and
ubiquitous products of secondary plant metabolism. Under normal
circumstances, they are often used by plants as a defense
mechanism against animal predation and the like. Additionally,
recent studies have begun to examine and heightened the role of
agriculture in the context of producing immunity against
illnesses.
One of such numerous illnesses that can be regarded as the
biggest challenge in the medical science field is AIDS which is
widely known to be caused by the virus HIV [Human Immuno-
deficiency Virus], which relates to the disruption of the immune
system. Evidently, many have lost their lives due to this
illness. At present time, massive amount of research has been

conducted to determine suitable drugs for the treatment of AIDS.
Naturally, plant-derived medicines comprising phenolics and
antioxidants are one of the most highly utilized categories of
drugs and thus have been taken into account in the possibility of
treating AIDS.
The HIV virus causing AIDS belongs to the retrovirus group of
viruses. Retroviruses are widespread in nature and can be
classified according to biological property, morphology and
genome size. Retroviruses are characterized by the presence of
the enzyme reverse transcriptase in the virions. Reverse
transcriptase is required for the unique retroviral type of
multiplication. After entering the cell, the uncoated viral RNA
is transcribed to double stranded DNA which is then incorporated
into the DNA of the host cell as a provirus. Reverse
transcriptase is required for early proviral DNA synthesis and is
therefore a prime target for anti-retroviral therapy including
AIDS.
Prior art of similar purpose includes ayurvedic compositions,
wherein they comprise pre-determined amount of plant extracts
and compositions which consist of a combination of well known
medicinal plants or herbs, for example ginseng, Radix Astragali
and the like. Apart from the above, other solutions comprise
pharmaceutically active compounds, having inhibitors.
Nevertheless, among these prior arts, more often than not, in the
case where inhibitors are the main compounds, there are
circumstances where the virus develop resistance against these
inhibitors, and therefore will defeat the whole purpose of the
compounds, in which this can be regarded as the main drawback in
using inhibitors to disrupt further propagation of the said
virus.

An exemplary of a composition which is related to the treatment
of AIDS is as disclosed in United States Patent Number 5529778 -
Ayurvedic Composition for the Prophylaxis and Treatment of AIDS,
Flu, TB and Other Immuno-Deficiencies and The Process Preparing
the Same (Surendra Ruhatgi et al.) which suggests a composition
containing the isolates and extracts of a variety of herbs, said
herbs including Phyllantus niruri, Tinosfora cordifolia,
phyllantus emblica, Terminalia beleria and Terminalia cherbulia.
Accordingly, the said composition stimulates the physiological
functions of the body for the treatment of AIDS. Evidently, the
essential compound of this invention is the extracts or isolates
from a plurality of herbs, which is distinctive from the
essential compounds of the present invention
Apart from the above, there have been numerous efforts and
research conducted to seek for effective broad spectrum antiviral
drugs. However, there have yet to be any antiviral drugs
successfully developed for treating mixed viral infections which
are as well non-toxic. Considering the appalling effect of mixed
viral infections which therefore lead to AIDS, it is highly
necessary to develop a non-toxic and effective broad spectrum
antiviral drug.
In regards to plant derivatives, previous studies have revealed
that coconut oils may provide the superiority function of
antiviral effects. In accordance to their disclosures, the
content of lauric acid which is formed into monolaurin in human
or animal body may provide the ability to eliminate virus
including HIV, herpes, cytomegalovirus, influenza and a variety
of pathogenic bacteria.
Another eminent exemplary of a plant having extremely beneficial
health properties is palm fruit. The oil palm contains compounds
which can be considered as highly effective in the treatment of
serious illnesses. Recent discoveries in regards to further

beneficial health properties of phenolics and antioxidants which
are eminently found in oil palm include providing treatments for
cancer, and for eliminating skin problems.
The present invention provides a new approach in using oil palm,
particularly in providing a treatment of AIDS by way of a
composition that promotes immunity against HIV infection.
Consequently, primary object of the present invention to provide
a composition based on a botanical extract of oil palm vegetation
liquor obtained from the palm oil milling process for promoting
immunity against HIV virus infection and thus for treatment of
AIDS.
It is a further object of the present invention to provide an
improved composition and method for producing a broad spectrum
antiviral which is non-toxic.
It is further the object of the present invention to provide an
improved composition and formulation for producing a composition
containing antioxidants and phenolics obtained from oil palm
devoid of chemically prepared adverse drug reactions in a patient
in need thereof.
It is a further object of the present invention to provide a
composition and formulation for producing a composition
containing but not limited to antioxidants and phenolics from
botanical extract of oil palm vegetation liquor obtained from the
palm oil milling process having anti-viral effects.
Summary of Invention
The present invention relates to an antiviral composition
comprising antioxidants extracted from the vegetation liquor of

the palm oil milling process for promoting immunity and for
providing inhibition against HIV infections.
The composition of the present invention may be provided with
pharmaceutically acceptable carriers.
In another embodiment of the present invention, there is provided
combination therapies involving HIV protease inhibitors and HIV
reverse transcriptase inhibitors are the cornerstones of
currently recommended therapies for HIV infection. The botanical
extract of vegetation liquor from the palm oil milling process as
well as its purified fractions have potent inhibitory action
against both HIV protease and reverse transcriptase as well as
hepatitis C (HCV) protease.
The present invention further relates to the use of
therapeutically effective amount of a composition in the
preparation of a medicament for promoting immunity and for
providing inhibition against HIV and HCV infections in a patient
in need thereof, wherein the composition may be administered
orally or as supplements or with additives.
The term "phenolics" refers to a class of compounds grouped
together due to their chemical structure wherein in this document
is extracted from vegetation liquor of the palm oil milling
process.
The term "flavonoids" refers to naturally occurring chemicals in
plants, wherein in this document is extracted from vegetation
liquor of the palm oil milling process
Detailed Description of the Present Invention
As mentioned, the present invention provides a composition based
on but not confined to the antioxidants and phenolics obtained

from vegetation liquor of the palm oil milling process promoting
immunity against HIV virus infection and thus for treatment of
AIDS.
Accordingly, the present invention is to provide a broad spectrum
antiviral composition which comprises but is not limited to
antioxidants and phenolics obtained from vegetation liquor of the
palm oil milling process. Essentially, the composition comprising
antioxidants, phenolic acids and flavonoids and the usage of the
said composition in a medicament for providing immunity against
and thus treatment of mixed viral infections.
The preparation of composition formulations according to the
present invention are described in detail by referring to the
experimental examples. However, the present invention is not
limited to these examples.
Methodology and Assay Design
Extraction
In accordance to a preferred embodiment of the present invention,
the method and preparation of the antiviral composition comprise
the steps of isolating the main compounds. The main compounds or
extract of the composition of the present invention can be
prepared in a variety of appropriate ways, however should not be
considered as limiting the scope of invention, but merely as
being illustrative. The isolated extracts mainly comprise
phenolic compounds, fruit acids, fruit sugars and glycerol from
the vegetation liquor from the palm oil milling process and the
prepared formulations containing these extracts. Isolation
methods include, and not limiting to, filtration, distillation,
crystallization and flash column chromatography. The main
requirement for the isolation is highly pure and concentrated
samples. The preferred isolation method in accordance to the

present invention is membrane filtration employing molecular
weight cut-off sieves. The resultant filtrate (botanical
extract) is further separated using flash column chromatography.
The said isolation method may consist of extracting the crude oil
palm product, with a predetermined yield rate.
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.
In addition to direct use of an extract, it is also possible to
use different fractions of the oil palm phenolic compounds. What
constitutes an effective amount of an extract, or an active
portion thereof, will depend on the purity of the extract
In accordance to the present invention, and for the purpose of
comparison, the preparations of extracts are preferably in the
liquid and dried forms as shown in Figure 2 of the present
invention.
Methodologies
Methodology-Flash Chromatography
The botanical extract of oil palm vegetation liquor obtained from
the palm oil milling process as described in US Patent
Application No. 20030031740 (Sambanthamurthi, Tan and Sundram
2004) were partially purified using flash chromatography and
preparatory high pressure liquid chromatography using among
others an Advanced Protein Purification System purchased from

Waters, USA). The extract was injected into a column and then
eluted with a methanol gradient, ranging from 0% to 100%. The
phenolic compounds were eluted in accordance with their
solubility in methanol.
Samples from different pooled fractions were concentrated using a
vacuum concentrator (Oligo Prep model OP120 purchased from
Savant, USA) before being analysed for activity.
Detailed Flash Chromatography Method
Four mL of extract were injected into a flash column (C18; 20
g/70 ml purchased from Jones Chromatography, USA) . The flow rate
was set at 5 mL/min. The column was eluted with water for 5 min.
Then, a linear gradient of methanol (0-100%) was applied for 25
min. The column was eluted a further 5 min with 100% methanol to
ensure the complete removal of the sample from the column. The
eluates were collected in test tubes (2.5 mL in each test tube)
and assayed for phenolic content. The phenolic content in the
samples were expressed as µg gallic acid equivalent (GAE) per mL
of sample or in parts per million (ppm) GAE. The eluates were
pooled into four fractions as shown in Fig 1.

Methodology-Reverse Transcriptase Assay: Inhibitor Determination
The preferred assay system for analyzing the human
immunodeficiency virus (HIV) replication activity for particle
associated in accordance to the present invention is the Reverse
Transcriptase system. According to studies in the relevant
field, the viral activity can be determined by way of a Reverse
Transcriptase Assay. Inhibition of reverse transcriptase is
thus indicative of anti-viral and more specifically anti-HIV
activity when HIV reverse trancriptase is used in the assay.
Two different commercial Reverse Transcriptase Assay Kits were
used to confirm the anti-HIV properties of the botanical extract
of oil palm vegetation liquor obtained from the palm oil milling
process.
RocheT Reverse Transcriptase Assay, Colorimetric
This is a colorimetric enzyme immunoassay for the quantitative
determination of retroviral reverse transcriptase activity by
incorporation of dioxigenein- and biotin-labeled dUTP into DNA.
The Reverse Transcriptase Assay, colorimetric takes advantage of
the ability of reverse transcriptase to synthesize DNA, starting
from the template/primer hybrid poly (A) x oligo (dT)15.
Digoxigenin- and biotin-labeled nucleotides in an optimized ratio
are incorporated into one and the same DNA molecule, which is
freshly synthesized by the RT. The detection and quantification
of synthesized DNA as a parameter for RT activity follows a
sandwich ELISA protocol: Biotin-labeled DNA binds to the surface
of microtiter plate (MTP) modules that have been precoated with
streptavidin. In the next step, an antibody to digoxigenin,
conjugated to peroxidase (anti-DIG-POD), binds to the
digoxigenin-labeled DNA. In the final step, the peroxidase
substrate ABTS is added. The peroxidase enzyme catalyzes the
cleavage of the substrate, producing a colored reaction product.
The absorbance of the samples can be determined using a
microtiter plate (ELISA) reader and is directly correlated to the
level of RT activity in the sample. For quantification of the
inhibitory effect of the botanical extract, a reverse
transcriptase, HIV-1 recombinant was used in conjunction with the
Reverse Transcriptase Assay, Colorimetric. The assay was carried
out according to manufacturers' instructions. (Cat. No. 1 468
120)
Accordingly, the extract was prepared at various concentrations
in both the dried and aqueous form. The four fractions from
flash chromatography were also prepared with varying
concentrations. Referring to Figure 2 of the present invention,
both the dried and aqueous extracts exhibited significant
inhibitory action against HIV-1 reverse trancriptase. The
activity was dose-dependent with both the dried and aqueous
extracts at 1500 ppm gallic acid equivalent exhibiting more than
90% inhibition in activity as seen by the decrease in absorbance
at these concentrations.

The inhibitory action of the four fractions from flash
chromatography is shown in Figure 3. Fractions 3 and 4 showed the
highest inhibitory effect. These fractions are high in cinnamates
and catechin. Fraction 2 which is rich p-hydroxybenzoic acid also
showed significant inhibitory effect.
The inhibitory effect of oil palm extract was compared with other
phenolic extracts such as roselle and grape seed extract (GSE).
The results are shown in Figure 4. Grape seed extract did not
have any inhibitory effect on reverse transcriptase activity.
Roselle however showed activity at 200 ppm gallic acid equivalent.
However, this activity was not as potent as that shown by oil palm
extract.

Molecular Probes EnzChek R Reverse Transcriptase Assay Kit ( E-
22064)
Molecular Probes EnzChek R Reverse Transcriptase Assay uses Pico
Green dsDNA quantitation reagent which preferentially detects
dsDNA or RNA-DNA heteroduplexes over single-stranded nucleic
acids or free nucleotides. In this assay reverse trancriptase
generates long RNA-DNA heteroduplexes from a mixture of a long
poly (A) template, an oligo-dT primer and DTTP. The RNA-DNA
heteroduplexes formed are then detected by the PicoGreen reagent.
For determining inhibitory action against reverse transcriptase
activity, a fixed amount of reverse transcriptase is incorporated
in the assay and the reduction in activity quantified. The
detailed experimental protocol was carried out according to
manufacturer's instructions.

The results using this method again showed potent inhibitory
action against reverse transcriptase activity. At 1000 ppm
gallic acid equivalent, reverse transcriptase activity was almost
completely inhibited. Even with the concentration as low as 250
ppm has exhibited about 80% inhibition of reverse transcriptase
activity.
In virus systems, proteases are enzymes that aid in the
maturation of other viral proteins, making them functional. For
example, without the work of protease, HIV will not mature
properly and therefore will not be able to make copies of itself
and infect other cells. This is also true for other virus such as
the Hepatitis C virus (HCV).
Inhibition of protease activity is indicative of anti-viral
activity. Protease Inhibitors are medications that block the
functioning of the enzyme protease. Without functioning
protease, HIV and HCV are unable to mature and therefore cannot
make more copies of themselves thus lowering the viral loads
carried by AIDS and HCV patents.
Three different commercial Protease Assay Kits were used to
confirm the antiviral properties of the botanical extract of oil
palm vegetation liquor obtained from the palm oil milling
process.
EnzoLyte™ 620 HCV Protease Assay
The EnzoLyte™ 620 HCV Protease Assay kit which is optimized for
detecting the activity of hepatitis C virus NS3/4A protease was
used to test the inhibitory effect of the extract. The NS3/4A
protease of hepatitis C virus (HCV) is required for the cleavage
of viral nonstructural polyprotein at the NS3-NS4A, NS4A-NS4B,

NS4B-NS5A sites. Three cleavage sites are essential for the
maturation of the viral proteins. This protease is one of the
key targets for developing anti-HCV drugs.
The kit utilized a HiLyte Fluor™ TR/QXL™ 610 fluorescence
resonance energy transfer (FRET) peptide substrate which could be
monitored at Ex/Em=591nm/622nm upon proteolytic cleavage. The
FRET peptide was derived from the cleavage site of NS4A/NS4B. In
the FRET peptide, the fluorescence of HiLye Fluor T was quenched
by QXL1M610. Upon cleavage into two separate fragments by the HCV
NS3/4A protease, the fluorescence of HiLyteFluor™ was recovered
and monitored at excitation/emission=591/622. Inhibitors would
thus reduce the fluorescence.
The detailed experimental protocol was carried out according to
manufacturer's instructions.

Fig.4A Protease Activity of Hepatitis C Virus NS3/4A protease in
the Presence of Botanical Extract of Oil Palm Milling Process(fil
1k ), Fractions 3 and 4 and a commercial preparation of black tea
all at concentrations of 1000 ppm stock solution.

Fig 4B Protease Activity of Hepatitis C Virus NS3/4A protease in
the Presence of Botanical Extract of Oil Palm Milling Process(fil
1k ) Fraction 3 (F3) and Fraction 4 (F4) and a commercial
preparation of black tea (Tea)at concentrations of 1000(1k) or
2000(2k) ppm stock solution. Positive control refers to protease
assay without any inhibitors
All the extracts showed significantly higher protease inhibitory
action compared to even the commercial inhibitor (inhibitor)
supplied with the kit.
The SensoLyte™ 520 HIV Protease Assay
The SensoLyte™ 520 HIV Assay kit used a new FRET peptide
substrate that incorporated HiLyte Fluor™ 488 (fluorophore) and
QXL™ 520 (quencher) for measurement of enzyme activities. In the
intact FRET peptide, the fluorescence of HiLyte Fluor™ 488 was
quenched by QXL™ 520. Upon cleavage of the FRET peptide by HIV
protease, the fluorescence of HiLyte Fluor™ 488 was recovered and
monitored at excitation/emission = 490 nm/52 0 nm
The detailed experimental protocol was carried out according to
manufacturer's instructions.
As in the HCV protease assay, the botanical extract of vegetation
liquor from the palm oil milling process showed significantly
higher inhibitory action against HIV protease.
EnzChek Protease Assay
The Molecular Probes' EnzChek Protease Assay Kit was used to
further test for protease inhibitory action of oil palm extracts.
The kit used a fluorescence-based assay for detecting metallo-,
serine and sulfydryl proteases. It contained casein derivatives
which were heavily labeled with the pH-insensitive green
fluorescent BODIPY FL or red-fluorescent BODIPY TR-X dyes
resulting in almost total quenching of the conjugate's
fluorescence. Protease-catalysed hydrolysis released highly
fluorescent BODIPY FL or BODIPY TR-X dye-labeled peptides. The
accompanying increase in fluorescence was measured in a
microplate reader. Inhibitors thus would decrease the
fluorescence. The inhibitory action of botanical extracts of
vegetation liquor of the palm oil milling process as well as
partially purified fractions on various proteases including but
not confined to chymotrypsin (serine protease). Pepsin (acid
protease) Elastase (metalloprotease) was investigated using the
EnzChek Protease Assay.
The detailed experimental protocol was carried out according to
manufacturer's instructions.
In all cases significant inhibitory action was observed and Fig 5
shows the results obtained using the protease chymotrypsin and
casein derivative labeled with green fluorescent BODIPY FL dye as
substrate

Fig. 5A Protease Activity in the Presence of Botanical Extract of
Oil Palm Milling Process (Fil 2000 ppm ) Fraction 3 (F31k) and
Fraction 4(F41k) all at concentrations of 2000 ppm stock
solution. Positive control refers to protease assay without any
inhibitors. Inhibitor refers to the commercial inhibitor
provided with the kit

Fig. 5B Protease Activity in the Presence of Botanical Extract of
Oil Palm Milling Process (Fil 2000 ppm ) Fraction 3 (F31k) and
Fraction 4 (F41k) all at concentrations of 2000 ppm stock
solution. Positive control refers to protease assay without any
inhibitors. Inhibitor refers to the commercial inhibitor provided
with the kit
The botanical extract of palm and the partially purified
fractions showed significantly higher protease inhibitory action
compared to even the commercial inhibitor (inhibitor) supplied
with the kit.
The conducted experiments using five different methodologies have
shown that the compositions having but confined to the said oil
palm phenolic compounds can effectively inhibit various viruses,
further supporting oil palm compounds as one of the broad
spectrum antiviral drugs. The results also indicate that roselle
and tea extracts also possess inhibitory action against reverse
transcriptase suggesting that the extracts from palm can be used
on their own or in combination with roselle and tea extracts for
anti-viral function. Current HIV and HCV therapies are often
contraindicated or poorly tolerated, underscoring the need for
safer and more effective drugs. The botanical extracts of
vegetation liquor of the palm oil milling process as well as
partially purified fractions showed potent inhibition against HIV
reverse transcriptase and protease and HCV protease in vitro and
hence are potentially useful for development of future antiviral
therapies including hepatitis C virus (HCV) therapies.
Dosage and Administration
In accordance to a preferred embodiment of the present invention,
the compounds of the present invention may be formulated in a
wide variety of oral administration dosage form and carriers. It
can be in the form of tablets, capsules, solutions, emulsions,
syrups or suspensions. Nevertheless, composition of the present
invention may be efficacious when used together with one or more
additives in conventional proportions.
The foregoing invention has been described in some detail by way
of examples; however it will be obvious to one skilled in that
changes and modifications may be practiced within the scope of
the appended claims.

We Claim:
1. A composition comprising extracts from oil palm vegetation
liquor for use in inhibiting the replication of human
immunodeficiency virus (HIV) and thereby preventing HIV
infections; characterized in that the composition provides
inhibitory action against protease and reverse
transcriptase activity.
2.The composition as claimed in Claim 1 wherein the
composition is for use in inhibiting replication of
Hepatitis C virus (HCV) and thereby preventing HCV
infections.
3.The composition as claimed in Claim 1 wherein the extracts
from oil palm vegetation liquor comprises phenolic compounds,
fruit acids, fruit sugars and glycerol.
4. The composition as claimed in Claim 1 wherein they may be
provided as compounds with pharmaceutically acceptable
carriers.
5. Use of therapeutically effective amount of a composition as
claimed in Claim 1 in the preparation of a medicament for
prevention of HIV and HCV infections in a patient in need
thereof.
6. Use of therapeutically effective amount of composition as
claimed in Claim 1, in the preparation of a medicament for
preventing HCV infections.
7. The use as claimed in Claims 5 and 6, wherein the
composition may be administered orally or as supplements.
8. The use as claimed in Claims 5 wherein the composition may
be administered together with additives.

The present invention relates to a composition enriched in but not confined to
phenolic antioxidants obtained from oil palm, and more particularly this invention
relates to a composition for promoting immunity in general and most essentially
against Human Immunodeficiency Virus (HIV) and HCV related infections.
Accordingly, the composition having the said oil palm compounds can effectively
inhibit various viruses, further supporting oil palm compounds as one of the broad
spectrum antiviral drugs.