FIELD
The present invention relates generally to a composition comprising at least 5 major
compounds which include the isomers of caffeoylshikimic acid, para hydroxybenzoic acid
protocatechuic acid and hydroxytyrosol and a method for preparing the same from oil palm
including but not confined to the vegetation liquor of palm oil milling. This includes the
separation and purification of caffeoylshikimic acid from the aqeous vegetation liquor.
BACKGROUND
Shimikic acid and its derivatives, found naturally in some plants play a significant role as a
biochemical intermediate compound in plants and also microorganisms. It is known to be
an imperative precursor for the synthesis of aromatic amino acids, phenolics and alkaloids
amongst many others. One of the most pertinent advancements in relation to the
exploitation of shikimic acids in the pharmaceutical industry is the production of Tamiflu,
a type of drug for use against avian flu in the recent years.
The demand for shikimic acid is expected to increase dramatically with the increase in
world population and thus the need for various industrial and pharmaceutical uses e.g in
the use of shikimic acid to manufacture tamiflu or other anti-viral drugs in the event of
pandemic flu outbreak. At present, the world's demand for shimikic acid is met from
fruits of Chinese star anise; however it is generally found in substantially low
concentrations. The low availability of star anise has hampered the production of tamiflu
and stresses the need for other sources of shikimic acid. Accordingly, it would be
desirable to explore other sources for shikimic acid so as to aid in fulfilling the global

demand. Oil palm including the vegetation liquor of palm oil milling offers a source of
shikimic acid.
It is known that oil palm is the most important commercial crop in Malaysia and several
countries in South East Asia. It has been identified that phenolic compounds extracted
from oil palm exhibit diverse health benefits. Thus efforts are being made to further
explore these compounds and other extracts of oil palm, including the vegetation liquor of
the milling process and palm oil mill effluent (POME) for the development of functional
foods, medical, nutraceutical and pharmaceutical preparations in recent times.
This invention focuses on realizing the value and potential of the vegetation liquor from
palm oil milling and palm oil mill effluent (POME) as a source as shikimic acid , and
further the possibility of using extracts from oil palm, the vegetation liquor from palm oil
milling and and POME as functional foods, medical, nutraceutical and pharmaceutical
preparations.
SUMMARY OF INVENTION
In one embodiment, the present invention relates to a composition of at least 5 major
phenolic compounds including but not confined to isomers of caffeoylshikimic acids,
hydroxy benzoic acids protocatechuic acids and hydroxytyrosol from any part of oil palm,
the vegetation liquor from palm oil milling and and palm oil mill effluent.
In another embodiment, the present invention relates to a method for the separation and
purification of a composition containing caffeoylshikimic acids and their derivatives. The
said method comprises the steps of pre-concentrating an extract containing said
caffeoylshikimic acids and their derivatives and then isolating said caffeoylshikimic acids
and their derivatives from said extract by preparatory liquid chromatography. The elution
time of the fraction comtaining said caffeoylshikimic acids could vary depending on the
conditions of the mobile and stationary phases.

BRIEF DECSRIPTION OF THE FIGURES
This invention will be described by way of non-limiting embodiments of the present
invention, with reference to the accompanying drawings, in which:
FIG 1 shows a chromatogram containing peaks 6, 7 and 8 obtained based on an
embodiment of the present invention;
FIG 2 (a) to FIG 2 (c) show the analytical chromatogram for peaks 6, 7 and 8 respectively
obtained based on an embodiment of the present invention;
FIG 3 shows the MALDI spectra of peaks 6, 7 and 8;
FIG 4 shows the UV spectra for peaks 6, 7 and 8;
FIG 5 shows caffeoylshikimic acid, or a 3,4-dihydroxycinnamoyl ester of shikimic acid;
FIG 6 shows the carbon and proton chemical shifts;
FIG 7 shows the selected HMBC correlations;
FIG 8 shows the marker compounds; and
FIG 9 shows the HPLC profile (fingerprint) of the composition.
DETAILED DESCRIPTION
In line with the above summary, the disclosed description and examples is directed to a
composition, use and method thereof for use various health related purposes, in the form of
treatments and/or prophylaxis of diseases using extracts of oil palm and from the
vegetation liquor of the palm oil milling process.
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 based on, but
not confined to the steps involved in the method as disclosed in US Patent Application No
20030031740.

Although the extract may contain a variety of compounds including phenolic compounds,
fruit acids, fruit sugars and glycerol, starch, cellulose and hemicellulose , for purposes of
standardization the concentrations of the extracts used were measured in terms of phenolic
content i.e gallic acid equivalent.
Embodiments of the present invention are predicated in part on a composition comprising
caffeoylshikimic acids and other major phenolic compounds obtained from any part of the
oil palm including vegetation liquor of palm oil processing and palm oil mill effluent. The
composition of the present invention is prepared in accordance with several steps of a
method, which is another aspect of the present invention. It is expected that the preparation
is safe and said composition is suitable for use in, but not limiting to, daily consumption
including dietary supplements, nutraceuticals, health promoting purposes, therapeutic
applications, immunostimulating, immune system enhancing, neuroprotective, anticancer,
antiflammatory, antioxidant and as antiaging agents. It is further noted that the
caffeoylshikimic acids and their derivatives obtained based on the preferred embodiments
of the present invention is suspected to exhibit vascular protective and antidiabetic effects
and to lower blood pressure.
It would be apparent to a person skilled in the art that the raw extracts obtained from any
part of the oil palm, the vegetation liquor from palm oil milling and and palm oil mill
effluent for the purpose of the present invention may contain various other phenolic
compounds in addition to primary marker compound caffeoylshikimic acids, these
compounds may include hydrobenzoic acid, hydroxytyrosol, gallic acids, protocatechuic
acid, 2,3-dihydroxybenzoic acid, chlorogenic acid, caffeic acid, ferulic acid, flavonoids,
micronutrients and other natural plant components including cellulose and hemicellulose,
starch, sugar, lipids, amino acids and proteins.
Caffeoylshikimic acids and their derivatives are found in low concentration in nature. The
method of the present invention aims to provide caffeoylshikimic acids and their
derviatives in substantially purified form for the preparation of the composition of the
present invention.

The extracts obtained form oil palm including the vegetation liquor from palm oil milling
and and palm oil mill effluent when subjected to isolation and purification stages in
accordance with the method of the present invention is found to contain at least one of the
following compounds; hydroxytyrosol, p-hydroxybenzoic acid, 5-0-caffeoylshikimic acid,
4-0-caffeoylshikimic acid and 3-0-caffeoylshikimic acid.
The present invention extends, therefore to a method for preparation of purified
caffeoylshikimic acids, whereby the primary steps of said method are pre-treatment of raw
extracts obtained from any part of the oil palm, the vegetation liquor from palm oil milling
and and palm oil mill effluents and the isolation of caffeoylshikimic acids from said raw
extracts. This embodiment encompasses isolated or substantially purified caffeoylshikimic
acids. It should be noted that an "isolated" or "purified" caffeoylshikimic acid, or
biologically active portion thereof, is substantially free of other cellular materials or other
components or substantially free of chemical precursors or other chemicals.
The following examples serve to merely explain different methods of preparing
caffeoylshikimic acids and related compounds and should not be confined thereof.
The first step of the method for preparation of the composition is pre-treatment of the raw
extracts to obtain pre-concentrated or partially purified extracts. This may be performed
with low stringent conditions of subjecting the extracts to a flash chromatography or the
likes, or alternatively, subjecting said extracts to ethanol precipitation, prior to separation
by high performance liquid chromatography. The main steps involved for the first
approach is loading a "sep-pal? type column, removing impurities, eluting said extracts
with methanol or ethanol and subjecting said extracts for concentration stage in a rotary
evaporator. The second approach comprises the steps of adding an amount of extract to
three volumes of cold ethanol, storing said mixture overnight at a preferred temperature of
-20°C, centrifuging at 1500 Xg for at least 15 minutes, dissolving the precipitate obtained
from the previous step with a suitably amount of distilled water and concentrating by
rotary evaporator at 50° C to obtain the preferred final value of 3 ml. It should be
mentioned that these steps for both approaches may be substituted with alternative steps of
standard procedures known in the art however to achieve a similar objective.

The next imperative step of the method for the preparation of the composition comprising
caffeoylshikimic acids and their derivatives involves the isolation and purification of
caffeoylshikimic acids from the partially purified extracts. This can be carried out with the
conventional high performance liquid chromatography (HPLC) based on low stringent
conditions or parameters of preparing an econosil CI8 5 um particle size, with the
preferred column length of 25cm x 10mm id, flow rate of 3 ml per minute. The preferred
mobile phase gradient comprises two solvents, at least one solvent consisting of 0.1%
trifluoroacetic acid (TFA) with an amount of water and added with another solvent
consisting of 10/90 of 0-1% TFA/acetonitrile (ACN) v/v. The mixture is subjected to
isolation by HPLC and it is observed that there are three major peaks indicating the elution
of caffeoylshikimic acids at 40 minutes, 44 minutes and 48 minutes. The eluted
caffeoylshikimic acids collected in accordance with the present invention may have within
90% to 95% or more purity. It would be understood that the choice of columns and
parameters for HPLC may vary however to obtain a similar result of elution time as
described. Eluted fraction may be suitably collected and provided in powder or liquid form
for use in further analysis.
Further chemical analysis on determining the structure of caffeoylshikimic acids based on
the peaks as obtained in accordance with the method of the present invention may be
carried out based on conventional or standard procedures known in the art, for instance
based on well known the Nuclear Magnetic Resonance (NMR) analysis.
The method of the present invention may be carried out to isolate and purify
caffeoylshikimic acids and their derivatives from any plant or type of sources, including,
but not limited to dates (fruits of Phenix reclinata), olive {Olea europaea), oats, barley,
safflower, fruits, vegetables, juices, coconut (Cocos nucifera), corn (Zea mays), seeds,
wastes, and tissues obtained from plants.
The present invention is further described in the following non-limiting Examples.

EXAMPLE 1
Isolation and Pretreatment
Extract from oil palm, vegetation liquor from palm oil milling and palm oil mill effluent
was prepared based on a filtration procedure.
Pre-concentration Treatment
The sample was pre-concentrated with Sample Prep using "Sep pah?' cartridges for solvent
exchange. The selected chromatography used to concentrate the sample was preparative
high performance liquid chromatography (HPLC).
The solvent exchange was carried out based on the following steps:
a) Conditioning - involving the flushing the unused separation pack (CI8)
with methanol and subsequently with water.
b) Sample Injection - involving injecting an amount of 2 ml of sample into the
separation pack and then forced out with a suitable syringe.
c) Washing - involving injecting 1 ml of water through the pack to remove
any form of impurities from the sample.
d) Eluting - involving injecting 2 ml of methanol through the separation pack
so as to elute the remaining sample which had not been eliminated during
the previous washing stage.
In the next step, the eluted sample as concentrated three (3) fold using nitrogen. The clear
sample was then injected into the semi-prep column.
Semi-Prep High Performance Liquid Chromatography (HPLC)

The preferred column was C18 column with the length of 25 cm and Id of 10mm, used
with a flow rate of 3ml/min. In this study, the injection column was 1ml and readings were
taken at 280nm. The mobile phase gradient is shown below in TABLE 1:

A chromatogram obtained from the injection of concentrated sample is as shown in FIG 1.
The sample is seen to elute at 40 minutes, 44 minutes and 48 minutes thereby resulting to
peaks 6, 7, and 8. Each eluted fraction was then freeze-dried, whereby 1.8 mg, 1.6 mg and
1, 1 mg of dry sample were obtained from peaks 6, 7 and 8 respectively.
In the next step, the purities of the peaks were further analysed through Analytical HPLC,
at which it was observed that the purity of the peaks is better than 95%. FIG 2 (a) to (c)
provides the analytical chromatograms for peak 6, 7, and 8 respectively.
EXAMPLE 2
Chemical Structure of Peaks
The molecular weights of the three peaks obtained can be obtained using MALDI Mass
Spectra analysis. An amount of 1 ul of the sample was directly deposited onto the sample
holder to determind (M-H). Results obtained from this analysis are shown in FIG 3.
The UV spectrums of peaks 6, 7 and 8 were taken and it is found that the UV spectrums
for all these compounds were similar, as suitably shown in FIG 4.

NMR Analysis
The chemical structure of the compound based on these peaks was analysed using NMR
analysis. Results obtained indicate that each molecule from peaks 6, 7 and 8 has 16
carbons with two carbonyls (ester or carboxyl) ten sp2 carbons whereby one aromatic ring
with another two double bonds, three oxygenated methine carbons and a methylene
carbon. It is further observed that the aliphatic protons in the molecule are in two spin
systems. The chemical shifts of the carbons and the associated protons thereby indicate the
structure of caffeoylshikimic acid, also known as 3, 4-dihydroxycinnamoyl ester of
shikimic acid. This can be seen in FIG 5.
It is recorded that the compound has a molecular mass of 336, as confirmed by a prominent
peak at (M-H) = 335 in its mass spectrum. Hetero-nuclear multiple bond correlation
(HMBC) provides extensive two and three C-H correlations thereby confirming the
structure as shown in FIG 6 and FIG 7.
The confirm structure of peak 6 is 5-O-caffeoylshikimic acid (CieHieOs) with a molecular
mass of 336. The identified compound and two positional isomers of caffeoylshikimic acid
(3-O-caffeoylshikimic acid and 4-O-caffeoylshikimic acid) can be found in date fruits.
Those skilled in the art would be able to determine that these compounds may be formed
by the dehydration of the analogous 5-O-caffeoylquinic acid.
EXAMPLE 3
Profiling - Reverse Phase HPLC
As shown in FIG 8, the sample contains at least one of the following compounds,
hydroxytyrosol,protocatechuic acid, p-hydroxybenzoic acid, 5-0-caffeoylshikimic acid, 4-
O-caffeoylshikimic acid and 3-0-caffeoylshikimic acid .

In order to carry out further HPLC analysis on the compounds, the sample was prepared as
follows:
a) Preparing 10 mg of alpha -cyano-4-hydroxycinnamic acid, 99% was added to
lmlof ethanol;
b) The internal standard solution (20ul) was added to 980 ul of the sample; and
c) 300 ul of this mixture was used for HPLC analysis.
A reverse phase HPLC was conducted based on the above conditions whereby an Exsil
ODS 5 um SGE column (250x4.6) was used with a flow rate of 0-8 ml/min and a
photodiode detection of 280nm. The injection volume was 20 ul. The mobile phase
gradient for this analysis is shown below in TABLE 2 below:

FIG 9 shows the chromatogram of the sample, whereby it can be seen that there are 5
major peaks, indicating the presence of hydroxytyrosol, protocatechuic acid, p-
hydroxybenzoic acid and three isomers of caffeoylshikimic acid, amongst the minor peaks
representing several other phenolic compounds including gallic acid, ferulic acid, and etc.
It is further observed that the minor peaks may account for at least 20% of the phenolics of
the sample. Accordingly, the major peaks were substantially accounted for the quantitation

of the phenolics content for the sample. The standard used for this study was alpha-
cyanohydroxycinnamic acid with the retention time of 39 minutes for quantitation.
Those skilled in the art will appreciate that the invention described herein is susceptible to
variations and modifications other than those specifically described. It is to be understood
that the invention includes all such variations and modifications. The invention also
includes all of the steps, features, compositions and compounds referred to or indicated in
this specification, individually or collectively, and any and all combinations of any two or
more of said steps or features.

We claim:
1. A composition comprising caffeoylshikimic acids, protocatechuic acid,
hydroxytyrosol, hydroxybenzoic acid , said caffeoylshikimic acids and their derivatives
extracted from any part of oil palm including but not confined to the vegetation liquor of
palm oil milling and palm oil mill effluent.
2. The composition as claimed in Claim 1 wherein the composition comprises
caffeoylshikimic acids, protocatechuic acid, hydroxytyrosol, hydroxybenzoic acid , said
caffeoylshikimic acids and their derivatives extracted from the vegetation liquor of palm oil
milling.
3. The composition as claimed in Claim 1 wherein the composition further contains at
least one of the following: hydroxytyrosol, p-hydroxybenzoic acid, 5-0-
caffeoylshikimic acid, 4-0-caffeoylshikimic acid and 3-0-caffeoylshikimic acid.
4. A method for use in the preparation of a composition containing caffeoylshikimic acids
and their derivatives, said method comprising the steps of providing an extract from any
part of oil palm including but not confined to the vegetation liquor of palm oil milling and
palm oil mill effluent pre-concentrating said extract containing said caffeoylshikimic acids
and their derivatives and isolating said caffeoylshikimic acids and their derivatives from
said extract by liquid chromatography, wherein the elution activity of said caffeoylshikimic
acids and their derivatives results to a plurality of peaks on said liquid chromatography;
wherein the elution activity occurs at 40, 44 and 48 minutes within at least one fraction of
the extract, thereby producing at least three peaks by liquid chromatography.
5. A method for use in the preparation of a composition containing caffeoylshikimic acids
and their derivatives as claimed in Claim 4 wherein the pre-concentrating step further
comprising the steps of loading a column with the extract, removing the impurities, eluting
the extract with methanol or ethanol, concentrating in a rotary evaporator, adding an
amount of cold ethanol, storing at -20°C, centrifuging said mixture, dissolving the
precipitate obtained from centrifuging with an amount of water and concentrating said
mixture by rotary evaporator at 50 °C.
6. A method as claimed in Claim 5 wherein the amount of cold ethanol is at least 3
volumes of the extract.

9 A method as claimed in Claim 5 wherein the amount of water for dissolving the
precipitate is approximately 10 ml.
10. A method as claimed in Claim 4 wherein in the isolation step, the following parameters
are provided for liquid chromatography; providing a C18 column with 25 length and 10 mm
id, using at least one solvent containing an amount of trifluoroacetic acid with water and
another solvent containing another amount of trifluoroacetic acid with acetonitrile for
mobile phase gradient.
11. A method as claimed in Claim 4 wherein at least one solvent contains 0.1%
trifluoroacetic acid and another solvent contains 10/90 0.1% trifluoroacetic acid per
acetonitrile.
12.A method as claimed in Claim 4 wherein the percentage of purity of the
caffeoylshikimic acids and their derivatives obtained upon isolated is within 90 to 95%

The present invention discloses a composition comprising caffeoylshikimic acids,
protocatechuic acid, hydroxytyrosol, hydroxybenzoic acid , said caffeoylshikimic acids
and their derivatives extracted from any part of oil palm including but not confined to
the vegetation liquor of palm oil milling and palm oil mill effluent, and a method for
use in the preparation of a composition containing caffeoylshikimic acids,
protocatechuic acid, hydroxytyrosol, hydroxybenzoic acid , said caffeoylshikimic acids
and their derivatives ,, said method comprising the steps of pre-concentrating an
extract containing said caffeoylshikimic acids, protocatechuic acid, hydroxytyrosol,
hydroxybenzoic acid , said caffeoylshikimic acids and their derivatives and isolating
said caffeoylshikimic acids, protocatechuic acid, hydroxytyrosol, hydroxybenzoic acid
, said caffeoylshikimic acids and their derivatives from said extract by liquid
chromatography, wherein the elution activity of said caffeoylshikimic acids,
protocatechuic acid, hydroxytyrosol, hydroxybenzoic acid , said caffeoylshikimic acids
and their derivatives vary depending on the stationaryphase and the composition of
the mobile phase. In the present example, elution of the fractions containing
Caffeoylshikimic acid occurs between 40 and 48 minutes with C18 column and a
gradient mobile phase containing 0.1% TFA( in water) and acetonitrile.