**3. Biological source of the polysaccharide fraction**

216 The Complex World of Polysaccharides

between the species of fungi.

**OH**

**OH**

**Isolichenan**

**OH**

**OH**

**OH**

**OH**

**O**

**O**

**O**

**OH**

**O**

**Lichenan**

**O**

**O**

**OH**

**OH**

described that the fungal wall is a complex structure composed typically of chitin, 1,3-- and 1,6--glucan, mannan and proteins, although wall composition frequently varies markedly

**O**

**OH**

**O**

**OH**

**OH**

**HO**

**O**

**OH**

**OH**

**O**

**O**

**O**

**<sup>O</sup> HO OH**

**OH**

**Galactomannan**

**O**

**O**

**HO**

**OH**

**OH**

**Figure 1.** Structures of some polysaccharides contained in fungal cell wall

**O**

**O**

**OH**

For the separation and isolation of lichen polysaccharides, traditional methods and modern techniques are used. Traditional methods basically involved freezing and thawing of the material originally extracted with hot water. Dialysis and ethanol precipitation has been employed for further purification. Also, alkali solutions have been used to extract these compounds. The polysaccharides present in the thalli of *Umbilicaria mammulata* were isolated by an exhaustive method starting with successive extraction with hot water at 100oC followed by extraction of the residue with hot 2% KOH at 100 oC. The alkaline extract was further treated with Fehlings solution to yield the 1,3-β- glucan from the supernantant and a 1,6-β- glucan was obtained from the precipitated Fehlings complex (Carbonero *et al*., 2006). In our laboratory, we have used a similar alkaline extraction procedure to isolate a polysaccharide from *Usnea* cf. *cornuta* Körb. Chromatographic techniques and filtration devices were utilized to separate lichen polysaccharides (Paulsen *et al*., 2002). However, different column chromatographic methods, including GP-HPLC, HPLC and ion exchange chromatography have been used recently to separate polysaccharides from lichens. Olafsdottir *et.al* (1999), fractionated the polysaccharides from *Cetraria islandica* on DEAE Sepharose CL-6B anion exchange columns with a 0 -1 M NaCl gradient. Further purification was performed by Sephacryl S-400 gel filtration. To determine homogeneity and *M*r of these Whether these polysaccharides are produced by the mycobiont or photobiont separately or in symbiosis has been debated for a long time. Takahashi *et al*. (1979) showed that aqueous extracts from cultivated myco and photobiont had different monosaccharide composition and physical properties. It was also found that while the extracts of the mycobiont had a similar composition to that of the parent intact lichen, the photobiont fractions were different from those of the symbiotic thalli and its mycobiont. Complete structural analysis by Cordeiro *et al*. (2004) confirmed Takahashi's results, and showed that the nigeran, laminaran and the galactomannan, found previously in the symbiotic thalli of *Ramalina peruviana* (Cordeiro *et al*. 2003) were also generated by the aposymbiotically cultured fungal partner.

Three different polysaccharide structural types: β-glucans, α-glucans (linear or lightly substituted), and galactomannans (branched) (Carbonero *et al*, 2005a) are present in the fungal cell wall. According to Olafsdottir and Ingólfsdóttir (2001), all the polymers present in lichen thalli are categorized into glucan type [β-(1→3)(1→4)], lichenan type [α- (1→3)(1→4)] and pustulan [β-(1→6)]. Some of these types are depicted in Table 1. However, the recent discovery of a few additional complex heteroglycans, such as rhamnogalactofuranan (Olafsdottir, 1999), galactomannoglucans (Woranovicz-Barreira, 1999 and Carbonero *et al.*, 2002) and thamnolan (Carbonero *et al.*, 2005a) necessitated the closer examination of the origins of the polysaccharides. Studies carried out by Cordeiro *et al.,* (2005), revealed that the algal partner (*Trebouxia* sp.) also consists of some carbohydrates such as β-galactofuranan heteropolysaccharide. Later in 2007, new polysaccharides xylorhamnogalactofuranan and Xylan in the photobiont *Asterochloris* sp. were recorded by Cordeiro *et al.* Thereafter, Jensen *et al*. (2010), isolated another heteropolysaccharide called colleman from a cyanobacteria present in the lichen *Collema flaccidum*.

In the past, lichen polysaccharides have been extracted from the whole thallus without giving consideration to the origin of components such as the fungal partner or the photobiont (Gorin and Iacomini, 1984, 1985; Gorin *et al*., 1993; Teixeira *et al*., 1995). Later Cordeiro *et al*. (2005, 2007, 2008), found that, dissimilarities between the polysaccharides extracted from the cultivated photobionts *Trebouxia* and *Asterochloris* with those from their respective lichens could indicate that the photobiont or mycobiont was responsible for these differences. Galactoglucomannans were isolated from the mycobiont of *Parmotrema* species including *Parmotrema austrosinense, Parmotrema delicatulum, Parmotrema mantiqueirense,* 



**Table 1.** Summarized data of lichen polysaccharides and their origin

218 The Complex World of Polysaccharides

Lichenan Homoglucan

Isolichenan Homoglucan

Pustulan Glucan with

Everniin Glucan with

Acroscyphan Homoglucan

Galactoglucomannans

Xylorhamnogalactofuranan with β(1→3) (1→4) linkage

with α(1→3) (1→4) linkage

β(1→6) linkage

α(1→3) (1→4) linkage

with α(1→3) (1→4) (1→6) linkage

a (1→6)-linked main chain of �-

(1→3)-linked galactofuranosyl O-2 and O-4 by α-Galp and β-

galactofuranosyl units 5-*O* and 6- *O*-substituted, rhamnopyranos yl units 2-*O*, 3-*O*  and 2,3-di-*O*substituted in position 6.

Galp nonreducing end-units

Manp

**Polysaccharide Main unit Side chain Lichen species Mycobiont/**

**Photobiont** 

Whole thallus


*Evernia prunastri* -do- Shibata, 1973

*Cladina confusa* Photobiont Cordeiro



mycobiont Carbonero

*et al*., 2005b

*et al*., 2007

*Cetraria ilandica, C.* 

*nivaris, C. richardsonii*, *Usnea barbata, U. lingissima, U. bayleyi, Parmelia tinctorum, P. conspersa, P. hypotrypella, P. nikkoensis, Alectoria* 

*sulcata, A. sarmentosa*

*esculenta*

*Acroscyphus sphaerophoroides* 

*Parmotrema austrosinense, P. delicatulum, P. mantiqueirense, P. schindlerii, P. tinctorum and* 

*Rimelia (R. cetrata*  and *R. reticulata)*

*Lasallia pustulata, L. papulosa, Umbilicaria hirsuta, U. angulata, U. caroliniana, U. polyphylla,Gyrophora*

**reference** 

Shibata, 1973

*Parmotrema schindlerii, Parmotrema tinctorum* and *Rimelia cetrata* and *Rimelia reticulata* by Carbonero *et al*., in 2005b. These galactoglucomannans consisted of (1→6)-linked main chain of α-Manp units, which were substituted preferentially at O-2 and O-4 by α-Gal*p* and β-Gal*p* nonreducing end-units, respectively. Further, they also isolated two galactomannan fractions from the lichen, *Roccella decipiens*. One galactomannan fraction had a main chain with (1→4)-linked α-D-Man*p* units, substituted at O-2 with side chains containing a nonreducing end, 2-O- and 6-O-substituted α-Man*p* units. The other fraction had a similar α-D-Man*p* core structure, but with side chains containing nonreducing end, 5-O-, 6-O-, and 5,6-di-O-substituted β-D-Gal*f* units (Cordeiro *et al*., 2005). Another heteropolysaccharide xylorhamnogalactofuranan was isolated from the lichen *Cladina confusa.* It consisted of (1→3)-linked galactofuranosyl units with side chains of galactofuranosyl units 5-*O* and 6-*O*substituted, as well rhamnopyranosyl units 2-*O*, 3-*O* and 2,3-di-*O*-substituted at position 6. Nonreducing end units were composed of Xylose (Cordeiro *et al*., 2007). In 2006, (1→3)-and (1→6)-linked β-glucans, namely laminaran and pustulan and galactofuranomannan which have a main chain of (1→6)-linked α-mannopyranosyl residues, partially substituted at O-2, O-4 were isolated from another lichen *Umbilicaria mammulata* (Carbonero *et al*., 2006). Later in 2008, Cordeiro *et al*. found Galactofuranose-rich heteropolysaccharide from the lichen *Ramalina gracilis*. This polysaccharide has (1→5)-linked galactofuranosyl units at the main chain with very complex branched structures of side chains in position 6. They found that this polysaccharide arose from the algal symbiont *Trebouxia sp.* of the lichen *Ramalina gracilis*. An O-methylated mannogalactan was isolated from *Peltigera aphthosa* by Cordeiro *et al*. in 2010. This consisted of (1→6)-linked β-galactopyranose main chain partially substituted at O-3 by β-Gal*p*, 3-OMe-α-Manp or α-Man*p* units. The algal symbiont *Coccomyxa mucigena* of the lichen *Peltigera aphthosa* was thought to be the origin of this heteropolysaccharide since the lichen thallus yielded a polysaccharide of different structure. A colleman like heteropolysaccharide was isolated from the cyano lichen *Collema flaccidum*.

Colleman is a complex heteroglycan containing the unusual monosaccharides 2-OMe Man*p* and 2-OMe-Arap as well as Xyl*p* and Gl*cpA* (Jensen *et al*., 2010). The presence of uronic acids has been reported previously from cyanobacterial polysaccharides. Since the structural features and sugar content of colleman is representative of polysaccharides of cyanobacterial origin, it is proposed that colleman originates from the cyanobacterial partner. Ruthes *et al*. (2010) were able to isolate (1→4)-linked β-D-xylan (an EPS) and heteropolysaccharide with a complex structure of β-L-Ara*p* and β-D-Xyl*p*-(1→4)-linked units from *Peltigera canina*. Again it was opined that the photobiont, *Nostoc muscorum* was the source of this heteropolysaccharide.
