REVIEW OF LITERATURE:

The concept of "functional food" has been raised from the knowledge of a relationship

between diet and disease and thus it has led to the development of a new scientific discipline,

which is termed as "functional food science."[ Sadler M, Saltmarsh M.., 1998]. A food could be

accounted as "functional" if it contains food component that affects any identified body function

in a positive manner, and could be available in different forms or under different names viz.

dietary supplements, medicinal foods, vita foods, phytochemicals, and myochemicals and also

pharmafood, which could be used specifically to improve the health[ Hasler CM.,1996]. Among

the developed nations, there are many causes of death or disability that include heart diseases,

diabetes, obesity and cancer, which could be attributed to diet [Barasi M., 2003]. Millennia are

evident that mushrooms have been extensively used by human for consumption, which is due to

their flavor and medicinal properties. Hence, widely sold as nutritional agents, these mushrooms

are helpful to human health. [ Barbisan et al. and Bellini et al., 2003]. These mushrooms are

extensively known for their immunomodulatory, hepatoprotective, antinoceceptive, antidiabetic,

antiviral, and antimicrobial properties.[ Wasser SP, Weis AL., 1999].

It was recognized that many of edible and some non-edible, non-consumable mushrooms

could have valuable health benefits. The edible class of mushrooms that shows potential

medicinal and functional properties includes Lentinus, Auricularia. Hericium, Grifola,

Flammulina, Pleurotus, and Tremella. The other species known only for their medicinal

properties include Ganoderma, Trametes, etc. As they are coarse, harder in texture, and are bitter

in taste [Bensky D, Gamble A.,1993 and Hobbs B., 1995].

Mushrooms are found to be good source of vitamins especially thiamine [B 1 ], riboflavin

[B 2 ] niacin, biotin, and ascorbic acid (vitamin C). Vitamin A and D are rarely found but certain

species contain detectable amount of vitamin D when exposed to UV light. Crude fat content of

mushroom consists of lipids viz. mono, di, and triglycerides, sterols, phospholipids. [Breene

W.,1990]. In the present era, medicinal mushrooms are being focused for discovering such

compounds that could modulate the response of immune cells. Thus by doing so, they might be

useful for stimulating and enhancing the biological response of immune system. And therefore

they could be utilized for the treatment of tumor and other immunodeficiency status along as a

combination with antibiotics as well as vaccine adjuvant. [Jong SC., Birmingham JM., 1992]. In

case of immunosuppressive activity, they might be used in autoimmune diseases. [Badger

AM.,1983]. As stated earlier, being fungi, mushrooms contain a variety of constituents that have

various biological activities including antioxidant activity, antiinfective activity, anticancer

activity, as well as modulation of immune system.

CULTIVATION ON SOLID AGAR MEDIUM:

To examine the preliminary growth patterns of fungi, many researchers have investigated

the growth of fungi on various agar media. Bilay et al., (2000) investigated the growth rate of

thirty mushroom cultures on a variety of media with different pH. They found that the

commercial malt extract agar (MEA) and Potato dextrose agar (PDA) provided the maximum

growth rate of G.lucidium and Agaricus maskae respectively, whilst an experimental medium

that contained glucose, potassium phosphated and corn steep liquor, provided maximum growth

for fifteen cultures. This highlighted the fact that different fungal species have different nutrient

requirements. The diameter of the colony as a reliable measure of growth and this has been a

topic of controversy amongst investigators (Brancato & Golding, 1953; Worley, 1939). It has

been noted that under uniform environmental conditions, there is a high degree of radial

symmetry in fungal colonies, with all portions of the mycelial front extending at the same rate

(Edelstein & Segel, 1983) and with no acceleration of growth over time (Brancato & Golding,

1953). Hence, it can be assumed that the growth rate of fungi is constant (Trinic, 1971). As a

result, the rate and extent of growth (radial or diameter) has been a useful criterion for

comparison of fungi (Lonergan et al., 1993). The ratio of the area and growth over time were

also been used to measure the fungal growth.

CULTIVATION IN LIQUID MEDIUM

Propagation of mushroom mycelium in submerged culture was initially developed during

the 1950’s. This method was very successful in growing lower fungi (fungi that do not form

basidiocarp) in fermentors for economical production of various natural products (Yang & Liau,

1998). A review by Fang & Zhong (2002c) indicated that there were few investigations into the

development of higher fungi (basidiomycetes) bioprocess.

Since, it generally takes several months to cultivate the fruiting body of the fungus and it

is also difficult to control the quality of the products during cultivation, researchers are turning to

the liquid cultivation of mycelium to obtain useful cellular material to produce effective active

substances. Liquid or submerged culture has the potential advantage over solid substrate

cultivation because it requires minimal space, less chance of contamination, the mycelium can be

dispersed with in the substrate more uniformly, the media parameters can be easily manipulated

and the time taken to produce the first crop may be shortened (Bae et al.,).

GROWTH PARAMETERS IN LIQUID MEDIUM

Different growth factors can influence the quality and quantity of mycelial production.

Two important factors that should be considered when cultivating in liquid cultures are the

components of the culture medium (e.g. nitrogen, carbon and inorganic salts) and the environment

(e.g. temperature, pH aeration and stirring, illumination and inoculum amount). These factors

have minimum and maximum enhancing effects and quantification of these limits is necessary in

order to model and predict effects on growth of the fungus (Kundsen and Stack, 1991). The

quantity, quality, morphology and the nutrient consumption of the mycelium are considered

important criteria for determining the optimal conditions for fungal and synthesis of effective

components of interests.

EFFECT OF CULTURE MEDIUM ON MYCELIAL BIOMASS

The culture medium is extremely important as it provides the nutrients for growth of the

mycelium. There have been few investigations reported the effects of different carbohydrate on

the growth of fungal mycelium (Lin et al., 1973; Tseng et al., 1984). Lin et al., (1973) reported

that there was no difference in the production of biomass when glucose or sucrose was used,

while Lin and Yang (2006) who examined the growth of Agaricus blazei Murill in a suite of

different carbohydrates, including glucose, sucrose, soluble starch, maltose, malt extract and

arabinose, found that sucrose and soluble starch was used as carbon source and had high mycelial

growth, arabinose and maltose supplementation had an inhibitory effect on mycelial growth when

was used as the carbon source.

The presence of nitrogen in the culture medium is another important factor to enhance the

mycelial growth (Lin et al., 1973). Lin and Yang (2006) reported that organic nitrogen source was

better than inorganic nitrogen sources, except urea. In the organic source he found that yeast

extract was better than peptone, while NH4NO3 was the best inorganic source for mycelial growth

of A.blazei Murill M21.

In recent times, researchers have been investigating the alternate materials which could

increase biomass production including fatty acids (Yang et al., 2000) and plant oils (Schisler and

Volkoff, 1977), which have both been found to have a stimulatory effect on the mycelial growth

of some mushrooms.

EFFECT OF THE ENVIRONMENT ON MYCELIAL BIOMASS

Temperature is one of the most important environmental factor in mycelial growth,

requiring careful control (Brancato and Golding, 1953). Temperature affects enzymatic activity

and vitamin synthesis and may accelerate or inhibit fungus growth (Miles; Chang, 1997). The

temperatures reported for A. brasiliensis growth are dissonant and vary from 18 ºC to 30 ºC

(Okubo; Kuramoto; Ohqubo, 1991), 22ºC to 26 ºC (Iwade; Mizuno, 1997), 25 ºC to 28 ºC (Eira;

Braga, 2003) for compost cultivation and 30 °C for submerged cultivation (Kawagoe et al., 2004).

Quimio, Chang and Royse (1990) reported 28 °C to 30 °C as optimal temperature for Agaricus

bitorquis growth, a tropical basidiomycete, whereas for Agaricus bisporus, a fungus in which A.

brasiliensis cultivation is based on, the recommended temperatures are from 22 °C to 25 °C.

The initial pH of the growth medium is another important factor for cell membrane

function, cell morphology, for the uptake of various nutrients and product biosynthesis (Fang and

Zhong, 2002). It is well known that fungi generally metabolize acids to decrease the pH (Lin et

al., 1973) and that during the cultivation of mycelia, the media becomes acidic and when it

reaches this acidic level (pH 3 to 4) the growth of the mycelia is retarded (Lin et al., 1973). For

A. brasiliensis, pH value of 4.5 was considered ideal for mycelial growth in submerged

cultivation (Kawagoe et al., 2004) and pH from 6.5 to 6.8 was ideal for compost cultivation

(Iwade; Mizuno 1997).

MUSHROOM POLYSACCHARIDE:

Edible mushrooms are valuable a source of biologically active compounds. Some are

used in the prophylaxis and therapy of such diseases as cancer and cardiovascular disease. Their

antitumor mechanism is complex. The biologically active substances in mushrooms decrease

DNA damage, reduce carcinogen concentrations and their activation, inhibit the growth of cancer

cells by scavenging free radicals, stimulate the immune system, and induce tumor cell apoptosis.

The stimulation of the immune system by the biologically active compounds in edible

mushrooms protects against cold, flu, infections, well as AIDS by inhibition of viral replication.

Mushrooms contain effective substances which decrease the LDL fraction of cholesterol in

blood. They also prevent the accumulation of serum triaclyglycerols, thus decreasing the risk of

developing cardiovascular disease. The therapeutic properties of mushrooms result from the

specific polysaccharides, such as beta-glucans and chitosans,that are present in the fructification

of fungi.

Polysaccharides are a structurally diverse group of biological macromolecules of

widespread occurrence in nature. They are composed of repetitive structural features that are

polymers of monosaccharide residues joined to each other by glycosidic linkages.

Polysaccharides present the highest capacity for carrying biological information since they have

the greatest potential for structural variability. The amino acids in proteins and the nucleotides in

nucleic acids can interconnect in only one way while the monosaccharide units in

oligosaccharides and polysaccharides can interconnect at several points to form a wide variety of

branched or linear structures (Sharon and Lis, 1993). As a consequence, this enormous potential

variability in polysaccharide structure allows for the flexibility necessary for the precise

regulatory mechanisms of various cell-cell interaction in higher organisms

Many Basidiomycete mushrooms have been shown to contain biologically active

antitumour and immunostimulative polysaccharides. Reshetnikov et al. (2001) have listed 650

species and 7 intraspecific taxa from 182 genera of higher Hetero- and Homo-basidiomycetes

that contain pharmacologicaly active polysaccharides that can be derived from fruit-bodies,

culture mycelium and culture broths.

Recent study show that PSP of Coriolus versicolor, has immunomodulatory and

antitumor activity in both experimental animals and cancer patients (Ng.T.B.,1998). Wang et al.

(1996a) found that both mouse lymphocytes and macrophages were activated by preparations of

polysaccharopeptide from cultured mycelia and culture medium of C. versicolor.

Glucans including lentinan and schizophyllan and those extracted from other fungi such

as Ganoderma lucidum and Volvariella volvacea are also known to possess antitumor and

immunomodulatory activities. It is hoped that these polysaccharides and protein-bound and

peptide-bound polysaccharides can be developed into clinically useful drugs.

ANTIOXIDANT PROPERTY OF MUSHROOMS:

Free radicals are classified as highly reactive molecules having one or more unpaired

electrons, which are able to damage numerous biological substances including DNA, protein and

lipid membranes (Wu and Hansen, 2008; Tsai et al., 2007) leading to various diseases and

disorders, such as cancer, cardiovascular diseases, impaired immune system, atherosclerosis and

aging (Xu et al., 2009; Wu and Hansen, 2008). Molecules that are able to scavenge free radicals

and provide protection against diseases caused by them are called antioxidants. Several synthetic

antioxidants are being used currently in the preservation of foods, i.e., butylated hydroxyanisole

(BHA), butylated hydroxytoluene (BHT) and Tert-butylated hydroxyquinone (TBHQ), but they

could promote tumor formation (Cheung et al., 2003). Naturally occurring antioxidantsubstances

from fruits and vegetables have been well known, which included: ascorbic acid, tocopherol, B-

carotene, flavonoids and other phenol compounds (Wu and Hansen, 2008; Xu et al., 2009).

Mushroom fruiting bodies were investigated invitro for antioxidant activity. Extracts of

G. lucidum can apparently remove the hyperoxide radical believed to be a main factor in the

human ageing process (Liu et al., 1997), and the ageing mouse model (Pan et al., 1999). In a

clinical trial with 30 elderly people Ganoderma lucidum extract (GLE) was given oral 1.5 g 3

times daily for 30 days. Interleukin-2 and interferon (IFN) production by peripheral mononuclear

cells (PBMC) and NK cell activity in vitro were respectively measured. Production of IL-2 and

IFN were significantly increased after GLE treatment. Such results could suggest that GLE is a

possible treatment to raise the cellular immunological activity in ageing people (Tao and Feng,

1991; Tao, 1993).

A Ganoderma lucidum polysaccharide GLB7 decreased the production of oxygen free

radicals and antagonised the respiratory burst induced by PMA in murine peritoneal

macrophages (Li and Lei, 2000). Such observations could implythat the polysaccharide-induced

inhibition of oxygen free radicals in murine peritoneal macrophages play an important role in the

anti-ageing effect of Ganoderma extracts.

PSK in a cell-free system consisting of hypoxanthine-xanthine oxidase rapidly quenched

the superoxide radical, a property not shared by Schizophyllan (Sakagami and Aoko, 1991). PSK

further repressed the mimetic activity of superoxide dismutase (SOD) and promoted oxidative

stress relief for cancer-bearing hosts (Kobayashi and Kariya, 1994). PSK also gave protection to

macrophages from lipoperoxide accumulation and foam cell formation created by oxidatively

modified low-density lipoprotein (Yuan and Meiz, 1996). This protection is believed to be due to

the induction of gene expression of antioxidative enzymes (Chen and Zhou,1997). PSP showed

similar scavenging effects on superoxide and hydroxyl radicals (Hu and Chen, 1992). Significant

superoxide and hydroxyl radical scavenging activities have been demonstrated for several

mushroom antitumour polysaccharides (Liu et al., 1997).

Mushroom extracts of Armillariella melea, Daedea dickens, Formitella farinea and

Pleurotus cornuspirae markedly exhibited inhibition on lipid peroxidation of rat liver microsome.

A. mellea were separated using silica gel column chromatography and recrystallization. The

structure of the compound was determined by Nuclear Magnetic Resonance (NMR), Gas

Chromatography and X-ray crystallography (Kim et al., 1999).

The methanol extracts from Agrocybe aegerita mushroom were studied for

cyclooxygenase inhibitory assay and antioxidant assay. The methanol extracts were subjected to

the GC-MS analysis that confirmed the presence of fatty acid fraction (FAR) along with palmitic

acid, ergosterol, EED, mannitol and trehalose which exhibited cyclooxygenase and antimicrobial

activity (Zhang et al., 2003).

Total antioxidant property of Morchella conica pers using ethanol and methanol was

carried out and compared with appropriate standards like butylated hydroxyanisol and x-

tocopherol .The inhibitory effect was found to be more in ethanol extract of Morchella conica

pers .Presence of total phenol and total flavonoid content were correlated with the antioxidant

property (Turkoglu et al .,2006 ). Ethanol extract of Ramaria flava were also found to have a

potent antioxidant property when tested with appropriate standards (Gezer et al., 2006).

Invitro evaluation of the antioxidant property of Cordyceps militaris was studied

using methanol such as DPPH free radical scavenging, hydroxyl radical eliminating, iron

chelating, lipid per oxidation and ferrous reducing power. Eighty percent scavenging effect were

seen in method DPPH, eliminating hydroxyl radicals, chelating ferrous ions and also positive

results of lipid per oxidation and reducing power (Zhan et al .,2006) of DPPH was scavenged by

methanol extract of Cordyceps militaris (Zhan et al., 2006).

In a study to evaluate the antioxidant and anti-radical activity of the extracts of Xylaria

nigripes of family xylariaceae, also known as Wu Ling Shen, a less studied medicinal fungus.

Polyphenols and total polysaccharide content of the extracts were determined, which showed that

extracts of XN possess stronger antioxidant and anti-radical activity than XNP in all tested

models. Results showed that at concentration of 25μg/ml, the percentage of inhibition on

superoxide radical generation by XN-H and XN-E was 75% and 83%, respectively, which were

comparable to the positive controls Trolox (65%) and vitamin C (80%) at the same

concentration. The superoxide scavenging effect (Glucin, 2006) was found to increase with

increasing concentration of XN extracts regardless of the nature of preparation. Based on IC50

values, the activity of superoxide radical scavenging of XN-E (IC50 5.12μg/ml) appeared

slightly weaker than that of vitamin C (IC50 4.25μg/ml), but it was stronger than that of Trolox

(IC50 6.94μg/ml) (Table 1). XNP-E also exhibited potent superoxide radical scavenging activity

(IC50 8.89μg/ml).

Antioxidant activity was also evaluated for the crude polysaccharide from edible fresh

and dry mushroom fruit bodies of Lentinus sp. In which the Trolox equivalent values in

scavenging abilities of both crude polysaccharides against both ABTS and DPPH radicals as well

as reducing properties were in similar ranges. (Thetstimuang et al.,2011).

Methanol extracts of P. ostreatus and P. cystidiosus fruiting bodies possessed

antioxidant, reducing power, radical scavenging and iron chelating activities that were higher

than those of other commercial mushrooms (yang et al.,2002).

On the other hand, Elmastas et al and Dubost et al. reported that oyster mushroom

extracts possessed only moderate antioxidant activities compared to other edible mushrooms.

The antioxidant activity was positively correlated with total polyphenol content. Furthermore,

Lee et al. showed that P. citrinopileatus extracts prepared from fruiting bodies were more

effective than those from mycelium and fermentation broth filtrate presumably due to a higher

amount of total phenols. Methanol extracts of P. florida and P. pulmonarius fruiting bodies

showed similar antioxidant activities (70,71), and an ethanol extract from P. citrinopileatus

fruiting bodies had antioxidant activities comparable to those from P. eryngii, P. ferulae and P.

ostreatus mushrooms .

P. citrinopileatus fruiting body extracts have shown antioxidant activities in vitro and in

hyperlipidaemic hamster rats. Extracts added to a high-fat diet increased the activities of

antioxidant enzymes in rats . P. ostreatus mushroom extracts had antioxidant properties in aged

and CCl4-induced liver damaged rats, as indicated by significant increases in concentrations of

antioxidants and antioxidant enzymes .

Pleuran, a β-glucan isolated from P. ostreatus, had a positive effect on the antioxidant

status of rats and decreased precancerous lesions induced in rat colon (Bobek, P. and Galbavy,

S., 2001). A polysaccharide-peptide complex isolated from P. abalonus fruiting bodies

prolonged the lifespan of senescence-accelerated mice. Gene expression of antioxidant enzymes

was up-regulated and consequently their activities were increased (Li et al., 2007).

In a study conducted by Bhaskar et al., (2008) to investigate the free radical scavenging

activity of polysaccharide fractions extracted from the fruiting body of medicinal mushroom,

Ganoderma lucidum P.Karst using different antioxidant assays acetic acid-NaOH fraction

exhibited potent antioxidant activity and effectively scavenged free radicals in a dose dependent

manner from 50 to 250 µg/ml followed by hot water fraction and ammonium oxalate fraction.

Hence, the antioxidants present in the system is also said to contribute to the antitumor activity.

Bum Chun Lee et al., in 2003 has conducted an analysis in which he prepared five groups

of polysaccharides from mycelia extract and top and bottom fractions of filter precipitates by

submerged culture of Grifola frondosa at two different medium viz. Glucose and PMP, in which

the polysaccharides obtained showed different biological activities at the concentrations of 0.01-

0.2% (w/v) where G-2 (bottom fraction of filter precipitates from glucose

mediumMW770kDa)and G-3 polysaccharide (Mycelial extract from glucose medium, MW500

kDa) showed strong antioxidant activity. It is noteworthy that G-2 and G-3 polysaccharides have

higher ratios of proteins to carbohydrates, which is probably closely related to higher antioxidant

activity. Using these two polysaccharides, free radical scavenging activity after UV irradiation in

HDF cells was further studied in vitro.

Liu et al. (1997) reported extensive results on free radical scavenging activity of various

mushroom polysaccharides of diverse forms (e.g. mycelium extract, fruiting body extract, and

culture filtrate). They found that five mushroom polysaccharide extracts and a protein-bound

polysaccharides exhibited significant activities of superoxide and hydroxyl radical scavenging.

Total antioxidant property methanol and ethyl acetate extracts of Phellinus rimosus, Pleurotus

florida, Pleurotus sojar-caju, and Ganoderma lucidum using different methods like DPPH,

ABTS, FRAP, lipid peroxidation and ORAC. Among this methanol extract of Pleurotus florida

and Phellinus rimosus shows significant antioxidant property of around 70% (Jose and

Janardhanan, 2000, Lakshmi et al., 2004

Methanolic and water crude extracts of Lentinus edodes and Volvariella volvacea tested

for their antioxidant activity and total phenolic content using three different methods. Among

these extracts, water extract from Lentinus edodes showed potent radical scavenging activity in

all the methods. Total phenolic in the water extracts was higher than that of the methanol ones

(Cheung et al.,2003).

Alpha amanitin and phalloidin are powerful hepatotoxins belong to the main class of

toxins namely amatoxins and phallotoxin, isolated from deadly poisonous mushroom Amanita

phalloides. Studies showed that alpha amanitin and phalloidin could inhibit catalase activity that

makes to study the invitro behaviour of both toxins on SOD activity using spectrophotometry.

And established that toxins having SSA (Superoxide anion scavenging activity) (Zheleva et al.,

2004).

Ganoderma lucidum and Phellinus linteus two medicinally potent mushroom

were investigated for the expression of antioxidant enzyme invitro and invivo. The catalytic

activity of catalase in the liver and brain was significantly increased after the oral treatment of

mushroom for 2 months. The tissue homogenate of liver and brain analyzed through western

blot revealed the expression level of catalase significantly increased compared to that of control

mice (Park et al., 2001).

Invitro evaluation of antioxidant activity of Auricularia auricula showed

significant inhibiton of lipid peroxidation a potent hydroxyl radical scavenging when compared

with standard drug catechin having potential therapeutic use (Acharya et al., 2004).

Fruit body of Phellinus linteus extracted with seven percent ethanol showed strong anti-

angiogenic activity detected using chick embryo chorio allantoic membrane (CAM) assay. The

invitro antioxidant activities of Phellinus linteus evaluated using two different bio assay DPPH

and lipid peroxidation concentration dependent method with standard vitamin-C (Song et al .,

2003).

The methanolic extracts of macro fungus Pleurotus rimosus possessed significant in

vitro superoxide anion hydroxyl radical and nitric oxide scavenging and lipid peroxidation

inhibiting activities. The extract showed remarkable anti inflammatory activity compared with

standard reference drug diclofenae. The results obtained shows anti-inflammatory activity of

P.rimosus which is possibly attributed to its free radical scavenging activity property (Ajith et

al., 2001).

The methanolic extract of Medicinal mushrooms Ganoderma lucidum, G. tsugae

and Coriolous versicolor were studied for their antioxidant property G.lucidum and G.tsugae

showed an excellent antioxidant activity (2.30-6.41 lipid peroxidation) whereas C.versicolor

showed only 58.56. In DPPH radical scavenging assays Ganoderma lucidum (67-74.4%)

showed more antioxidant property when compared with C.versicolor (24.6%). The antioxidant

property was due to total phenol, was found to be a major naturally occurring antioxidant

components found in methanolic extract of mushroom(Mau et al., 2002).

The antioxidant activity invitro of three poly-substituted terphenyl compounds from

the edible mushroom Thelephora ganbajun was investigated. The IC50 values of three

compounds for lipid peroxidation in rat liver homogenate were 400, 48, 54mm respectively; the

compounds increased superoxide dismutase (SOD) activity with EC50 values of 182, 74, 20mm.

They were also assessed on the DPPH radical scavenging activity with EC50 values of 49, 1233,

54mm (Yang et al., 2004).

The methanolic extracts of Grifola frondosa, Morchella esculenta and Termitomyces

albuminosus mushrooms were studied for the antioxidant activity. The reducing powers,

scavenging effect and chelating ability were assayed and their EC50 values that proved the

extracts to have a good antioxidant property. The total phenols were the major naturally

occurring antioxidant components found in methanolic extracts along with ascorbic acid and

tochopherols in all the three mushrooms (Mau et al., 2004).

The ethanolic and hot water extracts of Agaricus blazei, Agrocybe cylindracea and

Boletus edulis. The extracts were more effective in reducing the hydroxyl radicals and chelating

the ferrous ions at a lower EC50 values. The presence of naturally occurring antioxidant

compounds like tocopherol and phenols were found in the extracts, associated with EC50 values

of antioxidant properties (Tsai et al., 2007).

The methanolic extracts of Leucopaxillus giganteus, Sarcodon imbricatus and Agaricus

arvensis were screened for their antioxidant property by the following the standard methods like

DPPH- radical scavenging and β-carotene linoleic acid method The high contents of phenolic

compounds found in all three samples accounts for the antioxidant property, among which

Leucopaxillus giganteus had the highest content, proved to be more active, presenting lower EC50

values in all the antioxidant assays (Barros et al., 2007).

Protein bound polysaccharide k (PSK) from the mushroom Cordyceps versicolor was

tested against haematological malignant cells to induce apoptosis,out of 33 haematological

malignant cell line (HMC) test, PSK induced apoptosis was neutralized by the addition of

galactose to the culture medium, the apoptosis was augmented by treatment with β-galactosidase,

indicating the inhibitory involvement of galactose in the mechanism of action. These results

provide initial evidence of the direct cytotoxic activity of PSK in a haematological malignant cell

line (Hattori et al., 2004).

The ethanolic extracts of Morchella esculenta were studied for anti-inflammatory

and antitumor activity. The extract showed potent anti-inflammatory activity against acute and

chronic inflammation, compared with the standard drug, diclofenac. In addition on antitumor

activity against DLA cells and EAC cells were also observed (Nitha et al., 2007).

The fruiting body of Inonotus obliquus, a medicinal mushroom called Chaga, has

been used as a traditional medicine for cancer treatment. Free radical scavenges from the

methanolic extracts of this mushroom, inonoblins A(1), B(2) and C(3) were isolated along with

the known compounds PhelligridinsD(4), E(5) and G(6). This compounds exhibited significant

scavenging activity against the ABTS radical cation and DPPH radical and showed moderate

activity against superoxide radical anion (Lee et al., 2007).

The petroleum ether extract (PE) and acetone extract (AE) of Pleurotus ostreatus were

studied for phytochemistry, antioxidant and antimicrobial potencies. By agar well diffusion

method PE showed anti gram positive bacterial and inflammatory property than that of the AE,

whereas AE have high amount phenolic content (Iwalokun et al., 2007).

The Antioxidant activity of five Agaricus sp mushroom, followed by different chemical,

biochemical and electrochemical assay exhibited oxidation potentials more positive than the

standards. Agaricus silvaticus was the most efficient species contained the lowest EC50 values in

the chemical and biochemical assays and the highest antioxidant power in the electrochemical

assay (Barros et al., 2008).

The wild mushroom species proved to be less energetic than the commercial

species containing higher content of protein and lower fat concentration. The commercial species

have high sugar content than wild species, α –tocopherol was detected in wild species, whereas it

is not found in commercial species. Wild samples revealed a higher content in phenols, but a

lower content in phenols, but a lower content in ascorbic acid than commercial mushrooms.

There were no differences between the antimicrobial properties of wild and commercial

mushrooms (Barros et al., 2008).

Different stages in fruiting of Lactarius piperatus was analyzed for the amounts

of phenols, flavonoids, ascorbic acid, β-carotene and lycopene present in the immature, mature

and degraded fruiting bodies. The highest antioxidant contents and the lowest EC50 values for

antioxidant activity were obtained in the mature stage with immature spores (Barros et al., 2007).

A white mutant of Hypsizigus marmoreus a new edible mushroom was studied for

its antioxidant properties of ethanolic extract from fruit bodies and mycelia. Phenol was the

major antioxidant component in hot water and tocopherol in ethanolic extract. Correlation

content of total antioxidant components and total tocopherols(plus β-carotene) with EC50 values

in all antioxidant activity assay were established and total phenol was correlated only with

scavenging ability on hydroxyl radicals((Lee et al., 2007).

The medicinal mushroom Leucopaxillus giganteus grown under different nitrogen

sources in relation to the antioxidant and antimicrobial property and growth, the growth rate of

mycelium was same in different sources of nitrogen amended medium. Since (NH4)2HPO3 prove

to be the most appropriate nitrogen source to enhance the highest phenol content which is

directly proportional to the effective antioxidant property (Barros et al., 2007).

Methanolic extract of commercially available mycelia in Taiwan, namely Grifola

frondosa (maitake), Morchella esculenta (morel) and Termitomyces albuminosus (termite

mushroom) were studied for their antioxidant property. Total phenol from the three mycelial

extracts was relatively low. And EC50 value of reducing power, DPPH and chelating effect on

ferrous ions were below 10mg/mL, indicating that the three mycelia had good antioxidant

property except for the scavenging effect on hydroxyl radical(Mau et al., 2004).

Portuguese wild edible mushroom Leucopaxillus giganteus, Sarcodon imbricatus

and Agaricus arvensis were screened for their antioxidant property. All the three species contains

high phenolic content and low amount of ascorbic acid, β-carotene and lycopene. L. giganteus

revealed better antioxidant property when compare with other mushroom species, presenting

lower EC50 values in all the antioxidant activity assays and higher content of phenols(Barros et

al., 2007).

Free radical scavenging capacity of Lactarius delicious and Tricholoma

protentosum, wild edible mushrooms of north east Portugal, extracted using the solvent methanol

of cap, stipe and the entire mushroom. Scavenging effect was carried out by estimating total

phenol content, reducing power assay and DPPH. The two species showed potent antioxidant

property. The reducing power of Lactarius delicious is 2.41 and 2.71 for Tricholoma

protentosum. In DPPH scavenging, Lactarius delicious shown 8.52mg/mL, 22.9mg/mL for

Tricholoma protentosum. When compared with the stipe methanolic extract of cap and both the

mushrooms showed effective antioxidant property (Ferreira et al., 2007).

The two stages of maturity, young and mature of Agaricus brasiliensis was

evaluated for antioxidant and total phenolic content. Both the extract revealed minor difference

in total phenolic compounds and similar difference wass showed in antioxidant activities except

chelating ability for ferrous ions, higher in mature than young (Soares et al., 2009).

Oyetayo, V.O. in the year 2009 has carried out a study on the free radical scavenging

ability of the extracts of some wild mushrooms in which he has recorded that Lentinus subnudus

and Lenzites species displayed good scavenging ability against 2,2- Diphenyl-1-Picrylhydrazyl

(DPPH) and ferrous ion radicals at concentration of 2mg/mland Termitomycesclypeatus and

Termitomyces robustus exhibited better superoxide scavenging effect at 2mg/ml.Also he has

recorded that all extracts had comparable scavenging effect on hydroxyl radicals as butylated

Hydroxytoluene.(BHT)

Recently Yim.H.S. et al., in 2010 has evaluated Pleurotus ostreatus for its antioxidant

property for which the DPPH and ABTS radical- scavenging activity was found to be 63.20%

and 87.29% respectively . Antioxidant activity using Ferric reducing antioxidant power

(FRAP)at 1.45 mMFE/100g and B-carotene-linoleate bleaching assay was 83.51%, while the

total phenol content was found to be 798.55 mg GAE/100g.These antioxidant activities were

compared with synthetic antioxidants BHA and Ascorbic acid.

Invitro free radical scavengingactivity of wild edible mushroom, Pleurotus squarrosulus

was studied by Jaita Pal et al., (2010) in which allthree extracts viz., hot water , cold water and

methanolic extracts showed significant antioxidant activity in all systems of study such as

Hydroxyl radical , DPPH radical, Superoxide radical, Nitric oxide scavenging, Reducing power,

Ferrous ion chelatingability and B- carotene linoleic acid assay.

Methanol extracts of P. ostreatus and P. cystidiosus fruiting bodies possessed

antioxidant, reducing power, radical scavenging and iron chelating activities that were higher

than those of other commercial mushrooms (104).On the other hand, Elmastas et al. (109) and

Dubost et al. (110) reported that oyster mushroom extracts possessed only moderate antioxidant

activities compared to other edible mushrooms. The antioxidant activity was positively

correlated with total polyphenol content. Furthermore, Lee et al. (111) showed that P.

citrinopileatus extractsprepared from fruiting bodies were more effective than those from

mycelium and fermentation broth filtrate, presumably due to a higher amount of total phenols.

Methanol extracts of P. florida and P. pulmonarius fruiting bodies showed similar antioxidant

activities

(70,71), and an ethanol extract from P. citrinopileatus fruiting bodies had antioxidant activities

comparable to those from P. eryngii, P. ferulae and P. ostreatus mushrooms (111,112).

P. citrinopileatus fruiting body extracts have shown antioxidant activities in vitro and in

hyperlipidaemic hamster rats. Extracts added to a high-fat diet increased the activities of

antioxidant enzymes in rats (113). P. ostreatus mushroom extracts had antioxidant properties in

aged and CCl4-induced liver damaged rats, as indicated by significant increases in

concentrations of antioxidantsand antioxidant enzymes (114,115). Pleuran, a b-glucanisolated

from P. ostreatus, had a positive effect on the antioxidant status of rats and decreased

precancerous lesions induced in rat colon (116). A polysaccharide-peptidecomplex isolated from

P. abalonus fruiting bodies prolonged the lifespan of senescence-accelerated mice. Gene

expression of antioxidant enzymes was up-regulated and consequently their activities were

increased (117).

Yong Cui et al., in 2005 has carried out a study of the antioxidant peoperty of Inonotus

obliquus in which ha has evaluated four extracts of the fungus against DPPh, superoxide and

peroxyl radicals in which the Polyphenolics has a strong antioxidant activity and the estracts

containing triterpenoidsand steroids presented a relatively strong antioxidanteffect.

Chen XiaoPinget al., in 2009 has studied the antioxidant activity of polysaccharide from

Ganoderma lucidumin whichj the results have shown that G. licidum polysaccharides exhibited

higher DPPH-, O-, OH- free radical scavenging activity.

EPSs obtained as antioxidative exopolysaccharide from the submerged mycelia culture

of Ganederma resinaceum showed strong scavenging activities against superoxide and 1,1-

diphenyl-2- picrylhydrazyl (DPPH) radicals (Hyun Mi et al.,2006)

Abah in 2010 evaluated the antioxidant activity of Agaricus bisporus, an edible fungus

and its The antioxidant activity was analyzed using 2, 2-diphenyl-1-

picrylhydrazyl (DPPH) and hydroxyl (OH-) radical scavenging assays. The extract showed more

scavengingactivity on DPPH (IC = 0.139) more than OH (IC = 0.149)

Kin am yoon et al ., 2011 has studied the phenolic compound concentration and evaluated

the antioxidant activity of various extracts from the fruiting bodies of Lentinus edodesin which

the acetone extrats was more effective in scavenging DPPH radicals than the Methanolic and Hot

waterv extracts.Strongest Chelating effect of 86.45% was obtained from the acetonic extract at

1mg/ml methanolic extract showed strong b-carotene and linoleic acid inhibition and the hot

water extract showed a high reducing power of 0.96 .

MUSHROOMS AND IMMUNE MODULATION:

Bioactive compounds isolated from the medicinal mushroom exert antitumoral,

immunomodulator, antiproliferative, hypotensive, bioregulation (increasing of immunological

function), preserving homeostasis, preventing the prognosis of chronic generative diseases (Zha

et al., 2003) (Wasser et al., 1999) (Didukh et al., 2003). Several evidences show that the main

substances responsible for the immunomodulator effects of mushrooms is the polysaccharides β-

D-glycan (Wasser 2000).

Numerous bioactive polysaccharides or polysaccharide-protein complexes from

medicinal mushrooms are described that appear to enhance innate and cell-mediated immune

responses, and exhibit antitumour activities in animals and humans. Stimulation of the host

immune defence systems by bioactive polymers from medicinal mushrooms has significant

effects on the maturation, differentiation and proliferation of many kinds of immune cells in the

host. Many of these mushroom polymers were reported previously to have immunotherapeutic

properties by facilitating growth inhibition and destruction of tumour cells. Recent research has

also shown that some of these mushroom-derived polymers may possess direct cytotoxic effects

on cancer cells. While the mechanism of their antitumour actions is still not completely

understood, stimulation and modulation of key host immune responses by these mushroom

polymers appears central. Although somewhat controversial, recent evidence suggests that

mushroom polymers (-glucans) may trigger the stimulation of many kinds of immune cells in

animals and humans by binding to a specific cellular receptor known as complement receptor

type 3 or CR3.

The mushroom Coriolus versicolor (Yun Zhi) was recorded in the Compendium of

Materia Medica by Li Shi Zhen during the Ming Dynasty in China, as being beneficial to health

and able to bring longevity if consumed regularly. Various products derived from this mushroom

and claimed to have medicinal value are commercially available. Among them, PSK (Sakagami

et al., 1991) and PSP are the most prominent. Yang et al. (1993) classified PSP as a new

biological response modifier which is defined as an agent capable of modifying the host's

biological response by stimulating the immune system and thereby eliciting various therapeutic

effects (Tomada et al., 1987). PSP exerts immunomodulatory and antitumor activities (Yang et

al., 1992).Oral administration of PSP at 1.5 mg/kg to normal ICR mice brought about an

elevated production of interleukin-2.

Zeng et al. (1993) reported that oral administration of PSP at 1-2 g/kg per day for 15-20

days to nude mice inhibited growth of human lung adenocarcinoma by 50-70%. Wang et al.

(1993) found that PSP administered intraperitoneally (IP) at 50 mg/kg per day for about 3 weeks

produced approximately 45% inhibition of the growth of Lewis lung cancer. A reduced

inhibition of liver cancer was observed after treatment with PSP. Yang et al. (1993) noted an

inhibitory effect of PSP on incorporation of ~H-uridine and ~H-thymidine into nucleic acids in

Ehrlich ascites tumor cells. PSP exerted a greater inhibitory effect than PSK on P388 leukemia

cells. The antiproliferative potencies of PSP and PSK against human gastric cancer, lung cancer,

lymphoma and mononuclear leukemia cell lines were similar. Intraperitoneal or oral

administration of PSP likewise inhibited the growth of sarcoma 180 cells in mice, with the

fi)rmer route of administration being more effective. Xu et al. (1993) observed that PSP restored

the immune status in cyclophosphamide-treated rats as witnessed in serum lgG titer, lymphocyte

proliferation and NK cell flmction. Hence, PSP has been shown to manifest immunomodulatory

and antitumor activity in both experimental animals and cancer patients. Its ability to diminish

side effects of radiotherapy and chemotherapy make its inclusion as an adjunct for cancer

treatment worthy of consideration.

Recently, peptide-bound and protein-bound polysaccharides with immunomodulatory and

antitumor activities have also been purified from other mushrooms, for example, Tricholoma

species (Wang et al., 1995). From the culture filtrates of Tricholoma lobayense, a protein-bound

polysaccharide with a molecular weight of 154 kDa was isolated. It inhibited the growth of

sarcoma 180 cells that had been implanted in mice, restored the phagocytic function of peritoneal

exudate cells and mitogenic activity of T cells in tumor-bearing mice and induced gene

expression of some immunomodulatory cytokines in mice (Liu et al., 1995, 1996a,b). From a

submerged mycelial culture of another Tricholoma species, a peptidebound polysaccharide with

a molecular weight of 17 kDa, which exhibited more potent immunomodulatory and antitumor

activities than PSP, was purified (Wang et al., 1995). Another peptide-bound polysaccharide

with a molecular weight of 15.5 kDa was prepared from the cultured mycelia of Tricholoma

mongolicum (Wang et al., 1995). It possessed the properties of activating macrophages,

stimulating macrophage antigen-presenting activity, which in turn enhanced T-cell proliferation,

and inhibited the growth of sarcoma 180 cells implanted into mice. Wang et al. (1996a) found

that both mouse lymphocytes and macrophages were activated by preparations of

polysaccharopeptide from cultured mycelia and culture medium of C. versicolor. Glucans

including lentinan and schizophyllan and those extracted from other fungi such as Ganoderma

lucidum and Volvariella volvacea are also known to possess antitumor and immunomodulatory

activities. It is hoped that these polysaccharides and protein-bound and peptide-bound

polysaccharides can be developed into clinically useful drugs. Evidence has been accumulating

that mushrooms have nutritional as well as medicinal value.

Ganoderma lucidum has been known for its broad spectrum of health benefits from preventative

measures and maintainance of health to the regulation or treatment of chronic as well as acute

life threatening illness (Chang, 1995). The fungus is best known for its immunostimulatory effect

in managing certain types of cancer in combination with conventional therapy and for its anti-

HIV effect.A polysaccharide enriched GL-P fraction from G.lucidum significantly inhibited

fibrosarcomagrowth in mice when it was used in combination with bleomycin (Lee et al., 1984).

Likewise,a Ganoderma polysaccharide fraction, GLP(AI), greatly increased the life-span of

tumor- implanted mice when it was administered intraperitoneally with cytotoxic antitumor

drugs, such as Adriamycin, fluorouracil and cisplatin (Lee et al., 1994). Polysaccharides and

triterpenes are the two majorphysiologically active constituents in the fungus (Dharmananda

1988). The polysaccharide fractions and triterpenes were reported to have Anti-cancer, anti-HIV,

immunomodulatory, hepato-protective, hypoglycemic and cytotoxic tumor, anti-hyperlipidemic,

hypotensive, anti-platelet aggregation, analgesic activities respectively. The Ganoderma b-

glucanshave abroad spectrum effect via transduced cell surface receptors in the immune system,

lead to the release of cytokines and lymphokines (cell mediators) such as IL-1, IL-2, IL-4

(interleukins), interferon and TNF (tumor necrosis factor)Many immune parameters are

improved such as increaseof T-Cell function and antibody production. These mechanisms

account for the Antitumor, anti-inflammatory,and bacteriocidal effects (Chang

1994).Immunomodulatoryeffects of Ganoderma polysaccharides may be useful in autoimmune

disorders, AIDS and cancer prevention.Ling Zhi-8 a newly discovered BRM from the mycelium

of Ganoderma lucidum (Kino et al., 1989), wasshown to activate the defence systems in

promoting T-cell functions cytokine productionfor a host of health benefits.