Sporopollenin: The Ground Discussion

               

Nimish S. Khandekar, Rajesh S. Jagtap*, Sachin J. Sajane, Sneha R. Jagtap

Annasaheb Dange College of B Pharmacy, Ashta, Sangli - 416301 Maharashtra, India.

*Corresponding Author E-mail: rajeshjagtap10@gmail.com, nimishschool@gmail.com

 

ABSTRACT:

Sporopollenin is an ubiquitous and extremely chemically inert biopolymer .The surface of sporopollenin is richly sculptured, ornamented and porous and is species specific. It is stable in organic and aqueous solvents. It is insoluble in common acids and in most organic solvents. Sporopollenins have very similar chemical structures and to be identical in structure to the synthetic carotenoid polymes. The sporopollenins derived from angiosperms, gymnosperms and ferns and lower plant spores have similar chemical structures and also they are similar to synthetic polymers. Sporopollenins are strongly osmophilic. Sporopollenins react with basic dyes suggesting the presence of weakly amnionic groups such as acidic-enolic compounds. Sporopollenin can be isolated from spores or pollen grain by treating with solvents or enzymes that remove intine and cytoplasm. Purified sporopollenin retains the similar shape, size, and surface features as in its spore or pollen grain and remains an empty shell i.e sporopollenin microcapsules. The different methods developed to isolate sporopollenin from L. clavatum proved its exceptional stability and chemical inertness. Wiermann et al has proved that, the sporopollein survived a wide range of enzymes. This may explain why it does not easily submit to bacterial decomposition or digestion. Its principle function is to protect against oxidation and desiccation. The study led by Maak proved that sporopollenin by Chlorella vulgaris was harmless and could be rubbed on skin with no irritation, swallowed without any danger or even injected in blood stream. The sporopollenin particles were found to cause an antigenic reation and bind to antibodies. The sporopollenin remains unchanged when heated up to 3000C or treated with concentrated acids and bases. Sporopollenin appears to undergo carbonization and coalification with haet. The sporopollenin decomposes by chemolyses and ozonolysis. An unknown enzymatic sequence linked to the clotting cascade has also been discovered that degrades sporopollenin in the blood, both in vitro and in vivo.

 

KEYWORDS: Sporopollenin, Inert, Osmophilic,  Antioxidant, Ozonolysis.     

 

 


INTRODUCTION:

Sporopollenin is a ubiquitous and extremely chemically inert biopolymer that constitutes the outer wall of all land-plant spores and pollen grains. Sporopollenin protects the vulnerable plant gametes against a wide range of environmental assaults, and is considered as a prerequisite for the migration of early plants onto land.1 It was first observed and named as “sporonin” by John (1814) and latter characterized by Berzelius (1830). Fossil green algae dating back to Devonian period have been shown to contain sporopollenin.

 

The oldest sporopolleninous acritarchs occur in Pre-Cambrian rocks, 1.2-1.4 billion years old. The green algae are presumably responsible for the development of sporopollenin and its introduction into the armament of higher green plants, where its principal function is protection against oxidation and desiccation. Further Brooks and Shaw from their study have shown the presence of amorphous insoluble organic material which appears similar to present day sporopollenin. It forms the basic structure of the resistant wall of most palynomorphs, like spores, pollen, dinoflagellates, and acritarchs. It has also been recorded from the spores of Aspergillus niger, sexual (±) spores of Mucor mucedo, asexual spores of Pithophora oedogonia and several algae like, in the cell wall of Phycopeltis epiphyton (a subaerial green alga found growing on the leaves of vascular plants and bryophytes), Char a corallina, cyst of Prasinocladus marinus. A trilaminar sporopollenin sheath is also present in Chlorella, Scenedesmus, and Pediastrum. It is also distributed in the spores of Bryophytes, Pteridophytes, pollen of Gymnosperms and Angiosperms. In general it is limited to the outer wall, the exine however; fern spores and some gymnosperm pollen have an additional sporopollenin-bearing wall, the perine or perispore. The other sporopollenin-containing elements associated with spores/pollen are, viscin threads, elaters, perispore like bands attached to the Equisetum spores, Ubisch bodies or orbicules. The aquatic flowering plants have abandoned the production of sporopollenin while members belonging to the family Lauraceae, Cannaceae, etc. manage to operate with very little sporopollenin in their exines.48 A study led by Maak proved that sporopollenin by Chlorella vulgaris was harmless and could be rubbed on the skin with no irritation, swallowed without any danger or even injected in blood stream. Moreover, sporopollenin particles were found to cause an antigenic reaction and bind to antibodies. Importantly, direct enzymatic catabolism was also observed in the blood.2 Amongst other chemolyses, ozonolysis was a very common way of decomposing sporopollenin. Resulting ozonides were decomposed by hydrogen peroxides under acidic conditions. Oxidation products were found, by GC, HPLC or GC-MS, to be a mixture of carboxylic acids.2

 

Physical Nature of Sporopollenin:

Sporopollenin is the major component of exine of spores and pollen. It is most extraordinarily resistant material known in the organic world.  Sporopollenin remains unchanged when extant pollen grains are heated to 300°C or treated with concentrated acids and bases.3-4

 

 

Fig-1-Image of Sporopollenin from Scanning Electron Microscopy.

 

The surface of sporopollenin is richly sculptured, ornamented, and porous and is species specific. It is amphiphilic in nature and is selectively permeable. It is tough but elastic. The amount of sporopollenin in a given microspore varies from species to species. It is reported that Lycopodium clavatum spores contain a relatively high proportion (-20-25%) of sporopollenin.5

 

Chemical Nature of Sporopollenin:

Fourier-transform infrared spectroscopy analysis of the isolated sporopollenin showed the absence of polysaccharide and phenolic material and the presence of carboxylic acid groups joined to unsaturations and ether linkages.6 Sporopollenin is exceptionally stable in organic and aqueous solvents and in a wide range of chemical reagents. It is insoluble in common acids and in most organic solvents. Sporopollenin partially dissolves in organic bases, including 2-aminoethanol, 3-aminopropanol, and 4-methylmorpholine-N-oxide. It is degraded in fused potassium hydroxide and in oxidizing mixtures such as hypochlorite and hydrochloric acid, potassium dichromate and sulfuric acid, hydrogen peroxide and sulfuric acid, and ozone.5 Sporopollenin, which forms the outer wall of pollen and spores contains a chemical signature of ultraviolet-Bfluxvia concentrations of UV-B absorbing compounds (UACs), providing a proxy for reconstructing UV irradiance through time. The UACs in sporopollenin are the phenolic compounds p-coumaric acid and ferulic acid, which form cross-links between straight chain aliphatic compounds (the other major component of sporopollenin), and absorb solar UV-B through their aromatic ring structure.7 The spectra of sporopollenin obtained from an early, a middle post meiotic and the mature stage have been compared. The assumption that sporopollenin mainly consists of long chain aliphatic with varying amounts of aromatics is corroberated by our results Only slight changes in sporopollenin structure could be recognized during pollen wall development of Cucurbita maxima. In contrast to this the spectra of Tulipa sporopollenin of the three developmental stages exhibited significant differences concerning the aliphatic and aromatic components.8

 

I. Chemical Evidences:

Oxidative degradation (using ozone) of natural sporopollenins and of synthetic carotenoid polymers shows excellent correlation between the products9-10. Generally, the spectra of compounds are very similar showing sporopollenins to have very similar chemical structures and to be identical in structure to the synthetic carotenoid polymers. Detailed examination of the spectra of the di-carboxylic and mono-carboxylic acid oxidative degradation products derived from sporopollenins of different plant families show some minor. but significant differences that may have potential use in phytochemical correlations. Pyrolysis-gas chromatography of sporopollenins, synthetic carotenoid polymers and natural carotenoids10-13 gave almost identical chromatograms showing that the sporopollenins derived from angiosperm, gymnosperm and ferns and lower plant spores have similar chemical structures and also that they are similar to the synthetic polymers. A molecular structure for sporopollenin based upon an oxidative polymer of carotenoids and carotenoid esters has been reported.10 This structure is supported by elemental analysis, chemical, biochemical, radiochemical and cytochemical evidence that has been came out during the last few years. X-ray diffraction studies on the natural and synthetic polymers10 confirm the chemical structure of sporopollenin as an oxidative polymer of carotenoids.

 

II. Cytochemical Evidences:

Sporopollenins are strongly osmiophilic14, showing the material to contain aliphatic unsaturated C-C double bonds. The polymer absorbs U-V light (-290, 265 and 250nm)15 and its fluorescence suggests the presence of conjugated C-C double bonds adjacent to C-0 groups. Sporopollenin reacts with basic dyes (nile blue A; azure L3 and toluidine blue) suggesting the presence of weakly anionic groups such as acidic-enolic compounds. Sporopollenins can be distinguished from cutin and suberin by lack of stain with Sudan IV14 and from lignin by its insolubility in dioxane16 and its failure to stain with phloroglucinol17. These cytochemical observations clarify the postulated chemical structure of sporopollenin. The unsaturated C-C bonds provide the active sites for staining with osmium tetroxide and would show U-V and fluorescent spectra. The oxidation of the carotenoid structiire leads to the formation of enolic groups within long aliphatic carbon chains. In Sporopollctriti 9S corporation of radio-labelled sodium acetate. mevalonic acid and p-carotene compounds into developing pollen grains and spores during exine synthesis shows conclusively that the sporopollenin chemical structure is not related to lignin. Sporopollenins are oxidative polymers of care tenoids and carotenoid esters.

 

Biological properties of sporopollenin:

Spores and pollen grains can be found in many food sources such as mushrooms or honey. Additionally, bee pollen has been demonstrated to be good nutrients for athletes; they can provide energy, stamina and endurance. Pollen is being actively marketed for alleviating certain health afflicitions and as a beneficial dietary supplement. Pollen tablets are also used as desensitizing against allergy or as “natural food”, especially by athletes. Also some firms propose Chlorella Vugaris as neutraceutical. Pollen or spores may or may not be beneficial, but anyway a reasonably valid observation is that they are not harmful to humans even when ingested in large amount.

 

Fig-2-a) Before oral digestion b) After 30 minutes of ingestion c) After 60 minutes of ingestion

 

Sporopollenin is a naturally occurring polymer found in spores and pollen grains. From the previous observation, it can thus rightly be considered harmless to health, all the more that all of the allergens (proteins) have been stripped out by the extraction protocol. This is shown by combustion elemental analysis, sporopollenin was found to be virtually nitrogen-free. A study led by Maak proved that sporopollenin by Chlorella vulgaris was harmless and could be rubbed on the skin with no irritation, swallowed without any danger or even injected in blood stream. Moreover, sporopollenin particles were found to cause an antigenic reaction and bind to antibodies. Importantly, direct enzymatic catabolism was also observed in the blood.2

 

Ultra structure and nanochannels:

It is accepted that sporopollenin units form a spongy network ca. 70nm large in mesh. Different precise models have been proposed for the ultrastructure of exine: an arrangement of interconnected granules, a quasi-crystalloid molecular structure, a colloidal crystal self-assembled organization, or multi-helical units. Sporoderm, and a fortiori exine layer, “is not a barrier to penetration”, as Pettitt wrote. It is indeed traversed by monochannels.  Sporopollenin meshwork was found to intertwine with the array of channels, whose diameter was first reported at about 40-70nm, probably after broadening by oxidative extraction methods. A more recent work described those radial nanochannels as being approximately 25nm in diameter in mature pollen (but wider at a younger stage of spore development). This same study, based on a previous work over five species, proposed a model of ultrastructure endorsing most of the previous discoveries. Sporopollenin was said to polymerise into radial rod-like subunits. In the microspore growth, they are evident as columellae, from which tectum, foot layer and endexine are yielded. Each exine subunit seems to be centred on a nanochannel. Around this hollow core would be a corona (“binder zone”) composed of multi-helical sporopollenin subunits. The overall diameter of those substructures is 70-100nm in young microspores, and reaches up to 200-250nm on mature exines. Moreover, several studies demonstrate that the channels not only cross the exine (both ectexine and endexine) but also the intine. It was shown as well that they enable particulate material to enter the sporoderm, although colloid metals permeate through exine even where no evident nanochannels were found. The consistence of the studies from species to species in vascular plants (amongst which Lycopodium spp.) tends to firmly back up this hypothesis.2

 

Geochemical Studies of Sporopollenin:

Pollen grains and spores from different ranks of coal and different sediments show gradual colour changes from pale yellow, through light brown to dark brown-black, due mainly to different thermal histories. Considering experimental evidence, due to difficulties in understanding all the various parameters that may affect colour and chemical changes in pollen and spores, it seems that such changes occur by coalification and carbonisation processes18. The processes depend on the different rates of heating, different times of thermal alteration and the resulting chemical reactions. Thermal alterations are considered to be the most important parameter, but it is necessary to note the effects of hydrostatic pressure gradients ;ind shear pressure gradients in regions subject to deep burial 19. Laboratory experiments have been used to study the chemical changes in sporopollenins under carbonisation processes 20-23. The pollen and spore walls show colour and chemical changes that start at temperatures between 100" and 200°C. Few studies have been carried out on the progressive changes occurring with sequential heating of pollen and spores, but some results24 suggest that pollen and spores carbonise at different temperatures, depending on their previous geothermal history. Pollen grains and spores that are heated for varying lengths of time at different temperatures show very limited colour and chemical changes between 180'-200"C, but significant changes above 220°C and above 170'C further significant changes occur over shorter periods12,10,25,23. Laboratory experiments showed that when pollen and spore walls are located at lower temperatures (less than 180°C). it is from the evolution of small amounts of methane and carbon dioxide, the sporopollenin residue appears very little altered. Sporopollenin heated to higher temperatures (greater than 220°C) produces more volatile and soluble chemical products and shows significant alterations in both colour and chemical changes in a relatively short time26. Pollen and spores show changes in colour and chemical structure with temperature at different rates than dinoflagellates and acritarchs21 and they show earlier changes than chitinozoa. Pollen grains and spores are the most sensitive indicators of thermal changes in any particular region of a sediment, especially in areas of relative low thermal activities and gradients. These facts have resulted in palynomorphs-particularly pollen and spores-being used in geothermal studies on sediments. Although these techniques have wide applications.27 Their major current use is in petroleum exploration. Using relationships between pollen grain and spore colour, translucency and fluorescence, chemical changes, electron spin resonance (ESR) and thernial gravimetnc analysis (TGA) and geological conditions, it is now possible for organic geochemists and palynologists to determine the regional thermal history of sediments and their potential for hydrocarbon generation.28-29

 

 

Fig-3- Structure of Sporopollenin.

 


 

Fig-4- Chemical and Physical features of Sporopollenin.


 

 

Resilience of sporopollenin:

Faegri and Iversen described sporopollenin as “one of the most extraordinary resistant materials known in the organic world”. Brooks and Shaw stated “sporopollenins are probably the most resistant organic materials of direct biological origin found in nature and in geological samples”. This can be further reiterated by the survival of some intact exines in ancient sedimentary rocks, dating back from more than 500 million years old. Whereas the cytoplasmic, genetic and polysaccharide components are systematically destroyed by diagenesis , the resistant exine may remain unchanged and form part of the organic sediments (kerogen).

 

This illustrates sporopollenin’s (and exine’s) relative resistance to high pressure, mechanical stress and biological decay.

 

1) Chemical resilience:

The different methods developed to isolate sporopollenin from L. clavatum proved its exceptional stability and chemical inertness. Indeed, it is highly resistant to a variety of hot strong acids (including phosphoric acid, sulphuric acid and hydrofluoric acid), alkalis (e.g. concentrated sodium or potassium hydroxides) and organic solvents (e.g. acetone, methanol or dichloromethane). In keeping with this, the Erdtman’s acetolysis was shown not to degrade sporopollenin, In contrast, hydrofluoric acid solution was reported not to modify the chemical composition of sporopollenin and hence be useful for intine removal.

 

Also, in the past few years, the integrity of exine was checked after stirring 2h or 24h in DCM, ethanol, water, toluene, DMF or DMSO, at room temperature or 50°C. SEM pictures revealed that particles isolated from L. clavatum L. (25µm) were not soluble in these solvent and stayed undamaged. Sporopollenin exines from Lycopodium spec. (40µm particles) however were partly broken after 24h of stirring at room temperature or even 2h at 50°C. Nonetheless, numerous chemicals were found capable of degrading sporopollenin, including oxidisers (e.g. nitric acid, potassium permanganate, ozone, chromic acid or nitrobenzene) or fused potash, or of dissolving it, such as 2-aminoethanol. This was mostly explained by its high degree of unsaturation.

 

2) Biological resilience:

As aforementioned, sporopollenin was found able to resist decay after millions of years. The enzymatic extraction procedure developed by Wiermann et al has proved sporopollenin survived a wide range of enzymes (protease, amylase, lipase, cellulase and hemicellulase). This may explain why it does not easily submit to bacterial decomposition or to digestion. At a certain point, attack of sporopollenin by microorganisms was considered possible although complete biodegradability was still doubted.

 

However, it is important in nature that sporopollenin be biologically degraded, for instance to enable germination to occur and to recycle the material after fecundation. Above all, “all natural substances have some enzyme that will break it down, otherwise we would quite simply be submerged in these materials”, as Faegri stated. A number of bacteria were found able to degrade sporopollenin under certain conditions (e.g. pH or aerobic milieu). Some plant enzymes can also hydrolyse exine, for example those produced by fungal rhizoids to feed on pollen. Furthermore, after pollination, intine secretes an enzyme cocktail (acid phosphatase, ribonuclease, esterase and amylase) to break up the exine. In the same vein, a pollen esterase has been found in Hordeum vulgare L. (barley) pollen that hydrolyses sporopollenin, in order to form late pores. Incidentally, sporopollenin was also expected to be destroyed in the blood stream, as a foreign body. An unknown enzymatic sequence linked to the clotting cascade has also been discovered that degrades sporopollenin in the blood, both in vivo and in vitro.

 

3) Physical and mechanical resilience:

It must be highlighted that, whereas sporopollenin material resist chemical, physical and biological aggressions, exines themselves may be damaged under similar conditions. Indeed, some plant species develop spores or pollen grain with very thin exines or exine elements only loosely linked together. Removal of intine and/or sporoplasm may thus lead to exine fragmentation, collapsing or bursting. This was first noticed by Shaw and Yeadon on several species whose exine could not be recovered after potassium hydroxide treatment: Fraxinus excelsior L. (ash), Populus nigra L. (Lombardy poplar), Lupinus sp. L. (lupin) and Platanus sp. L. (plane). Recent experiments with Chlorella vulgaris cells, Ambrosia trifida pollen or Aspergillus niger conidia corroborated these results: the amount of sporopollenin in the wall did not suffice to hold it intact under mechanical or thermal stress after extraction of the other components. Moreover, many workers also mention disruption of pollen grains by stirring and/or subjection to ultrasounds. The species chosen for such treatments were therefore of a more fragile nature.

 

3) Thermal resilience:

Sporopollenin appears to undergo carbonisation and coalification with heat. Different changes occur depending on the temperature:

·       Under 100°C: no change;

·       100-180°C: slow colour changes;

·       180-220°C: darkening and evolution of water, methane and carbon dioxide;

Over 220°C: quick darkening and release of more volatile chemicals (hydrogen sulphide, dihydrogen and light hydrocarbons).

 

A more detailed investigation by IR and elemental analysis showed that sporopollenin was dehydrated by heat (loss of hydroxyls with creation of carbon-carbon double bonds), and, above 400°C, aromaticity drastically increased.2

 

 

Spore Wall Topology in Relation to the Sporopollenin Transfer Hypothesis.

 

 

Fig-5- Wall Layers of Spores and Pollen.

 

The SPTH posits that sporopollenin deposition on the zygote wall in charophycean algae was developmentally delayed during evolution to be later deposited on meiospore walls. Sporopollenin deposition is initiated as a secretory process in the cytosol of the developing zygote and deposition occurs centrifugally to the outside of the cell to produce an encysted zygote. .

 

The second stage in the SPTH, which is not seen in extant organisms, Series II, for both dyads (row B) and tetrads (row C). In this depiction of the model, Series II represents a hypothetical intermediate scenario in which sporopollenin deposition is retained both on the sporocyte wall and on the resultant meiospores. Meiospores are coated as centrifugal deposition of sporopollenin continues during meiotic cell divisions. The topology of sporopolleninous wall formation is constrained by the notion of centrifugal deposition: pulses of sporopollenin deposition can only occur as a coating of a single cell. In row B we have only shown sporopollenin deposition as occurring at the last stage of dyad development, resulting in a dyad pair, however, one could also envisage sporopolleninous wall formation as occurring prior to M2, in which case each of the resultant dyad pairs would also be enclosed by an envelope.

In any case, it is worth noting that the dispersed form of an enclosed dyad pair is not found in the post-Dapingian spore record. Thus, it is unclear whether or not the fossil record supports an intermediate phase in the SPTH in which sporopollenin is retained on both the sporocyte wall and the enclosed meiospore walls. For tetrads this determination of the retention of a sporopollenous sporocyte wall is bit trickier, because envelope-enclosed cryptospore tetrads (e.g., Velatitetras) are well known in post-Dapingian assemblages. Still, it is possible that an enclosing envelope in cryptospore tetrads may have been homologous to some form of perispore—a coating that was initiated centripetally from a surrounding tapetum, or tapetum-like inner sporangial lining.

 

Series III depicts more evidence of the SPTH, but without hypothesizing retention of sporopollenin deposition in the initial sporocyte wall. The model depicts latent sporopollenin deposition as either occurring prior to M2 (row D) to produce pseudodyads, or after M2 (row E), in which case enclosed dyads are produced. What is intriguing here is that both of the topologies seen in the model are common in post-Dapingian cryptospore assemblages. Pseudodyads have always been somewhat problematic because they often do not display clear separations between the members of the dyad pair; Strother and Traverse (1979) originally interpreted them as diacrodioid acritarchs, mistaking internal thickening demarcating thetwo internal spore-bodies for external, arcuate folds. Basically, the exact nature of a contact surface between spore-bodies in a pseudodyad is indeterminate. The individual spore-bodies are depicted here, as consisting only of a plasmalemma, although, in many instances a cell wall probably was also in place. The wall topology of enclosed dyads can vary as well, as the outer wall (envelope) can either be free from the contact region, or converge at the contact area as depicted here at the end of row E.

 

Sporopollenin transfer is considered complete where there are no longer any intermediate traces of sporopollenin deposition in the morphology/topology of dispersed cryptospores. That is, sporopollenin walls occur only on the walls of meiospores, without any further envelopes or enclosing membranes. Series IV, in which row F represents the production of free dyads, and row G represents that of free tetrads. These forms first occur in the Darriwilian and persist well into the Devonian where they are found in the sporangia of cryptophytes and tracheophytes.30

 


Fig-6- Diagram showing possible developmental pathways involved in producing common cryptospore topologies in relation to the sporopollen in transfer hypothesis (SPTH)

 


Series I A Model showing meiosis and zoöspore formation in Coleochaete. Here after DNA endoreduplication, 8–32 zoöspores are produced after cytokinesis occurs. Series II represents a hypothetical first step in the SPTH in which a sporopollenous wall is retained on the sporocyte initial. In row B, meiosis is successive, with the first meiotic division (M1) separated in time from the second meiotic division (M2). This results in an enclosed dyad pair. In row C, meiosis is simultaneous and an enclosed tetrad (=Velatitetras) results. Series III posits the SPTH in an intermediate stage in which sporopollenin-containing walls are produced at different stages in successive meiosis. In row D, sporopollenin deposition occurs on the two intermediate cells that result from meiosis 1, but is incomplete or missing after meiosis 2 (M2), resulting in a pseudodyad. In row E, sporopollenin deposition takes place on both of the spores of the dyad pair, in addition, to the surrounding, intermediate wall. The resulting enclosed dyads may be free from the synoecosporal wall or attached, as depicted here in the dispersed form. Series IV demonstrates the completed SPTH state in which free dyads (row F) and free tetrads (row G) are the only cells with sporopollenin walls.31

 

Isolation and purification of sporopollenin:

In order to obtain sporopollenin, the exine has to be separated from the cytoplasm, the intine and from the associated remnants in the interstices and on the exine surface. Sporopollenin can be isolated from spores or pollen grain by treating with solvents or enzymes that remove intine and cytoplasm. Purified sporopollenin retains the similar shape, size and surface features as in its spore or pollen grain and remains an empty shell i.e. sporopollenin microcapsule.5

 

Exine isolation:

Pollen exines were obtained by the procedure based on anhydrous hydrogen fluoride. A pollen sample of 100–200mg, dried under vacuum, was suspended in 1 or 2ml of a solution of anhydrous hydrogen fluoride in pyridine (Aldrich Chemical) in a plastic vessel sealed with parafilm (American National Can) and incubated for 5 h at 40°C. Pollen grains were pelleted by centrifugation at 3500g for 5 min at room temperature, and washed several times with distilled water until the pH was 6–7 (estimated by pH indicator strip). Two to three additional washes with ethanol and a final wash with methanol were performed prior to desiccation by evaporation. Glass lab equipment must not be used when working with hydrogen fluoride. Sporopollenin purification and dissolution Sporopollenin was first separated from phenolics and other components. Lignin like, phenolic material, was removed by refluxing the samples in 0.1 N HCl in 1,4 dioxane for 2 h (acidolysis). In addition, fatty acids and aromatic compounds linked by ester bonds were removed by refluxing in 1% KOH in methanol for 6 h (saponification). After each chemical treatment, samples were washed several times with ethanol, once with methanol, and desiccated by evaporation at room temperature. Sporopollenin purified from an aromatic domain as well as from phenolics and fatty acids was obtained after these procedures. Sporopollenin dissolution was achieved using an ozonolysis procedure similar to the one described by Metzger and Casadevall (1989). Purified sporopollenin samples (40mg) dissolved in 5 ml CH2Cl2 were ozonized under an oxygen stream containing 5% (v/v) ozone. The ozone flux was obtained using a Sander ozonisator model 300 (Germany). Initially, the samples were ozonized at 0°C for 3 h. However, this treatment did not completely dissolve the sporopollenin. Therefore, an exhaustive ozonolysis treatment was used in which samples were ozonized for 24 h at room temperature. Sporopollenins from the four species were completely dissolved by this treatment. After ozonolysis, the organic solvent was removed in a N2 stream and the ozonides decomposed by refluxing with H2O2 30% (1.5ml) and HCO2H (3ml) for 1.5 h. The acid mixture, extracted with Et2O, was derivatised using N,O-bis-(trimethylsilyl)- acetamide to form the corresponding trimethylsilyl esters. The esters were analysed by GC-MS in a Hewlett-Packard 5890 chromatograph with an HP-1 methyl silicon capillary column. Peak areas were recorded by electronic integration.6

 

Isolation and Purification Methods of Sporopollenin

Method

Details

References

Extraction hydrolysis

Extraction with organic solvents i.e. acetone, diethyl ether, etc. Treatment with methanolic KOH and phosphoric acid and in modified forms

Zetzsche, et al., 1928, 1931a, May, et al.,

Ultrasonication

Disruption of pollen wings (Pine pollen) by ultrasonication, centrifugation on a glycerol-water gradient, exhaustive extraction by solvents of increasing polarity, enzymatic hydrolysis

Schulze-Osthoff, et al., 1987

Hydration

Hydration, autoclaving the suspension, differential centrifugation on a discontinuous sucrose gradient

Southworth, 1988

4-methylmorpholine N-oxide treatment

Exhaustive extraction by acetone, incubation in 4-methyl-morpholine Noxide

Loewus, et al., 1985, Baldi, et al., 1987,

Disruption and enzymatic hydrolysis

Disruption of the pollen, treatment by enzymatic hydrolysis, extraction with solvents

Gubatz, et al., 1986, Herminghaus, et al.1988

Acetolysis

Boiling with 1:9 H2SO4 in acetic anhydride mixture

Hemsley, et al., 1993

 

Degradation of Sporopollenin:

Sporopollenin samples were successfully degraded by exhaustive 24-h ozonolysis at room temperature. Gentle ozonolysis (3 h at 0°C) did not completely degrade the biopolymer. The compounds obtained after exhaustive ozonolysis were analysed by gas chromatography-mass spectrometry.6

 

Ozonolysis:

Amongst other chemolyses, ozonolysis was a very common way of decomposing sporopollenin. Resulting ozonides were decomposed by hydrogen peroxides under acidic conditions. Oxidation products were found, by GC, HPLC or GC-MS, to be a mixture of carboxylic acids. Their exact composition appeared to vary from species to species and from workers to workers. Incidentally, ozonolysis was found not to affect the intine, which sometimes remained intact when it was not extracted first.2

 

Ozonolysis is known to cleave double bonds and ether linkages, thus releasing the corresponding aldehydes and acids, under appropriate conditions. This was confirmed on sporopollenin by IR monitoring: reduction of C=C stretching (ca. 1650- 1600cm-1) and C-O stretching peaks (ca. 1150-1100cm-1) parallel to an increase of C=O stretching bands (ca. 1700cm-1). Shaw and Yeadon reduced the acids resulting from ozonolysis so as to change oxygenated carbon to methylenes and obtained a mixture of fatty acids, dominated by palmitic acid. Brooks and Shaw and their co-workers1 showed similar oxidative products were obtained by ozonolysis of sporopollenins and polymers of several carotenoids.2

 

Applications or Advantages of Sporopollenin:

Sporopollenin can be an ingested nutritional supplement and has been reported to help reduce pain, symptoms of depression, and stress. Sporopollenin is non-toxic, non-allergic and biocompatible.5 One of its most profound attributes is that it is known to have a fantastic ability to act like a sponge around substances such as pesticides, toxins, chemicals and toxic metals, soaking them up and binding them together.32 Use of an exine shell of a naturally occurring spore (which term embraces a pollen grain and an endo spore of a micro-organism), or a fragment thereof, as an antioxidant.33 Sporopollenin protects a substance or composition against aerial and/or UV-induced oxidation. Sporopollenin is used as an antioxidant in a formulation containing an active substance. Wherein this active substance is selected from pharmaceutically and nutraceutically active substances, foods and food ingredients, food supplements, herbicides, pesticides and pest control agents, plant treatment agents such as growth regulators, antimicrobially active substances, cosmetics (including fragrances), toiletries, disinfectants, detergents and other cleaning agents, adhesives, diagnostic agents, dyes and inks, fuels, explosives, propellants and photographic materials.  Use of an exine shell of a naturally occurring spore (which term embraces a pollen grain and an endospore of a micro-organism), or a fragment thereof, in the manufacture of a medicament for delivering an antioxidant to a human or animal body.34 A method of screening for a composition comprising an active substance for use as, or inclusion in, a formulation, the method comprising, also measuring an initial oxidative stability value of the active substance. If the post-mixing oxidative stability value of the active substance or composition is equal to, or greater than, the initial value, selecting the composition for use as, or inclusion in, a formulation. A method for reducing the amount of a previously formed oxidation product from an active substance or formulation, the method comprising.

a)   Determining that the active substance or formulation has already undergone at least some oxidative degradation by measuring a property affected by oxidation;

b) Selecting an exine shell of a naturally occurring spore, or a fragment thereof, said exine shell being capable of reducing the amount of a previously formed oxidation product from an active substance or formulation; and

c)   Mixing the exine shell or fragment with the active substance or adding the exine shell or fragment to the formulation to reduce the amount of a previously formed oxidation product from the active substance or formulation.

 

From the above listed methods, wherein the property affected by oxidation is selected from the group consisting of:

1.     The chemical structure of the active substance;

2.     The purity of the active substance in the formulation;

3.     A physical property of the active substance or formulation;

4.     The activity of the active substance

5.     Bioavailability of the active substance;

6.     The taste of the active substance or formulation;

7.     The smell of the active substance or formulation;

8.     The appearance of the active substance or formulation;

9.     The concentration of an oxidation35 product of the active substance in the formulation; and/or

10.Shelf-life of the formulation. 36

 

Dosage form comprising an exine coating of sporopollenin:

A pharmaceutical or dietetic dosage form comprising an effective quantity of an active substance37 chemically or physically bound to, or encapsulated within, a support selected from: an exine coating of spores of a plant, moss, fungus, bacterium or algae or a fragment thereof, wherein: the active substance comprises one or more drugs, one or more dietetic substances, or a mixture thereof; the coating comprises sporopollenin derived from plant, moss, fungus, bacterium or algae spores; the dosage form is in the form of a foodstuff or a pharmaceutical selected from the group consisting of a tablet38,39, a capsule, a soft gel capsule40, an ovule, an elixir41, granules, an inhaleable formulation comprising an inhaleable carrier, a suppository42, a pessary, a gel, a hydrogel lotion, a cream, an ointment43,44, a dusting powder and a skin patch; and any spore protein present in the dosage form is present at less than 0.5% of the exine coating.  A pharmaceutical or dietetic dosage form comprising an effective quantity of an active substance chemically or physically bound to, or encapsulated within, a support selected from: an exine coating of spores of a plant, moss, fungus, bacterium or algae or a fragment thereof, wherein: the active substance comprises one or more drugs, one or more dietetic substances, or a mixture thereof; the coating comprises sporopollenin derived from plant, moss, fungus, bacterium or algae spores; the dosage form is in the form of a foodstuff or a pharmaceutical selected from the group consisting of a tablet, a capsule, a 2 soft gel capsule, an ovule, an elixir, granules, an inhaleable formulation comprising an inhaleable carrier, a suppository, a pessary, a gel, a hydrogel lotion, a cream, an ointment, a dusting powder and a skin patch; and the exine coating is obtainable by a process comprising treating a spore with a solvent, an alkali45 and an acid46 and is formulated without additional spore protein.47

 

CONCLUSION:

In this review article, we come to know that, the sporopollenin is an ubiquitous and extremely chemically inert biopolymer.  Sporopollenins have very similar chemical structures and to be identical in structure to the synthetic carotenoid polymers. Sporopollenins are strongly osmophilic. The sporopollenin remains unchanged when heated up to 3000C or treated with concentrated acids and bases. The different methods developed to isolate sporopollenin from L. clavatum proved its exceptional stability and chemical inertness. Ozonolysis is known to cleave double bonds and ether linkages, thus releasing the corresponding aldehydes and acids, under appropriate conditions.

 

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Received on 30.08.2019                                   Modified on 17.09.2019

Accepted on 04.10.2019                                 © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(3):1555-1564.

DOI: 10.5958/0974-360X.2020.00282.6