Chapter 2. Review of Literature Review of Literature

chapter 2. Review of Literature Review of Literature

Peter Adolf Karsten inl881 used Polyporus lucidum (described in 18"^ century by William Curtis) to establish the Genus Ganoderma with the type species G. lucidum. Number of species has been described in the genus Ganoderma thereafter. Patouillard in 1889 specified the double wall character of the basidiospore and also published a monograph of 48 species. He also transferred several names to this genus and defined the spherical and sub-spherical spores of specimens from the section Amauroderma. The genus Ganoderma was divided in two sub-genus, Ganoderma and Elfvingia by Karsten in 1889 (Steyaert, 1980). The sub-genus Elfvingia was based on the name Boletus applanatus (described by Persoon) and the section was dedicated to the species with non-laccate pileated fruit body.

Different taxonomic characters have been used for identification, such as Murill (1902, 1903) studied the species from the subgenus Ganoderma and Eljvingia using micro and macromorphology of the basidiocarp. Atkinson (1908) used the geographical distribution along with the morphology to separate the Polyporous lucidus and allied species from Europe. Coleman (1927) has discussed the spore structure with special reference to the spore wall, especially from Ganoderma applanatum, G. tsugae, G. tornatum, G. lobatum and G. lionettii. .

Comer (1947) studied the variation in basidiospore, basidia and cystidia in basidiomycetes. He discussed the close interliiik between the Ganoderma applanatum and Amauroderma using the basidia and spore characters. Steyaert (1962, 1967a, b, 1972, 1980) worked extensively on the genus from nearly each continent of the world, he created many new species or transferred many names to this genus and also removed many synonyms.

Steyaert (1980) divided the subgenus Ganoderma in to Sections Ganoderma and Section Charcoderma, and also made two new subgenera Anamixoderma and Plecoderma, Section Ganoderma characterized with hymenioderm with swollen element (fastigiated), more or less cylindrical, each up to 4 - 5 times the diameter of the terminating hypha. Section Charcoderma characterized with hymenioderm with non swollen elements, each equal to or slightly thicker than the diameter of the hyaline hypha which terminates;

17 Review of Literature

sometimes a few or the majority are capitate by a spherical or by a spheroid inflation. Sub genus Elfvingia was maintained separate to incorporate the species with dark red brown context concolourous with the tube layer with Ganoderma applanatum, type species. Apart from these sub-genera, other two sub-genera have been mentioned one with hyaline hyphal ends intermix with brown hyphae, whole embedding in melanoid substances namely sub genus Anamixoderma. While the other sub-genus Plecoderma with cutis of hyaline hyphae densely entwined and impregnated by melanoid substances, forming a layer sharply distinct from the context.

Stalpers (1978) used the cultural characters for identifying the wood inhabiting aphyllophorales, describing 15 species o{ Ganoderma. He referred to a work similar to that carried out by Nobles in 1958.

Bazzalo and Wright (1982) surveyed and reported the species from Ganoderma lucidum group of Argentina. He used the basidiospores and gasterospore for identifying the species. The cultural characters of the studied specimens alone were insignificant to distinguish them.

Zaho and Zhang (1995) have given the distribution of various species from the different environmental conditions of China. Ganoderma haninanense, G. atrum and all species of Amauroderma grow in tropics and subtropic; G. sinense and G. lucidum in the temperate regions with comparatively less rain; G. tsugae in low temperature regions with less rains while in the fourth class, variety of species tolerant to wide environmental conditions are distributed through out the country e.g. G. applanatum. Various species of Ganoderma generally grow between latitude 45° N and 25° N. Ganoderma tsugae is dominant in the North east of China, where as G. lucidum in Yellow river valley, but it is not at all found in Heilongjian region (Zaho and Zhang, 1995).

Baxter and Eieker (1995) listed the Ganoderma taxa from southern Africa. According to them 38 epithets have been recorded from the said region and only 20 species of Ganoderma actually exists, considering the synonymies.

18 Review of Literature

The Ganodermataceae of New Zealand is represented by only four species (Buchanan and Wilkie, 1995), out of which one non laccate species is misidentified as Ganoderma tropicum, one unidentified and the other two are G. australe and G. cf. applanatum.

Ryvarden (1995) questioned the morphology of Ganoderma. He studied 53 specimens of G. lucidum from Norway for the morphological variations. He concluded in judging the following morphological characters: a. Shape and size of basidiocarp are doubtful and atleast 3-5 collections should be examined. b. Colour of pileus and stipe changes with age and should be carefully considered. c. Pore size is a valuable taxonomic character as it is constant. d. Colour of pore surface and context changes over time so specimens of different age should be examined. e. Hyphal system is of less help as majority species of G. lucidum group has Trimitic hyphal system (but latter on Ryvarden (2000) reported 15 species from lucidum group with dimitic hyphal system). f Size and shape of apical pilear cells (cutis/ dermal elements) is another reliable and constant character and valuable for taxonomic separation of species in at least lucidum group. g. Basidiospore may vary in size and shape and hence should be used carefully.

The important thing noted by Ryvarden (1995) is 1-2 collections are insufficient to describe a species unless there are striking microscopic characters coupled with distinct macromorphological features.

Gottlieb & Wright (1999a; b) carried out their studies using the macro and micromorphology, of the subgenus Ganoderma and Elfvingia respecively. In the study of subgenus Ganoderma spore index and spore ornamentation were used along with good characterization of cutis in three major types: a) Hymeniodermis vera (composed of cylindrical, claviform and smooth elements); b) Hymeniodermis formed by diverticulated hyphae (lateral branching, acanthophyses-like) and c) Hymeniodermis spheroid pedunculate or capitate knobs at apex of the element.

19 Review of Literature

Recently modem methods like isozyme analysis are used to prominently for phylogenetic studies. Park et. al. (1994), Gottlieb et. al. (1995, 1998), and Gottlieb & Wright (1999a) used this technique to discrete the species of Ganoderma. Smith and Sivasithamparam (2000) studied isoenzymes of five Australian species using Cellulose acetate gel electrophoresis (CAGE) and PAGE. Another modem biotechnological tool used is Random Amplified Polymorphic DNA/Restriction Fragment Length Polymorphism (RAPD/RFLP) and DNA sequencing, RADP analysis is thought to be helpful for systematic at lower taxonomic levels to distinguish isolates from each other. Moncalvo et. al. (1995 a, b, c) have used ribosomal DNA sequencing as tool for analyzing phylogenitic relationship in Ganoderma lucidum complex. Hseu et. al. (1996) used RAPD - Polymerase chain reaction (PCR) and intemal transcribed spacer (ITS) sequences to differentiate the isolates of G. lucidum complex, the authors concluded that ITS sequences can be used for identifying isolates of G. lucidum complex, whereas RAPD can be used to differentiate between isolates having identical (ITS) sequences. Moncalvo and Ryvarden (1997) published a world list of Ganoderma species. The study considered the species described in last 200 years i.e. from 1781, species described William Curtis up to the study. The authors did not described any species but recorded the nomenclatural and taxonomic data as have been reported by various authors. In India, Bakshi (1971) contributed to study of this genus, he prepared key to Indian species, describing five species, which till date have not yet been updated, viz., Ganoderma lucidum, G. applanatum, G. philippi, G. australe and G. colossum. Bilgrami et. al (1991), recorded 'Fungi of India,' which included seven species, viz., Ganoderma lucidum, G. leucophaeum, G. colossus, G. australe, G. applanatum, G. annulare and G. adspermum, but these species have not been described by them. Gade (1992) and Hiremath (1993) worked on the pathological aspect of Ganoderma lucidum on the Arecanut (Areca catechu L.) from the Konkan region. The identification of the species reported was doubtfully described as G. lucidum. Whereas some of the above said specimens are also reported in various databases/ checklists of various states, e.g., G. lucidum reported in the list of fungi from Tamil Nadu (http://thallsvr.tn.nic.in/envis/CheckListFungi.htm^) and also in the biodiversity list of Punjab (http://www.punjabenvironment.com/bdJist.htm^). Many times the species used are reported as G. lucidum, in various publications other than taxonomy, their authenticity is doubtful.

^ Online Reference 20 Review of Literature

B. ANALYTICAL STUDIES:

Modem molecular and analytical tools and techniques have enabled researchers to study the biochemistry of Ganoderma. More than 300 reports have been published concerning the chemical constituents of Ganoderma and their pharmacological activities (Gao and Zhou, 2003). The general composition has been studied as nutritional components, non-volatile components or proximate analysis (Mau et. al; 1998a and b; Mau et. al; 2001; Cheung, 1997; Gao and Zhou; 2003 and Tseng et. al; 2005). There are many reports giving complete biochemical/ nutritional composition of Ganoderma. The table 2-1 represents the general composition of G. lucidum as in Gao and Zhou (2003).

Table 2-1: General nutritional components of Ganoderma lucidum (gm lOOgm"' dry matter)

Components Wild Cultured Extracted Moisture 10 9.8 4.0 Crude protein 11.82 9.71 23.70 Crude fat 2.51 6.21 2.40 Total carbohydrates 25.5 22.9 59.10 Crude fibers 48.80 41.0 ND* Crude ash 1.63 1.41 4.2

*ND: Not detected.

G. tsugae has been studied by Tseng et. al. (2005), the report considered two stages of basidiocarp (mature: Ling chih and immature: baby Ling chih), mycelia and filtrate for analysis. Variation in component concentration was also observed in the different stages of G. tsugae. Moisture content was higher in mycelia than that of basidiocarps, carbohydrate content was found to be high in the filtrate followed by mycelia. Ash content was less than 2gm lOOgm'', whereas crude fiber was found high in mature basidiocarps. The study also comprised of quantitative analysis of amino acids (17 different amino acids considered, consisting of 8 essential amino acids) using HPLC and based on the amino acid composition taste characteristics was determined. Apart from this 5' nucleotide contents were determined (5'AMP, 5'CMP, 5'GMP, 5TMP, 5'UMP and 5'XMP). Mau et al.

21 Review of Literature

(2001) studied similar parameters of several medicinal mushroom namely Ganoderma lucidum (mature and antler stage), G. tsugae and Coriolus versicolor. Mau et. al (1998b) studied flavor compounds of Pleurotus eryngii wherein 7 volatile compounds were reported along with 15 amino acids and other parameters as stated above (Mau et. al; 2001), while in a similar study non volatile taste components from 3 species of Auricularia and from Tremella fuciformis were analyzed and based on the amino acid composition taste characteristics was determined (Mau et. al; 1998a).

Cheung (1997) studied the dietary fiber content and composition of some edible mushrooms including Ganoderma lucidum. The author compared the results obtained using two different methods. The main component focused was total dietary fibers (TDF), along with proximate analysis of the samples was also carried out. The TDF calculated by Uppsala method was by addition of neutral and aminosugars, uronic acids and Klason lignin, whereas by the AOAC (Association of Official Analytical Chemists) method TDF was calculated by addition of fiber residue (crude fiber), proteins and ash (which is followed in the analytical studies of medicinal mushrooms).

Amino acid composition of several cultivated mushrooms from Finland has been reported by Mattila et. al. (2002). The mushrooms studied were Agaricus bisporus (white and brown strains), Pleurotus ostreatus and Lentinula edodes. The study considered 17 different amino acids including 8 essential amino acids. A comparative account of essential amino acids from different vegetables and mushrooms has been reported, showing that majority essential amino acids are observed in higher concentrations in mushrooms than vegetables, with a significant difference in case of tyrosine, leucine, cystine, phenylalanine and threonine. The study also reports the proximate values of the studied mushrooms. Mattila et. al. (2001) studied vitamins, minerals and some phenolic components from the above said mushrooms. The minerals considered were Ca, K, Mg, P, Na, Cu, Fe, Mn, Zn, Se, Pb and Cd. Whereas Mizuno et al. (1988) screened 30 different Ganoderma lucidum samples (wild as well as cultivated) along with Fomitopsis pinicola, Ischnoderma resinosus, Grifiola frondosa, Polyporus confluens, Agaricus blazei, A. bisporus, Hericium erinaceum and Pleurotus ostreatus. The mineral elements considered were same as above

22 Review of Literature

with additional three viz.. Mo, B and Ge. According to the report germanium content of aphyllophorales mushrooms is the highest as compared with other herbal drugs.

Petropavlovskii et. al. (2001) reported moisture content along with the extractive values of some cultivated wood rotting mushroom which included two strains of Ganoderma applanatum. \\ was the only reference found on the extractive values of Ganoderma samples. The authors used water, mixture of alcohol and benzene, and 6% NaOH. In pharmacognostic studies of crude drugs (Kokate, 1997; Daniel, 1982) generally use of alcohol is recommended separately and instead of benzene (now banned for its toxicity) solvent ether is used wherein crude fats are separated. For NaOH fraction the concentration mentioned is 5%, but Petropavlovskii et. al. (2001) used 6% NaOH.

Apart from the analysis of general composition, Ganoderma samples have been analyzed for vitamins, alkaloids fatty acids etc (Huie and Di, 2004). Choline and betaine, two known alkaloids have been isolated from spores of Ganoderma lucidum, whereas two novel pyrrole alkaloids (Ganoine and ganodine) have been reported from the mycelia of G. capense.

C. CULTURAL STUDIES:

Ganoderma being medicinally important, lot of work on cultural studies could be found. The cultural work was initially focused on the cultural characteristics to discriminate the species, taxonomical approach. Adaskaveg and Gilbertson (1986) describe the cultural relations between Ganoderma lucidum and G. tsugae, while Hseu et. al. (1988) used the incompatibility studies of these species to identify these species. Similarly, Stalpers (1978) segregated numerous wood rotting aphyllophorales using more than 100 different culture characters.

But in latter 90's the commercialization of the mushroom boosted and cultivation technology needed to be modernized looking at the global market. Use of different media compositions, development of commercial strains using different tools like protoplast fusion (Cha and Yoo, 1997b) etc. were used. Used of submerged culture and solid state fermentation was the major focus in the last decade of the last century and in the last few

23 Review of Literature

years (Hsieh and Yang, 2004). There are some reports of growth responses to different cuUural conditions (Jung et. al; 1996, Sung, et. al; 1996, Garibova, et. al; 2003, Gong and Wang, 2002) but were in local language, i.e. Chinese, Korean, Russian etc. and hence could not be considered. Growth responses of Ganoderma and other wood rotting aphyllophorales have been well studied by Stalpers (1978). He described the culture morphology and physiology including the growth rate. The highest was observed in G. resinaceum, (5.5 to 7.0cm per week). Buzzalo and Wright (1982) studied culture characteristics oi Ganoderma lucidum, G. resinaceum, G. zonatum and G. subamboinense.

Wagner et. al. (2003) reviewed the techniques used in production of biomass, polysaccharides and Ganoderic acids from Ganoderma lucidum. They have summarized the published data of the submerged fermentation and solid-state fermentation and reported variety of substrate compositions (54 different compositions) used in 15 different referred articles. Apart from submerged cultivation of Ganoderma, solid-state fermentation has been regularly reported from Ganoderma, but only one large scale fermentation tank with 20m^ volume has been successfully erected using G. tsugae (Wagner et al; 2003). While all other experiments were less than lOL volume. According to them, out of the reports, half of the trials were conducted in Erlenmeyer flasks and some in bioreacters. According to the review the best biomass yield till date was 22.1gm L''.

Production of biomass is secondary in many studies; the studies are mainly focused on enhancement of production for polysaccharide and ganoderic acids. The best results observed by Wagner et al. (2003) is 1.7gm L"' extra cellular polysaccharide, 2.49gm L"' intracellular polysaccharide and 582mg L'' Ganoderic acids.

Lomberh et al. (2002) studied different parameters of submerged cultivation of some medicinally important mushrooms including Ganoderma lucidum and G. applanatum. The optimum pH was in the range of 4.75 -6.0 in selected media. Out of the four media studied for optimal time to achieve inoculums showed varied results for Ganoderma strains with 6 and 7days on PDA and MEA respectively for G. lucidum, while 8 and 7days for G. applanatum on MEA and Oat agar media respectively. The maximum biomass for G. lucidum obtained was 21 to 25gm L" .

24 Review of Literature

Cultivation of medicinal mushroom has been a tradition in Japanese or Chinese culture. Cultivation of shiitake mushroom {Lentinm edodes) has been carried out for hundreds of years (Bhosle and Vaidya, 2001). Unlike shiitake, Ganoderma does not have extensive cultivation history, the reasons could be its non edible form or it caught the attraction of scientist very late in 20^'^ century. The scientific record of Ganoderma cultivation atleast in India is nearly a century old, Bose (1929), isolated pure culture of 6". lucidum from spores, reported the in vitro carpogenesis and basidiosporogenesis and also cultivated sporocarp in vitro (on wood logs).

Trirantana et. al. (1991) discussed the selection of sawdust for substrate formulation in Ganoderma lucidum cultivation. The authors used composition of various sawdust-agar media to assess the growth response and also screened different supplements along with the sawdust. Recently Stamets (2000) discussed the culture and cultivation details of some commercial strains of G. lucidum.

Though the cultivation history of Ganoderma is short but the technology has developed very fast in last 3 -4 decades. Many commercial companies developing nutraceuticals from various forms of Ganoderma (sporocarp, mycelial biomass, broth etc) exists in South East Asian countries, China is the leading country for production of G. lucidum with respect to yearly production. Acquiring the exact figure of annual production oi Ganoderma is very difficult (Chang and Buswell, 1999). Chang and Buswell (1999), described in brief the cultivation methodology, according to them, the process involves two stages. The first stage involves production of fruiting and stock cultures along with mother spawn and planting spawn. The second stage involves the preparation of growth substrate. There are arrays of commercially used substrates, viz., wood long, short wood segments, tree stumps and a variety of combination with saw-dust as basic ingredient. Chen (2001, 2002 a,b,c,d) wrote series of articles on Ganoderma cultivation discussing the strains isolation from natural habitant; different media composition for maintaining Ganoderma cultures; strategies for strain selection; different substrate formulations with variety of supplements; use of different inoculation types (grain culture, liquid culture); management of growth parameters (RH, Light, CO2. O2. Temperature etc) and added the list of resources in Ganoderma cultivation. The cultivation parameters changes according to the use of

25 Review of Literature

strains and substrate adopted for cultivation (Chang and Buswell, 1999; Chen, 2001; 2002 a,b,c,d).

Cha and Yoo (1997a) discussed the effect of temperature, carbon and nitrogen sources and pH on the growth of G. lucidum and G. japonicum. The authors reported the effect of different sawdust samples in substrate formulation, concluding that Oak wood sawdust is better than the poplar and also noted the effect of different supplements in substrate formulation. In case of log cultivation, it was reported that sand is the best covering material as compared to sandy loam, clay loam and peat. The efficiency of different Ganoderma lucidum strains has also been discussed. Cha and Yoo (1997b) discussed the strain improvement using protoplast fusion technique in Ganoderma lucidum. Kim et. al. (2001) cultivated nine species oi Ganoderma (including G. lucidum) of different geographical, comparing the biological efficiency and the morphology of the basidiocarps cultivated in the bottles using sawdust medium.

Croan and Kim, (1997) and Croan (1997) have studied the carpogenesis (Carpogenesis: production of basidiocarp from vegetative mycelium in vitro) and basidiosporgenesis of several basidiomycetes including members of aphyllophorales (Trametes versicolor, Antrodia carbonica, Gloeophyllum trabeum). Effect of different carbon and nitrogen source was studied along with light and incubation period on induction of carpogenesis and basidiosporogenesis. Though no report of carpogenesis was found in case of Ganoderma.

D.BIOACTIVE COMPOUNDS I. Polysaccharides:

Polysaccharides are the best known and most potent bioactive compounds in mushrooms with antitumor and immune modulating properties (Wasser, 2002; Mizuno, 1999; Wasser and Weis, 1999). The historic evidence for the use of mushroom polysaccharide could be as old as the tradition of mushroom hot water extract in the eastern countries. Large number of basidiomycetes have been screened for polysaccharides, 651 species and 7 intraspecific taxa from 182 genera of higher hetero and homo basidiomycetes have been studied (Wasser, 2002). An exhaustive list of basidiomycetes studied for the

26 Review of Literature

antitumor activity against. Sarcoma 180 solid cancer and Ehrlich solid cancer have also been listed by Wasser (2002). Numbers of promising candidate still are to be worked out for the potential polysaccharide source and their biological activities. Further, there is an urgent need to study the down stream mechanism of action at molecular level that occur in specific immune modulation such as receptors and the various events triggered by the binding of these polysaccharides to the target cells (Daba and Ezeronye, 2003).

Mizuno (1999) reported a procedure for extraction of polysaccharide, fractionation and purification. This method has been established for isolation of polysaccharides from basidiocarp, spores, culture mycelia and broths. In general, the extraction scheme involves, initial boiling of mushroom material with 80% ethanol to eliminate low molecular weight substances. The residue is then successively extracted with water (100°C, 3hrs), 1% ammonium oxalate (100°C, 6hrs) and 5% sodium hydroxide (80°C, 6hrs). The fractions thus obtained are purified by various methods, v/z., ethanol precipitation, acid precipitation, ion exchange chromatography, gel filtration and affinity chromatography (Mizuno, 1999; Wasser, 2002).

Three polysaccharide based carcinostatic (immuno therapeutic) agents viz., Lentinan, Kerstin and Schizophyllan have already been commercialized from mushrooms namely, Lentinus edodes, Coriolus versicolor and Schizophyllum commune respectively. These are used currently in the treatment of cancer of the digestive organs, lungs, breast, stomach and cervical cancers (Daba and Ezeronye, 2003).

a. Lentinan: Lentinan is a P-D-glucan, shown by electrophoresis and ultra-centrifugation, as well as by other chemical techniques and instrumental analysis and isolated from the mushroom Lentinus edodes {= Lentinula edodes). Lentinan has been studied more extensively than other similar substances. The molecular formula of Lenfinan is (C6Hio05)n, the mean molecular weight is about one million - 5 X lO' Da, [a] D + 20° - 22° (NaOH) (Wasser and Weis, 1999). An efficient method of fractionation and purification of Lentinan given by Yap and Ng (2001) resulted in 87.65% pure polysaccharide. The p-glucan was isolated by ethanol precipitation and freeze drying in liquid nitrogen. Lentinan showed prominent

27 Review of Literature antitumor activity not only against allogenic tumors, but also against various synergic and autochthonous tumors, and prevented chemical and viral oncogenesis. Wasser and Weis (1999) and Bhosle and Vaidya (2001) have discussed the extraction, structure and activities of lentinan in details.

Several species belonging to the polyporaceae are also studied as multipurpose medicines that are not only carcinostatic but also antiinflammatory, antiviral (against AIDS), hypoglycemic and anti thrombotic (Daba and Ezeronye, 2003).

b. Structural features of Polysaccharides: Polysaccharides are a structurally diverse group of macromolecules, these are polymer of monosaccharides arranged in chains with glycosidic linkages. These are linear and also have side branches of either same monosaccharide or may also possess side chain of one or more variety of monosaccharides. The polysaccharides have a much higher capacity of carrying biological information, than nucleotide in nucleic acids and amino acids in proteins, for the reason that these possess great variation in the structure. The monosaccharides can interconnect in various ways to form the polysaccharides (Wasser, 2002).

The major group of polysaccharides are glucans with different types of glycosidic linkages, viz., 1-3, 1-6 p-glucan and a-glucan. (3-glucans are the polysaccharides which yields D-glucose on acid hydrolysis (Mizuno, 1999). Variation also observed in chemical conformation, the structure of schizophyllan (P-D-glucan) has a triple strand right winding, whereas the acidic glucoronoxylomannan from Tremella fuciformis shows left handed three fold helical back bone (Wasser, 2002).

Apart from the variation in backbone, the side chains also vary, the PsG of Ganoderma lucidum (Hsu et. al; 2002). Lentinan of Lentinus edodes possess single type of monosaccharides as a side chain. Whereas some polysaccharides may possess one or more than one type of monosaccharides as the backbone and side chain as well, these are called as heteroglycan. GL-1 from G. lucidum, has branched arabinoxyloglucan backbone and side chain involving D-glucopyranosyl, (l-4)-a and -P or (l-6)-P and xylose as side chain. Similarly

28 Review of Literature heteroglycan are reported from G. lucidum (Miyazaki, 1982 and Sone et. al; 1985) and also from submerged culture of Tremella mesenterica and from T. aurantia, respectively.

In addition to the heteropolysaccharides, proteoglycans i.e., protein polysaccharide complex have also been reported from various mushrooms. These types of complexes are reported to possess more bioactivity than polysaccharide. Herein the backbone of polysaccharide is attached with side chains of peptides as in case of Ganoderan B and C (Tomoda et. al; 1986 and Cao and Lin, 2004). Wasser (2002) has reported various polysaccharides and their structures from different mushrooms having antitumor and immunostimulant activity.

Several polysaccharides have been isolated from the water and alkali extract of the fruiting body of Ganoderma lucidum. Studies even reveal that glucans when modified to D- glucan polyols by periodate oxidation and borohydrate reduction, exhibited higher antitumor activities. Thus, attachment of polyol groups to (1-3) linked backbone significantly enhances the host mediated antitumor effect. Some studies have been carried out to increase the activity or productivity of the P-D-glucan by different chemical modifications which have been discussed in depth by Wasser and Weis, (1999); Wasser (2002).

Overall more than 200 polysaccharides have been isolated from various parts of basidiocarp, spores, culture mycelia or culture broth of G. lucidum (Huie and Di, 2004). Wasser (2002) has reported that seven species of Ganoderma have been studied for their polysaccharide. Some of the polysaccharides from Ganoderma and their activity has been represented in table 2 -2.

The antitumor activity of the polysaccharide is also dependent on its structure, i.e. the activity is influenced greatly by the degree of branching, at least in case of Ganoderma lucidum and few other mushrooms. The activity of polysaccharides decreases with the decrease in the branching. Moderately branched structures show high anti-tumor activity, than that of less branched ones. The reason for the branching affecting the antitumor activity may be partly due to the molecular rigidities of the (l-3)-P-D-glucans in triple helix form. The lower branching is also related to structure of fruit body, which may affect the intermolecular hydrogen bonding and would be characteristic of rigid, hard type of fruit

29 Review of Literature

body as that of Ganoderma lucidum (Sone et. al; 1985). According to Bao et. al. (2001b), degree of substitution on the main chain and the length of side chains are important factors in determining the conformation and the biological activities, whereas the linear structures showed decreased activities. Water solubility of the polysaccharides also plays important role in the activity of the polysaccharides (Bao et. al: 2001b).

Protein bound polysaccharides GLhw, GLhw - 01, GLhw - 02, GLhw - 03 have been isolated from the water extracts of Ganoderma lucidum fruit body. These compounds showed antiviral activity against Herpes Simplex Virus Type - 1 and Herpes Simplex Virus Type - 2. The GLhw - 02 exhibited the most potent antiherpetic activity among the above said compounds (Eo, et. al; 1999).

The polysaccharides being the main active component, the study of characteristics of these polysaccharides, the composition, the molecular weight of these polysaccharides is important in drug designing. Similar data has been developed in case of Ganoderma lucidum. The composition and characteristics of the polysaccharides isolated from Ganoderma lucidum has also been well understood.

Apart from Ganoderma, polysaccharides from some other mushrooms have been listed along with their activity in table 2 -3. A weak antitumor (l-3)-p-D-glucans (T-4-N and T-5-N) have been isolated from Dictyophora indusiata (Hara and Ukai, 1995). The compounds were isolated with alkaline solutions and water. These p-D-glucans showed weak activity than the water extracted (l-3)-P-D-glucans isolated from the other mushrooms like Lentinus edodes, Ganoderma lucidum, Hericium erinaceus.

Table 2 -2-: Different polysaccharides and their biological activities from various species of Ganoderma.

Name of Compound name Activity Reference Mushroom G. applanatum G-Z Antitumor Sasaki er. a/; 1971. G. applanatum G-l-la-p,G-I-2a-p, Antitumor Usui e/. a/,- 1981a. G. applanatum Fucogalactan & — Usuier. al; 1981b. Mannofucogalactan G. applanatum F-I-la, F-I-lalp & F-I- Antitumor Usuie/. al; 1983 la2-p

30 Review of Literature

G. japonicum G-A Antitumor Ukai et. d; 1982; 1983. G. lucidum GL-I Antitumor Miyazaki & Nishijima, 1981. G. lucidum Arabinoxyloglucan — Miyazaki, 1982 G. lucidum Exo-polymer Hepato protective Song e/. a/; 1998. G. lucidum GPS Antigenotoxic and anti tumor Kim et. al; 1999 G. lucidum Fraction A, B, C, D & E Immune stimulatory Habijanic et.al; 2001 (mycelia) G. lucidum GLE «&GLB Antitumor Lin, 2001 G. lucidum GLP Antitumor Lee e/. a/; 2001 G. lucidum GLPP Protection of Macrophages and You & Lin, 2002. other cell organelle G. lucidum GLPs Antiulcerogenic Gao et. al; 2002 G. lucidum PSG Antiapoptotic on neutrophils Hsu et. al; 2002 G. lucidum GLPP Antitumor and anti angiogenic Cao & Lin, 2004 G. lucidum GIPs Neuro protective Zhao et. al; 2004a G. lucidum Glycoprotein Immuno-Modulating, Wang et. al; 2002 Antitumor G. lucidum GLIS Immune stimulatory Zhang et al; 2002b G. lucidum (broth CW-I, CW-II, HW-I, HW-Il, Antitumor Sonee/. a/; 1985. & basidiocarp) CA-ICA-II,HA-I&HA-I1 G. lucidum GLP Hepatoprotective Zhang et. al; 2002a (mycelia) G. lucidum (spores) PGL Immuno-stimulant Baoe/. a/; 2001a. G. lucidum (spores) PSGL-1 Immuno - stimulant Bao et al; 2002 G. lucidum (spores) SP«&SP1 Immune stimulatory Baoe/. a/;2001b. G. tsugae (mycelia) GM3 — Peng and Zhang, 2003 G. tsugae (mycelia) EPFl &EPF2 Antitumor Peng et al; 2003 Ganoderma Ganoderan A, B & C Hypoglycemic Tomodae/. a/; 1986 lucidum

31 Review of Literature

Table 2 -3: Polysaccharides from different mushrooms and their biological activities.

Mushroom Compound name Activity Reference Calvatia gigantea Calvacin Antitumor Wasser&Weis, 1999 Schizophyllum schizophyllan Antitumor communae Wasser «fe Weis, 1999 Antitumor, anti-cancer, Lentinan immunostimulating, antiviral Antitumor, interferon KS-2 (glycoprotein) Wasser&Weis, 1999 Lentinus edodes induction, cytocidal Kime/. a/; 1999 Immunoactivation, Yap and Ng, 2001 LAP Antitumor

LEM, EP3 Immunoactivation, Antitumor Immunostimulating, Tremetes versicolor PSK, Krestin, CPS Antiviral, Antitumor, Kime/. al; 1999 antigenotoxic Mizuno, 1996, Miles Innonotus obliqus Befungin Antitumor & Chang, 1997 Agaricus blazei ATOM. AB-FP Antitumor Wasser, 2002 Auricular ia auricula Antitumor Sonee/. al; 1985 Pestalotia species Antitumor Soneer. al; 1985 Volvariella Antitumor SoneeA al; 1985 volvaceae Antitumor, Sasaki er. a/; 1971, Phellinus linteus Immune modulation Kim et. al; 2004 Antitumor, Falmmulina EA-6, Proflamin Wasser&Weis, 1999 veluticeps Immunostimulating

Dictyophora T.4-N, T-5-N Weak antitumor Ukaier.a/; 1983 indusiata Antitumor, Wasser&Weis, 1999 Grifiola frondosus Immunostimulating Kiho et. al; 2000 Antidiabetic Tremellafusiformis Vinogradov et. al; T. messenterica GXM 2004

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Anticancer, CS F30, CI-P and KihoeA al; 1996 Cordyceps sp. Hypoglycemic, CI-A Immunostimulating Wang & Shiao, 2000

c. Mechanism in antitumor activity of Polysaccharides: The polysaccharides are referred as Biological Response Modifier (BRM; Wasser & Weis, 1999; Wasser, 2002). It means that, these compounds do not cause any harm and place additional stress on body, these help the body to adapt to various environmental and biological stresses and these compounds support some or all of the major body systems, including nervous, hormonal and immune systems. The general mode of mushroom P-D- glucan is it binds to lymphocyte surfaces or serum specific proteins, which activate macrophage, T-helper, NK, and other effector cells. All these increase the production of antibodies as well as interleukins (IL-1, IL-2) and interferon (IFN-y), which are released upon activation of effector cells. Thus the carcinostatic effect of p-D-glucan results from the activation of the host's immune system. The mode of action for Lentinan proposed by Chihara in 1981 represented in fig. 2 -1 (Wasser & Weis, 1999; Mizuno, 1999; Wasser, 2002).

Studies demonstrating the antitumor activity of various polysaccharides from Ganoderma, by induction or stimulation of one or different immune regulatory components have been carried out. (Lin, 2001; Chang, 1996; Habijanic et. al; 2001; Lee et. al; 2001). A similar study in polysaccharide from Phellinus linteus (Kim et. al; 2004) suggests that nitric oxide (NO) levels along with tumor necrosis factor (TNF-a) increases and the cytotoxicity was due to the up regulation of NO and TNF-a. Similarly increase in NO levels due to Ganoderma polysaccharide has been proved to be effective against BCG induced immune liver injury (Zhang et. al; 2002a). According to this study the liver protective mechanism is due to the influence of polysaccharide on NO production.

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Apart from antitumor and anticancer activities, Ganoderma polysaccharides shows variety of other biological activities hypoglycemic, hepato-protective, anti genotoxic, anti ulcerogenic, anti angiogenic etc. (table 2 -2).

Colony General Potentiation o C3a Anaphylatoxin Stimulating Host Resistance Factor

Serum Protein LA, LB, LC etc.

Live> L Target LENTINAN tumor cell

TUMOR ANTIGENS

Figure 2-1: Possible mode of action of Lentinan as host defense potentiator (Chihara, 1981, Redrawn from Wasser & Weis 1999)

II. Terpenoids: Terpenoids are the compounds having isoprene units as their structural units. The general classification is based on the number of isoprene units. C5: Hemiterpene; Cio Monoterpene; C15: Sesquiterpene, C20: Diterpenes, C25: Sesterterpene; C30 Triterpene; while C40 are Tetraterpenes.

Ganoderma has been known for its bitter taste afforded by the various triterpene present in it (mostly lanostane type, fig. 2-2, a). The bitterness of the mushroom is related to the structure of the triterpenes. The hydrophobic methyl group and their relation with the three functional oxygen atoms are the key role players in generating the bitterness. Apart from the functional groups, the structural conformation (boat or chair) also has role for bitterness in G. lucidum (Nishitoba et. al; 1988). In 1982, Kubota and his coworkers reported triterpenes from Ganoderma lucidum for the first time and since than, till date

34 Review of Literature more than 130 oxygenated triterpenes have been isolated from various parts of fruit bodies, culture mycelia and culture media (Huie and Di, 2004). Though a large number of triterpenes have been isolated, only few have been worked for their activities. These triterpenes show activity against an array of ailments (table 2 -4). The triterpenes are called as Ganoderic acids (C30), Lucidenic acid (C27), etc., which are the acid type of triterpene (fig, 2-2, b and c respectively), whereas the triterpene alcohols are called as Ganoderiol or Lucidumol etc (Jong and Birmingham, 1992).

c MYe

C27

Figure 2-2: a. Lanostane Terpenoid skeleton; b. C30 terpenoid and c. C27 terpenoid (redrawn from Jong and Birmingham, 1992)

Apart from the bioactivities of the triterpenes, these have been reported as an important tool for quality assurance. A method using FT (Fourier Transformation) RP - HPLC (reverse phase) based triterpenoid profile for seven commercial samples has been reported (Chen et. al; 2001). Gao et. al; (2004), considered 19 triterpene constituents (16 triterpene alcohol and 3 triterpene acids) for HPLC analysis for evaluation of the Ganoderma products. Ten different products were considered for qualitative determination of constituents. The results suggested that an appreciable difference was observed in various samples considered.

The triterpenes have also been useful in chemotaxonomy. Chen et. al; (1999), used 3 strains of each G. lucidum and G. tsugae, coupled with RP-HPLC studies of triterpenes. Difference was observed in the RP-HPLC triterpene pattern when all the strains of G. lucidum and G. tsugae were considered. Whereas there was no difference in patterns of all G. tsugae strains. But a difference was observed in strains of G. lucidum RP-HPLC pattern questioning the authenticity of the strains. Ganoderic acid A, B, C and D were used as marker compounds. Actually, Nishitoba and coworkers in

35 Review of Literature

1986 reported that the HPLC pattern of triterpenes could be useful as a chemotaxonomic tool. A similar study was carried out by Hirotani et. al, in 1993 using Ganoderic acid A and H as the marker compounds to analyze three local G. lucidum strains.

Table 2 -4: Different triterpenes isolated from various sources of Ganoderma lucidum only.

Compound name Source Activity Reference Lucidenic acid A, B & C Basidiocarp — Nishitobae/. a/; 1984 Ganoderic acid C Basidiocarp — Nishitobae/. a/,i985 Methyl ganoderate A, B — Basidiocarp Hirotani e/. a/; 1985 &C Ganoderic acid A, B, C & Basidiocarp Histamine Release Inhibitor D KoMa et.al; 1985 Hirotani & Furuya, Ganoderic acid E & F Basidiocarp — 1986 Cultured Ganoderic acid T, S & R Antihepatotoxic Hirotani e/. a/; 1986 Mycelia Ganoderal A, Ganoderol Angiotensine Converting A & B, Ganoderic acid K Basidiocarp Morigiwae/. al; 1986 Enzyme Inhibitor &S Methyl ganoderate M, N, 0, K, H, E Basidiocarp — Nishitobae/. al; 1987c Methyl lucidenate H, I J, K, L M & Ej Ganoderic acid Ma, Mb, Cultured Mc, Md, Me and Mf and — Nishitoba e/. a/; 1987a Mycelia W, R&T Ganoderic acid Mg, Mh, Cultured — Nishitobae/. a/,i987b Mi, Mj & Mk Mycelia Ganodermic acid Ja, Jb, Cultured — Sh\ao et.al; 1988 P1&P2 Mycelia Ganoderic acid V| Basidiocarp — Hirotani e/. a/; 1993 Lucidumol A & Spores HIV -1 protease inhibitor Min et.al; 1998 Ganoderic acid p Ganoderic acid a. A, B, AntiHIV-l andantiHIV-1 El Makkawy et. CI, H; Ganoderiol A, B, Basidiocarp protease a/; 1998 F; Ganodermanontriol Calf DNA polymerase Lucidenic acid 0, Basidiocarp inhibitor and HIV-1 reverse Mizushina et. al; 1999 Lucidenic lactone transcriptase inhibitor

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Compound 1 & 2 Basidiocarp Anticancer Ha et. al; 2000 Ganoderic acid 7, 5, e, (,, Antitumor against Meth-A Spores Min et. al; 2000 and LLC tumor cells Prostaglandin Ei induced Ganoderic acid S — CaMP elevation in human Su et. al; 2000a platelet Lucidenic acid SPl; Spores Anti complement activity Mine/, a/; 2001 Ganoderic acid A, B & C Induction of apoptosis in HL Compound i, ii & iii Basidiocarp Hu et al; 2002 -60 cells Lucialdeiiyde A, B, C Basidiocarp Anticancer activity Gao et al; 2002 Inhibitory activity on Lucidenic acid P & Q; Basidiocarp Epstein Barr Virus Iwatsuki et al; 2003 Methyl lucidenate P activation

In general two solvent extraction schemes are used for isolation of triterpenes from Ganoderma (Huie and Di, 2004). The first scheme is for extraction of total triterpenes using organic solvents and water, e.g. extraction with methanol by refluxing; these are generally called as triterpene alcohols. Whereas the second scheme is for the selective isolation of acidic triterpenes from the total triterpene fraction. According to which the material is extracted with 95% ethanol under reflux, the ethanol is evaporated under reduced pressure. The residue thus resulted is suspended in water and extracted in chloroform. A saturated aqueous solution of NaHCOs is added to the concentrated chloroform fraction. The aqueous layer is re-extracted with chloroform after the acidification of the suspension (pH 3-4, using 6N HCl).

The extracts are separated and purified using various chromatographic techniques, generally the silica gel column chromatography. The solvent system (mobile phase), which has been preferred by many workers is Chloroform : Methanol. Recently, Huie and Di (2004) described different HPLC techniques useful in separation and purification of triterpenes from Ganoderma, along with other chromatographic procedures used by different authors.

Ganoderic acids A, B, C and D from G. lucidum, showed inhibition of histamine release from the rat mast cells, induced by Concanavalin A and compound 48/80 proving the anfiinflammatory or anfi allergic activity of the ganoderic acids. Ganoderic acids C and

37 Review of Literature

D showed the distinctive inhibitory activity on histamine release (Kohda et. al; 1985). Other than the cytotoxic effect the triterpenes also have a harmonizing effect on the body, on the immune system and on circulatory system. These triterpenes have an adaptogenic effects, it helps the body to adapt to wide range of environmental, biological and sociological stress (Willard 1990). The triterpenes also show anti-hypertensive (high blood pressure) action and the mode of action is through inhibition of Angiotensin Converting Enzyme [ACE] (Morigiwa et. al; 1986). Komoda et. al. (1989) showed that ganoderic acids B & C from the fruit body of Ganoderma lucidum to be an active inhibitor of cholesterol biosynthesis. The oxygen position present in the structure is the key to the cholesterol biosynthesis inhibition.

Ganoderic acid -a has been proved to be Anti-HIV - 1 and Anti-HIV - 1 protease. Ganoderiol F and Ganodermanotriol has anit-HIV-1 activity at a dose of 7.8|ig/ml. Ganoderic acid B, ganoderiol B, ganoderic acid CI, 3P-5a dihydroxy - 6p - methoxy ergosta - 7, 22- diene, ganoderic acid a, ganoderic acid H and ganoderiol A are moderate active inhibitors against HIV-1 Protease (El-Mekkawy et al; 1998). Min et. al. (1998) screened 10 different triterpenes from Ganoderma lucidum for HIV -1 protease inhibitory activity where in ganoderic acid P showed highest activity, followed by Lucidumol B, Ganodermanontriol and Ganolucidic acid, with IC50 values between 20 -90^m. Lucidenic acid O and ketone, have been isolated from the fruit body of G. lucidum which prevented activities of calf DNA polymerase alpha and rat DNA polymerase beta, besides it has also shown to prevent the HIV - 1 reverse transcriptase (Mizushina, et. al; 1999).

Apart from Ganoderma lucidum, many other Ganoderma species and other mushrooms have been screened for triterpenes and evaluated for various bioactivities (table 2-5).

A single report of sesquiterpene from the genus recorded four cadinene type sesquiterpenes from Ganoderma mastoporum (Hirotani et al; 1995). The sesquiterpenes were named as Ganomastenol A, B, C and D were isolated from the cultured mycelium. No assay was carried out. The sesquiterpenes were not tested for any bioactivity.

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Table 2 -5: Terpenoids from different mushrooms and their activities.

Name of Mushroom Compound name Activity Reference Ganoderma Applanoxidic acid A, B, Antitumor Cha\ru\et. al; 1991 applanatum C«&D 4 lanostane type G. applanatum — Shim et. al; 2004 terpenoids Ganoderic acid F; Ganoderenic acid A, G. applanatum — Boh et al; 2000 Lucidenic acid Dl & applanoxidic acid A Tsugaric acid A, B & C; G. tsugae Anticancer Su et al; 2000b Tsugarioside A, B & C Ganodermadiol, G. pfeifferi Lucidadiol & Antiviral Mothana et al; 2003 Applanoxidic acid Applanoxidic acid A, C, G. annulare Antifungal Samania et. al; 2003 F,G&H Cryptoporous Hashimoto & Asakawa, Cryptoporic acids Immunomodulator volvatous 1998 Cytotoxic, Omphalatus olearius Illudin S & M Wasser& Weis, 1999 antitumor Lampteromyces Cytotoxic, UludinA&B Wasser& Weis, 1999 japonicus antitumor Mycena leaiana Illudin like compound Cytotoxic Wasser&Weis, 1999 Porta cocos — Sedative Giner-Larza et. al; 2000 Laetiporous Versisponic acids A, B, — Yoshikawa et al; 2000 versisporus C, D&E Inotodiol, trametenolic Inonotus obliquus acid and triterpene — Shin et al; 2000 lactone Naematoloma Sublateriol A, B & C — Yaoita et al; 2001 sublateritum

III. Proteins and Amino acids:

Proteins have been isolated and analyzed from various species of Ganoderma. Many bioactive proteins have also been isolated from different medicinal mushrooms, e.g. LZ -8 (Ling Zhi -8), which is an immunomodulatory protein isolated from the mycelia of Ganoderma lucidum. The complete amino acid sequence of this protein has also been

39 Review of Literature carried out (Tanaka et. al; 1989). Similarly another immunomodulatory protein has been isolated from Ganoderma tsugae; (Fungal immunomodulatory protein) Fip -gts (Lin et. al; 1997b). Other Pips have been isolated form Flamulina veltipes (Fip -fve) and Volvariella volvacea (Fip -wo), Lin et. al. (1997b). Apart from these, proteins with relatively low sugar content (lectin) have been isolated from the mycelia of Ganoderma lucidum (GLL - M) and from fruit body (GLL -F), Kawagishi el. al. (1997) & Huie and Di (2004).

Though LZ -8 is not referred as lectin but it shows the biological characteristics of lectins, viz., mitogenic and haemagglutinating activities (Wang et. al; 1998). A thermostable lectin was isolated from Ganoderma capense with mitogenic and antiproliferative activity towards leukemia cells and hepatoma cells (Patrick et. al; 2004). Various authors have reported proteins in form of enzymes. Sripuan et. al. (2003) isolated a -galactosidase from fruiting body of Ganoderma lucidum, which is believed to be involved in the production of D-galactose in bioactive polysaccharides such as Ganoderan C (Tomoda, 1986; Huie and Di, 2004). Several enzymes are used for identification of different species of Ganoderma by applying isozyme analysis (Smith and Sivasithamparam, 2000; Gottlieb et. al; 1995; Gottlieb et. al; 1998 and Gottlieb and Wright, 1999a). U^TO^

A variety of amino acids have been reported from various species of Ganoderma. The spores are observed to contain higher amounts of amino acids as compared to fruiting bodies (Huie and Di, 2004). It is also reported that the percentage of amino acids varies from sample to samples of the same species (Huie and Di, 2004). Mau et. al. (2001) analyzed different amino acids including 8 essential amino acids, using HPLC techniques from Ganoderma lucidum, G. tsugae and Coriolus versicolor. While Tseng et. al. (2005) analyzed amino acids from different forms of Ganoderma tsugae, its mycelia and culture filtrate. The results reveal that the filtrate contained highest amino acids than mycelia, whereas the antler form was superior to that of mature form of G. tsugae for amino acid content.

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IV. Nucleosides, Nucleotides and RNAs:

Shimizu, et. al; (1985) reported the inhibition of platelet aggregation from the water extracts of Ganoderma lucidum with the active component identified as adenosine. Kawagishi (1993) also reported that 5' -Deoxy - 5' - methylsulpinyladenosine, which inhibited the platelet aggregation. Different methods for the extraction / isolation and analysis of the nucleosides have been reported Huie and Di (2004). Similar, platelet aggregation inhibitory activity has also been reported from Lentinus edodes, few species of Auricularia (Agarwal, et. al; 1982) and from Cordyceps (Shiao, et. al; 1994). RNA exhibits interferon inducing activity as well as antiviral activity, while uridine/ uracil shows neuro muscular restorative effects (Willard, 1990). Recently, micellar electrokinetic chromatographic (MEKC) method with selected base (uracil) and nucleoside (adenosine, uridine) as markers were used to analyze Ganoderma lucidum, G. japonicum extracts and Ganoderma capsules. This technology can be useful as sensitive tool for quality control and assurance, and extended with more spectrums of Ganoderma components as marker. It will also be more effective tool for quality assurance of Ganoderma products (Cheung, et. al; 2001). Mau et al. (2001) and Tseng et al. (2005) analyzed different 5' nucleotides from various form of Ganoderma tsugae, its culture and culture filterate, and also from G. lucidum and Coriolus versicolor. Based on the flavour 5 'nucleotides, the flavours of the said mushrooms were identified.

V. Germanium: Germanium in organic form (Ge02) is found in many plants and fungi. The organic form is known to possess anticancer and immune enhancer / stimulator (^http://www.positivehealth.com/permit/Articles/Nutrition/Germanium/chapter7.htm). It induces interferon and activates macrophages, also enhances the natural killer cell activity (Goodman, 1988). There are many reports of germanium analysis from Ganoderma, Mizuno et. al; (1988), screened nine mushroom apart from Ganoderma lucidum and G. applanatum, for their mineral content, which included germanium. The results infers that G. applanatum was the highest germanium containing organism with 182ppb, while other studied samples were recorded to be less thanlOOppb, while Pleurotus ostreatus with least of 16ppb only.

^ Online Reference 41 Review of Literature

The study also covered various local samples of artificially cultivated Ganoderma lucidum. Patocka (1999) highlighted the importance of the germanium from Ganoderma lucidum, and also supported the above mentioned biological activities of germanium. Wang, (1991) reported the germanium content of Ganoderma lucidum, G. tsugae and the log used for cultivation from Taiwan. A spectrophotometric method with phenylflurone was used for analysis of germanium.

Selenium is an essential trace mineral, which functions mainly through its association with proteins, known as selenoproteins. Several selenoproteins defend against oxidative stress. The selenium content of food depends upon the selenium content in the substrate. Anonymous (2004) reported that the mushrooms provide more selenium than other foods in the produce category, the report also comments on the use of selenium in various cancers such as prostate and colorectal cancer.

Selenium was recognized as an essential nutrient on the basis of its ability to serve interchangeably with vitamin E in prevention of vascular or muscular symptoms. Zhao et. al. (2004b) referred to the essentiality of selenium for it is necessity in the 21^' amino acid selenocysteine, which is useful for the synthesis of selenoenzymes like glutathione peroxidase, iodothyronine deiodinases, and thioredoxin reductasaes. The authors also referred to that consumption of selenium is safer through natural food materials (organic form of selenium) than that of inorganic form. Zhao et. al. (2004b) also reports that there is less information about selenium enrichment of Ganoderma. The experimental results of Zhao et. al. (2004b) suggests that Ganoderma lucidum could biotransform inorganic selenite from the substrate into organic form by integrating selenium into proteins and polysaccharides. They also reported that low concentration of selenium in substrate facilitated the synthesis of total protein and amino acids in Ganoderma with reverse role is played in presence of high concentration of selenium (Zhao et. al; 2004b).

There are few reports of detection and quantitative analysis of selenium from mushrooms. Stijve (1977), reported selenium content of 83 species of wild mushrooms with a range of 0.012 -20.0mg /kg dry weight (ppm). Further he reported that selenium content was species dependent; Agaricaceae with high concentration while certain members of Boletaceae. Whereas Russulaceae, Amanitaceae and Cantharellaceae were reported to be

42 Review of Literature

the selenium deficient species. The highest selenium content was reported in Boletus (Tubiporus) edulis. Piepponen et. al. (1983) reported selenium content of 142 mushrooms from Finland, confirming the highest selenium content in Boletus edulis.

VI. Sterols: Several sterols have been isolated from different mushrooms; ergosterol and its derivatives being the major group having biological activities hence are focused generally. These are fungal specific sterols and important components of mevalonic acid pathway. Ergosterol is a fungal specific sterol and is a precursor for vitamin D2 (ergocalciferol). Windaus in 1928 first reported the conversion of ergosterol to vitamin D2 on ultra violet irradiation, who received the Noble prize in chemistry for his work on sterols and their relations with vitamins. Windaus (1928) also reported that 5mg of irradiated ergosterol possessed equivalent action that of 1 liter good quality cod-liver oil in curing rickets.

Further, the research has proved that ergosterol has been accepted as a tool to measure the fungal biomass (Volker, et. al; 2000). Ergosterol being fungal specific sterol found in the cell membrane makes it an important target component in antimycotic drug development (Arthington-Skaags et. al; 1999 & 2000). It is also used as a measurement tool for quantifying fungal biomass from a substrate, as it is a fungal specific sterol (Padgett and Posey, 1993).

There are several methods reported for ergosterol extraction from fungi. Padgett and Posey (1993), evaluated several such extraction techniques, concluding that use of alcoholic KOH substantially enhanced ergosterol recovery. Currently HPLC methods are regularly practiced for the estimation/ determination of ergosterol, but a spectrophotometric method is also sensitive for semi micro-determination of ergosterol. The method is based on the specific absorption spectrum of ergosterol (4 headed peak, fig. 2 -3), which was mentioned by Windaus (1928). Thereafter Breivik and Owades (1957) developed the basic spectrophotometric semi micro-determination protocol of ergosterol from yeast, which is still used with some modifications (Arthington-Skaags et. al; 1999 & 2000).

43 Review of Literature

i^-iV-t c i^jt-.t-—~ (

1.6- ' "^l ~-[^ ^•- ^ t 4j ^ it irf (/jd i / 1.0-

i ,

0.5- \ / 1 \ / ' ^&5

300_ Ji 25z0 : 30!i^-0 . SSOnifj Figure 2 -3: Absorption coefficient of Ergosterol (0.04% in ether), reproduced from Windaus(1928)

Further with some more modification are made in present study so as to make efficient extracfion and determination from aphyllophorales members (chapter 3. D-I).

Ergosterol derivatives Hke ergosta - 7, 22 - dien - 3a-yl Unoleate; 5a, 8a - epidioxyergosta-6, 22-dien-3p-yl linoleate and ergosta-7, 22-diene-2P, 3a, 9a-triol from Ganoderma lucidum exhibit potent antitumor activity against KB cells and human PLC/PRF/5 cells in vitro (Lin et. al; 1991). Strigina et. al. (1971), isolated few steroid metabolites from Ganoderma applanatum, concluding that ergosterol is not found in G. applanatum, but reported A 8(14)-ergostenol.

Dominguez et. al. (1971), reported ergosterol and L-mannitol from various mushrooms which included G. lucidum. Jain and Gupta (1984), reported ergosterol palmitate; ergosta -7, 22-dien-3-one and ergosterol from the mushroom G. australe. Kac et. al. (1984) reported several sterols from G. applanatum, G. lucidum and Polyporus sulfurous, the sterols included ergosterol, confirming that G. applanatum do not contain

44 Review of Literature

ergosterol. Lin et. al. (1993) reported various ergosterol derivatives from G. amboinense. Couple of terpenoids, sterols including ergosta-7, 22-dien-3p-ol has been reported by Lin et. al. (1997a). Keller et. al. (1997) reported ergosterol peroxide and ergosta-7, 22-dien-3p- ol from G. carnosum.

While the ergosterol peroxide and ergosterol from Polyporus species (Yazawa, et al; 2000) strongly inhibited (100% inhibition) the bladder tumor promoters, that to in a dose dependent manner. This not only inhibited the promotion of the carcinogenesis in bladder but also inhibited the effects of other carcinogenesis promoter. According to (Yazawa, et al; 2000) only ergosterol had the ability to inhibit two or more tumor promoters having different mechanism. Cordyceps sinensis show antitumor activity. Ergo peroxide was found to be greater inhibitor to the proliferation of K562, Jurkat, WM - 1341, HL - 60 and RPMI - 8226 tumor cell lines (Bok et al; 1999). The ergo peroxide from Meripilus giganteus was identified as immunosuppressive component (Narbe, et. al; 1991).

E. BIOASSAY: Fungi are considered to be a good source of various biologically active compounds, especially antibiotics. After the discovery of Penicillin, number of ftingal species were screened for antimicrobial activity. The soil was considered as the best source of ftangi and actinomycetes producing antibiotics, and hence many potential higher fungi, especially basidiomycetes were ignored.

Suay, et. al. (2000), screened 317 isolates from 204 species of basidiomycetes including Ganodermataceae (11 isolates), collected in Spain. All the isolates of Russulales were active against tested bacteria but not against fungal test organisms 73% of isolates of Ganodermataceae were active against bacterial organisms whereas no activity was observed against fungal test organisms. 15mm or more, zone of inhibition was considered as strong, less than 15mm was considered as moderate, while hazy inhibition was considered week activity. Recently, Rosa et. al (2003) evaluated the antimicrobial activity of 103 isolates from basidiomycetes representing 84 species of Brazil, which included one Ganoderma species (not identified). In both the studies crude extracts were used for assay. They have considered 12mm and more inhibition zone as significant.

45 Review of Literature

The Ganoderma species have been screened for various biological activities afforded mainly by triterpenes, polysaccharides, sterol and nucleic acid derivatives. These activities are generally focused on using animal system, in vitro and in vivo and human trials. The in vitro assays were mainly against various kinds of tumor or cancer cells, while in some studies, pathogenic viral, bacterial and fungal cells were also screened for antagonism.

Yoon, et. al. (1994), screened the aqueous extracts oi Ganoderma lucidum against fifteen species of bacteria, in vitro. The method used was minimum inhibitory concentration (MIC). The aqueous extract was most active against Micrococcus luteus (0.75mg/ml). The extracts were also tested along with standard antibiotics.

Mothana et. al. (2000), isolated and characterized two famesyl hydroquinones (Ganomycins A and B) from Ganoderma pfeifferi. These compounds were tested against gram positive and negative bacteria {Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Micrococcus flavus). Disc diffusion method was used to assess the activity along with MIC. Both the compounds were moderately active against S. aureus, B. subtilis and M. flavus. None of the compounds were active against Candida albicans and C. malsotsa, even at 100|j,g per disc.

Sudirman^(www.kyotan.com/lectures/lectures/Lecturel 2.html) screened methanolic extracts of seven Ganoderma species (not identified) against B. subtilis using disc diffusion method. The extracts were also tested using bioautography by TLC. Not more than three spots were detected to be active from all the species studied. The Rf values of the active spots were identified.

Samania et. al. (2003) reported antifungal activity of eight compounds including sterols and triterpenes from Ganoderma annulare. Microsporum cannis and Trichophyton mentagrophytes were the test organisms. The compounds showed antifungal activity but were not as strong as the standard antibiotics used in treatment of the said test organisms.

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