Chemical, physical and biological features of Okra pectin

In Thailand, many plants have been used as vegetables as well as for traditional
medicine. Okra, Abelmoschus esculentus (L.) Moench, is an example of such a plant.
Examples for the medical use are treatment of gastric irritation, treatment of dental
diseases, lowering cholesterol level and preventing cancer. These biological activities are
ascribed to polysaccharide structures of okra in particular pectin structures. However, the
precise structure of okra pectins and also of other polysaccharides in okra pods have been
lacking so far.
In order to obtain detailed information of the different polysaccharides present in
okra, okra cell wall material was prepared from the pulp of okra pods and was then
sequentially extracted with hot buffer, chelating agent, diluted alkali and concentrated
alkali. The sugar (linkage) composition indicated that okra cell wall contained, next to
cellulose, different populations of pectins and hemicelluloses.
The pectic polysaccharides were mainly obtained in the first three extracts having
slightly different chemical structures. The okra pectin extracted by hot buffer was almost a
pure rhamnogalacturonan (RG) I with a high degree of acetylation (DA), covalently linked
to a minor amount of homogalacturonan (HG) having a high degree of methyl esterification
(DM). The chelating agent extractable pectin and the diluted alkali extractable pectin
predominantly contained HG with only minor amounts of RG I. Okra pectins extracted by
hot buffer and with chelating agent had in common that both contained highly branched RG
I with very short side chains containing not more than 3 galactosyl units attached to the
rhamnosyl residues in RG I backbone. Chelating agent extracted okra pectins also carried
arabinan and arabinogalactan type II as neutral side chains and these side chains were even
more abundantly present in the diluted alkali extracted okra pectin.
The hemicellulosic polysaccharides ended up in concentrated alkali extract. From
the sugar (linkage) composition and enzymatic degradation studies using pure and well
defined enzymes, it was concluded that this fraction contained a XXXG–type xyloglucan
and 4-methylglucuronoxylan. The cellulosic polysaccharides were retained in the residue.
The okra hot buffer extractable RG I having a high level of acetyl substitution
appeared to be very well degradable by rhamnogalacturonan hydrolase which was known to
be hindered completely by acetylated substrates. In contrast, an acetylated galacturonic
acid-specific rhamnogalacturonan acetyl esterase was unable to remove acetyl groups from
the RG I molecule of hot buffer extracted okra pectin. For these reasons, the precise
position of the acetyl groups present on enzymatically released oligomers were determined
by Electron Spray Ionization Ion Trap Mass Spectrometry (ESI-IT-MS) and Nuclear
Magnetic Resonance (NMR) spectroscopy. The acetyl groups were found to be
predominantly located at position O-3 of the rhamnosyl moiety, while the methyl esters
seemed to be present only on the HG part of the hot buffer extracted okra pectin. Another
novelty of okra RG-I was the presence of terminal alpha-linked galactosyl substitution at
position O-4 of the rhamnosyl residues within the RG I backbone. These specific features
(acetylated rhamnosyl- and alpha-galactosyl-substitutions) were almost absent in the
chelating agent extracted okra pectin where more commonly known substitutions were
present, including acetylated galacturonosyl residues in the RG I backbone. The unique
structure features of hot buffer extracted okra pectin led to the assumption that these
features may contribute to the rather typical physical properties as well as to the biological
properties found for okra pectin.
In order to understand the effect of the specific structural features of RG I on its
physical properties, the rheological properties of hot buffer extracted okra pectin were
determined and compared to those found for chelating agent extracted okra pectin and for
pectins from other plant materials as reported in the literature. The solutions of hot buffer
extracted okra pectin showed a high viscosity and predominant elastic behaviour which
most probably is caused by strong hydrophobic associations through its acetylated
rhamnosyl residues rather than by methyl esterified galacturonosyl residues as is commonly
the case for pectins. The removal of acetyl groups and methyl esters decreased the
association of the pectin molecules as observed by the light scattering experiment, meaning
that not only viscosity and rheological properties but also association of pectin molecules
were as result of both hydrophobic interactions and charge effects.
The effect of the position of acetyl groups on the bioactivity of okra pectin was
also determined. The complement-fixing activity of okra pectins was found to be affected
by many factors like e.g. the presence of acetyl groups, the size of RG segments and the presence of terminal alpha galactosyl groups and even the three dimensional conformation
of the molecules. The hot buffer extracted okra pectin was also examined for its potential to
modify surfaces of medical devices and implants. The results showed that okra pectin can
be used in coating medical device since it promotes cell apoptosis and shows no
macrophage activation.
The knowledge described in this thesis provided us with novel information on the
unique structures of okra pectins and may lead to a better understanding of the functional
properties of okra polysaccharides in general and okra pectin in particular and to optimize
the use of okra pectins within the food industry and in medical applications. However,
despite our efforts, at the moment the dependency of the (bio) functionality of okra pectins
on the precise chemical structure are not yet completely understood.