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/

A review on development and application of plant-based bioflocculants and grafted bioflocculants

Flocculation is extensively employed for clarification through sedimentation. Application of eco-friendly plant-based bioflocculants in wastewater treatment has attracted significant attention lately with high removal capability in terms of solids, turbidity, color, and dye. However, moderate flocculating property and short shelf life restrict their development. To enhance the flocculating ability, natural polysaccharides derived from plants are chemically modified by inclusion of synthetic, nonbiodegradable monomers (e.g., acrylamide) onto their backbone to produce grafted bioflocculants. This review is aimed to provide an overview of the development and flocculating efficiencies of plant-based bioflocculants and grafted bioflocculants for the first time. Furthermore, the processing methods, flocculation mechanism, and the current challenges are discussed. All the reported studies about plant-derived bioflocculants are conducted under lab-scale conditions in wastewater treatment. Hence, the possibility to apply natural bioflocculants in food and beverage, mineral, paper and pulp, and oleo-chemical and biodiesel industries is discussed and evaluated

Characterisation of cell wall polysaccharides from okra (Abelmoschus esculentus (L.) Moench)

Okra pods are commonly used in Asia as a vegetable, food ingredient, as well as a traditional medicine for many different purposes; for example, as diuretic agent, for treatment of dental diseases and to reduce/prevent gastric irritations. The healthy properties are suggested to originate from the high polysaccharide content of okra pods, resulting in a highly viscous solution with a slimy appearance when okra is extracted with water. In this study, we present a structural characterisation of all major cell wall polysaccharides originating from okra pods. The sequential extraction of okra cell wall material yielded fractions of soluble solids extractable using hot buffer (HBSS), chelating agent (CHSS), dilute alkaline (DASS) and concentrated alkaline (CASS). The HBSS fraction was shown to be rich in galactose, rhamnose and galacturonic acid in the ratio 1.3:1:1.3. The degree of acetylation is relatively high (DA = 58) while the degree of methyl esterification is relatively low (DM = 24). The CHSS fraction contained much higher levels of methyl esterified galacturonic acid residues (63% galacturonic acid; DM = 48) in addition to minor amounts of rhamnose and galactose. The ratio of galactose to rhamnose to galacturonic acid was 1.3:1.0:1.3 and 4.5:1.0:1.2 for HBSS and CHSS, respectively. These results indicated that the HBSS and CHSS fractions contain rhamnogalacturonan type I next to homogalacturonan, while the latter is more prevailing in CHSS. Also the DASS fraction is characterised by high amounts of rhamnose, galactose, galacturonic acid and some arabinose, indicating that rhamnogalacturonan I elements with longer arabinose- and galactose-rich side chains were part of this fraction. Partial digestion of HBSS and CHSS by pectin methyl esterase and polygalacturonase resulted in a fraction with a lower Mw and lower viscosity in solution. These samples were subjected to NMR analysis, which indicated that, in contrast to known RG I structure, the acetyl groups in HBSS are not located on the galacturonic acid residues, while for CHSS only part of the acetyl groups are located on the RG I galacturonic acid residues. The CASS fraction consisted of XXXG-type xyloglucan and 4-methylglucuronoxylan as shown by their sugar (linkage) composition and enzymatic digestion

Okra pectin contains an unusual substitution of its rhamnosyl residues with acetyl and alpha-linked galactosyl groups

The okra plant, Abelmoschus esculentus (L.) Moench, a native plant from Africa, is now cultivated in many other areas such as Asia, Africa, Middle East, and the southern states of the USA. Okra pods are used as vegetables and as traditional medicines. Sequential extraction showed that the Hot Buffer Soluble Solids (HBSS) extract of okra consists of highly branched rhamnogalacturonan (RG) I containing high levels of acetyl groups and short galactose side chains. In contrast, the CHelating agent Soluble Solids (CHSS) extract contained pectin with less RG I regions and slightly longer galactose side chains. Both pectic populations were incubated with homogeneous and well characterized rhamnogalacturonan hydrolase (RGH), endo-polygalacturonase (PG), and endo-galactanase (endo-Gal), monitoring both high and low molecular weight fragments. RGH is able to degrade saponified HBSS and, to some extent, also non-saponified HBSS, while PG and endo-Gal are hardly able to degrade either HBSS or saponified HBSS. In contrast, PG is successful in degrading CHSS, while RGH and endo-Gal are hardly able to degrade the CHSS structure. These results point to a much higher homogalacturonan (HG) ratio for CHSS when compared to HBSS. In addition, the CHSS contained slightly longer galactan side chains within its RG I region than HBSS. Matrix-assisted laser desorption ionization-time of flight mass spectrometry indicated the presence of acetylated RG oligomers in the HBSS and CHSS enzyme digests and electron spray ionization-ion trap-mass spectrum showed that not only galacturonosyl residues but also rhamnosyl residues in RG I oligomers were O-acetylated. NMR spectroscopy showed that all rhamnose residues in a 20 kDa HBSS population were O-acetylated at position O-3. Surprisingly, the NMR data also showed that terminal a-linked galactosyl groups were present as neutral side chain substituents. Taken together, these results demonstrate that okra contained RG I structures which have not been reported before for pectic RG I

Physicochemical properties of pectins from okra (Abelmoschus esculentus (L.) Moench)

Okra pectin obtained by hot buffer extraction (HBSS) consists of an unusual pectic rhamnogalacturonan I structure in which acetyl groups and alpha galactose residues are substituted on rhamnose residues within the backbone. The okra Chelating agent Soluble Solids (CHSS) pectin consists of slightly different structures since relatively more homogalacturonan is present within the macromolecule and the rhamnogalacturonan I segments carry slightly longer side chains. The rheological properties of both okra pectins were examined under various conditions in order to understand the unusual slimy behaviour of okra pectins. The viscosity of the okra HBSS pectin was 5–8 times higher than the viscosity of the okra CHSS pectin. The okra HBSS pectin showed an elastic behaviour (G' > G¿) over a wide range of frequencies (10-1–10 Hz), at a strain of 10%, while okra CHSS and saponified okra HBSS/CHSS pectin showed predominantly viscous responses (G' <G¿) over the same frequency range. The results suggest that the structural variation within the okra pectins greatly affect their rheological behaviour and it is suggested that acetylation of the pectin plays an important role through hydrophobic associations. Dynamic light scattering was used to study the association behaviour of both okra pectins at low concentration (0.001–0.1% w/w). Results showed that the saponified okra pectins did not exhibit a tendency to aggregate in the concentration range studied, whereas both non saponified samples showed a substantial degree of association. These results suggest that the unusual slimy behaviour of the non saponified samples may be related to the tendency of these pectins to associate, driven by hydrophobic interactions

Antiproliferative and proapoptotic actions of okra pectin on B16F10 melanoma cells

The proliferation and apoptosis of metastatic melanoma cells are often abnormal. We have evaluated the action of a pectic rhamnogalacturonan obtained by hot buffer extraction of okra pods (okra RG-I) on melanoma cell growth and survival in vitro. We added okra RG-I containing an almost pure RG-I carrying very short galactan side chains to 2D (on tissue culture polystyrene, tPS) and 3D (on poly(2-hydroxyethylmethacrylate), polyHEMA) cultures of highly metastatic B16F10 mouse melanoma cells. We then analyzed cell morphology, proliferation index, apoptosis, cell cycle progression and the expression of adhesion molecules. Immunostaining and western blotting were used to assay galectin-3 (Gal-3) protein. Incubation with okra RG-I altered the morphology of B16F10 cells and significantly reduced their proliferation on both tPS and polyHEMA. The cell cycle was arrested in G2/M, and apoptosis was induced, particularly in cells on polyHEMA. The expression of N-cadherin and 5 integrin subunit was reduced and that of the multifunctional carbohydrate-binding protein, Gal-3, at the cell membrane increased. These findings suggest that okra RG-I induces apoptosis in melanoma cells by interacting with Gal-3. As these interactions might open the way to new melanoma therapies, the next step will be to determine just how they occu