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. <br/