U.S. patent application number 12/670398 was filed with the patent office on 2010-11-04 for anti viral composition.
Invention is credited to Ravigadevi Sambanthamurthi, Kalyana Sundram, Yew Ai Tan.
Application Number | 20100278943 12/670398 |
Document ID | / |
Family ID | 40281998 |
Filed Date | 2010-11-04 |
United States Patent
Application |
20100278943 |
Kind Code |
A1 |
Sambanthamurthi; Ravigadevi ;
et al. |
November 4, 2010 |
ANTI VIRAL COMPOSITION
Abstract
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.
Inventors: |
Sambanthamurthi; Ravigadevi;
(Selangor Darul Ehsan, MY) ; Sundram; Kalyana;
(Selangor Darul Ehsan, MY) ; Tan; Yew Ai; (Kuala
Lumpur, MY) |
Correspondence
Address: |
FOLEY & LARDNER LLP
P.O. BOX 80278
SAN DIEGO
CA
92138-0278
US
|
Family ID: |
40281998 |
Appl. No.: |
12/670398 |
Filed: |
July 23, 2008 |
PCT Filed: |
July 23, 2008 |
PCT NO: |
PCT/MY08/00075 |
371 Date: |
June 21, 2010 |
Current U.S.
Class: |
424/727 |
Current CPC
Class: |
A61K 31/047 20130101;
A61K 31/192 20130101; A61P 31/12 20180101; A61P 31/18 20180101;
A61K 36/889 20130101; A61K 31/353 20130101 |
Class at
Publication: |
424/727 |
International
Class: |
A61K 36/889 20060101
A61K036/889; A61P 31/18 20060101 A61P031/18 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 23, 2007 |
MY |
PI20071192 |
Claims
1-7. (canceled)
8. 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.
9. The composition as claimed in claim 8 wherein the composition is
for use in inhibiting replication of Hepatitis C virus (HCV) and
thereby preventing HCV infections.
10. The composition as claimed in claim 8 wherein the extracts from
oil palm vegetation liquor comprises phenolic compounds, fruit
acids, fruit sugars and glycerol.
11. The composition as claimed in claim 8 wherein they may be
provided as compounds with pharmaceutically acceptable
carriers.
12. A method of preventing HIV or HCV infections in a patient, said
method comprising administering a therapeutically effective amount
of a composition of claim 8 to said patient.
13. A method of preventing HCV infection in a patient, said method
comprising administering a therapeutically effective amount of a
composition of claim 8 to said patient.
14. The method of claim 12, wherein the composition is administered
orally or as supplements.
15. The method of claim 13, wherein the composition is administered
orally or as supplements.
16. The method of claim 12, wherein the composition is administered
together with additives.
Description
FIELD OF INVENTION
[0001] 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
[0002] 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.
[0003] 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 heighten the role of agriculture in the
context of producing immunity against illnesses.
[0004] 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.
[0005] 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.
[0006] 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.
[0007] 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.
[0008] An exemplary of a composition which is related to the
treatment of AIDS is as disclosed in U.S. Pat. No.
5,529,778--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
[0009] 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.
[0010] 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.
[0011] 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.
[0012] 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.
[0013] 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.
[0014] 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.
[0015] 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.
[0016] 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
[0017] 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.
[0018] The composition of the present invention may be provided
with pharmaceutically acceptable carriers.
[0019] Combination therapies involving HIV protease inhibitors and
HIV reverse transcriptase inhibitors are the cornerstones of
currently recommended therapies for HIV infection. In accordance
with one embodiment of the present invention, 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.
[0020] 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.
[0021] 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.
[0022] 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
[0023] 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.
[0024] 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.
[0025] 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
[0026] 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.
[0027] The said isolation method may consist of extracting the
crude oil palm product, with a predetermined yield rate.
[0028] 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
[0029] 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 FIG. 2 of the present
invention.
Methodologies
Methodology-Flash Chromatography
[0030] 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.
[0031] 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
[0032] 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 .mu.g 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
[0033] 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 transcriptase is used in the assay.
[0034] 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.
Roche.TM. Reverse Transcriptase Assay, Colorimetric
[0035] 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).times.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)
[0036] 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 FIG. 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.
[0037] The inhibitory action of the four fractions from flash
chromatography is shown in FIG. 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.
[0038] 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 FIG. 4. Grape seed extract did not
have any inhibitory effect on reverse transcriptase activity.
Roselle however showed activity at 200 ppm gallic acid
equivalent.
Molecular Probes EnzChek.RTM. Reverse Transcriptase Assay Kit
(E-22064)
[0039] Molecular Probes EnzChek.RTM. 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.
[0040] 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.
[0041] 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).
[0042] 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.
[0043] 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.TM. 620 HCV Protease Assay
[0044] The EnzoLyte.TM. 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.
[0045] The kit utilized a HiLyte Fluor.TM. TR/QXL.TM. 610
fluorescence resonance energy transfer (FRET) peptide substrate
which could be monitored at Ex/Em=591 nm/622 nm upon proteolytic
cleavage. The FRET peptide was derived from the cleavage site of
NS4A/NS4B. In the FRET peptide, the fluorescence of HiLye Fluor.TM.
was quenched by QXL.TM.610. Upon cleavage into two separate
fragments by the HCV NS3/4A protease, the fluorescence of
HiLyteFluor.TM. was recovered and monitored at
excitation/emission=591/622. Inhibitors would thus reduce the
fluorescence.
[0046] The detailed experimental protocol was carried out according
to manufacturer's instructions.
[0047] All the extracts showed significantly higher protease
inhibitory action compared to even the commercial inhibitor
(inhibitor) supplied with the kit.
The SensoLyte.TM. 520 HIV Protease Assay
[0048] The SensoLyte.TM. 520 HIV Assay kit used a new FRET peptide
substrate that incorporated HiLyte Fluor.TM. 488 (fluorophore) and
QXL.TM. 520 (quencher) for measurement of enzyme activities. In the
intact FRET peptide, the fluorescence of HiLyte Fluor.TM. 488 was
quenched by QXL.TM. 520. Upon cleavage of the FRET peptide by HIV
protease, the fluorescence of HiLyte Fluor.TM. 488 was recovered
and monitored at excitation/emission=490 nm/520 nm
[0049] The detailed experimental protocol was carried out according
to manufacturer's instructions.
[0050] 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
[0051] 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.
[0052] The detailed experimental protocol was carried out according
to manufacturer's instructions.
[0053] 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
[0054] 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.
[0055] The conducted experiments using five different methodologies
have shown that the compositions having but not 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
[0056] 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.
[0057] 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.
* * * * *