Results and Discussion

44.. RREESSUULLTTSS AANNDD DDIISSCCUUSSSSIIOONN

The results of the present investigation entitiled, “Volvariella volvacea (Bulliard

ex Fries) Singer: A therapeutic fungal biofactory” are discussed as follows:

4.1 Evaluation of the antioxidant constituents in the fruiting bodies and mycelia of

Volvariella volvacea

4.1.1 Evaluation of enzymic and non-enzymic antioxidants

Free radicals are generated in normal pathological cell metabolism from

external factors such as pollution, exogenous chemicals and UV radiation. Body

posses defense systems against free radical-induced oxidative stress, which involve

preventative mechanisms, repair mechanisms, physical defenses and antioxidant

defenses. A variety of antioxidant defense systems are operative, including enzymatic

and non-enzymatic antioxidants (Yadav et al., 1997).

Enzymatic antioxidant defenses include superoxide dismutase (SOD),

glutathione peroxidase (GPx), catalase (CAT) etc. Non-enzymatic antioxidants are

ascorbic acid (vitamin C), α-tocopherol (vitamin E), glutathione (GSH), carotenoids,

flavonoids, etc. These antioxidants possess mechanisms as reducing activity, free

radical-scavenging, potential complexing of pro-oxidant metals and quenching of

singlet oxygen. Enzymes directly involved in detoxification of ROS are superoxide

dismutase, catalase, glutathione peroxidase and glutathione-S-transferase (GST) and

small molecules such as reduced glutathione, vitamin C, vitamin E and uric acid

(Revenanen et al., 1998). Some endogenously synthesized low molecular weight

compounds are also involved in antioxidant defense (Halliwell and Gutteridge, 2007).

Enzymic (SOD, CAT, GR, GST, GPx, G6PDH and PPO) and non-enzymic

antioxidants (vitamin C and GSH) in the fruiting bodies and mycelia of the paddy

straw mushroom, V. volvacea were assessed. The results are discussed as follows:

Superoxide dismutase and catalase are two major scavenging enzymes that

remove the toxic-free radical in vivo. The enzyme SOD scavenges superoxide radicals

by catalysing the conversion of two of these radicals into hydrogen peroxide and

molecular oxygen (Fridovich, 1973). The hydrogen peroxide formed by SOD and other

processes is scavenged by CAT, a ubiquitous heme protein that catalyses the dismutation

of hydrogen peroxide into water and molecular oxygen (Sumanth and Rana, 2006).

SOD activity in the fruiting bodies of V. volvaea was observed as

20.54 ± 0.60 U/mg protein and V. volvaea mycelia was 10.42 ± 0.02 U/mg protein.

The activity of CAT was found to be 35.48 ± 1.43 U/mg protein and 6.41 ± 0.55 U/mg

protein in the fruiting bodies and in mycelia respectively (Table 1).

Glutathione peroxidase (GPx), a selenium enzyme, plays a major role in

regulating the peroxides and prevents lipid peroxidation and protects the cell

membrane from oxidative damage (Sies, 1991). Glutathione reductase (GR), a

NADPH flavoprotein is concerned with the maintenance of cellular level of GSH

(especially in the reduced state) by effecting fast reduction of oxidized glutathione to

reduced form (Venukumar and Lattha, 2002). GPx activity was found to be

2.53 ± 0.12 U/mg protein in fruiting bodies and 1.78 ± 0.09 U/mg protein in mycelia

of V. volvacea. The GR activity (Table 1) was found to be 24.90 ± 1.36 U /mg protein

in fruiting bodies and 7.30 ± 0.15 U/mg protein in the mycelia.

Glutathione-S-transferase (GST) catalyzes the conjugation of the tripeptide,

glutathione (GSH) to many compounds bearing a sufficiently electrophilic center. The

electrophilic compounds are mainly xenobiotics, some endogenously generated toxic

compounds and many environmental pollutants (Hayes and Pulford, 1995). GST offers

protection against LPO by promoting the conjugation of toxic electrophiles with GSH.

GST activity in the fruiting bodies and mycelia of V. volvacea was found to be

14.76 ± 0.60 U/mg protein and 5.66 ± 0.24 U/mg protein respectively.

Glucose-6-phosphate dehydrogenase (G6PDH) is the first and key enzyme of

pentose phosphate metabolic pathway and it is widespread in all tissues and blood

cells, catalyzing the conversion of glucose-6-phosphate to 6-phosphoglucono-δ-lactone

in the presence of NADP+. This reaction yields NADPH and D-ribose 5-phosphate.

NADPH protects the cell against the oxidant agents by producing reduced glutathione.

G6PDH activity was evaluated in V. volvacea and the results are presented in Table 1.

G6PDH activity was 1.43 ± 0.04 U/mg protein in fruiting bodies and

2.23 ± 0.10 U/mg protein in mycelia.

Polyphenol oxidase (PPO) is an oxygen transferring enzyme. Besides using O2

to catalyze the dehydrogenation of catechols to orthoquinones and the

orthohydroxylation of phenols to catechols, a peroxidase activity has been reported by

Strothkamp and Mason (1974). PPO activity was 16.17 ± 1.89 U/mg protein in

fruiting bodies of V. volvacea and 6.73 ± 0.45 U/mg protein in mycelia (Table 1).

Glutathione (GSH) is the strongest antioxidant produced by the body. It is a

tripeptide, whose antioxidant property is facilitated by the sulphydryl group of cysteine

and it is known to have key functions in protective processes. GSH is the reducing

agent that recycles ascorbic acid from its oxidised to its reduced form by the enzyme

dehydroascorbate reductase (Loewus, 1988). It also participates in the detoxification of

xenobiotics, as a substrate for the enzyme GST. GSH acts directly as a free radical

scavenger by neutralizing OH-, restores damaged molecules by hydrogen donation,

reduces peroxides and maintains protein thiols in the reduced state (Sies, 1986).GSH is

also the precursor of the phytochelatins that act as heavy metal binding peptides in

plants. The GSH content of the mycelia was observed to be 31.56 ± 1.75µg/g and

101.34 ± 4.56 µg/g in fruiting bodies of V. volvacea (Table 2).

Vitamin C is a very effective free-radical scavenger. It acts as a chain breaking

scavenger for peroxyl radicals and acts in synergy with vitamin E. Vitamin C is an

outstanding antioxidant in biological systems and powerful reducing agent, also

involved as cofactor in numerous metabolic processes and protects biological

membranes against reactive oxygen species (ROS) (Frei et al., 1989). 2.10 ± 0.08 µg/g

and 0.294 ± 0.01 µg/g of vitamin C was reported in the fruiting bodies and in mycelia

respectively (Table 2).

4.2 Bioactive components

4.2.1 Total yield, protein and carbohydrates in aqueous extract of fruiting bodies

and mycelium of V. volvacea

The functional properties of mushrooms have been attributed to the presence of

bioactive compounds in mushrooms. Table 3 presents the total protein and total

carbohydrate content of V. volvacea.

The fruiting bodies were found to possess higher protein content (68.50 ± 2.15

mg/g) as compared to the mycelia (49.02 ± 1.16 mg/g).

The major bioactive compounds found in mushrooms are the polysaccharides.

Mushrooms represent an unlimited source of polysaccharides with ability to enhance

good health in man (Lindequist et al., 2005). Some mushroom metabolites, such as the

glucans like - lentinan and schizophyllan are used clinically for immune therapy

(Ooi and Liu, 2000; Cui and Chisti, 2003) and have been developed as

pharmaceuticals in Japan and are now commercially available worldwide.

The total carbohydrate content in the fruiting bodies was 63.72 ± 3.50 mg/g

and 39.19 ± 1.57 mg/g in the mycelia. The polysaccharide yield of the fruiting bodies

was higher than the mycelia. The yield of aqueous extract of fruiting bodies was

48.15 % and mycelia were 40.03 % (Table 3).

From the results obtained it could be said that V. volvacea as a good source of

carbohydrates.

4.2.2 Mineral content of fruiting bodies and mycelium of V. volvacea

The content of ash, iron, magnesium, calcium and phosphorous in

V. volvacea are presented in Table 4.

Mineral elements are essential for human health. The concentration of elements

has an important physiological effect on different organs and cellular mechanisms.

The calcium content was 2.93 ± 0.07 mg/g d.wt in the fruiting bodies which

was almost 50 % higher as compared to mycelia. Magnesium content was

1.10 ± 0.04 mg/g d.wt in fruiting bodies and 0.83 ± 0.06 mg/g d.wt in mycelia. The

content of phosphorous and iron was 9.64 ± 0.27 mg/g d.wt and 3.12 ± 0.19 mg/g d.wt

in the fruiting bodies. The mycelia possessed 1.49 ± 0.21 mg/g d.wt of phosphorous

and 1.54 ± 0.03 mg/g d.wt of iron.

The results suggest that the fruiting bodies and mycelia of V. volvacea are good

sources of minerals.

4.2.3 Analysis of phytochemicals in the extracts of V. volvacea

The aqueous extract of the fruiting bodies and mycelia of V. volvacea were

screened for the presence of phytochemicals and the results are represented in Table 5.

A positive result indicating the presence of protein, carbohydrates, phenols,

alkaloids, flavonoids, tannins and steroids were observed in the aqueous extracts of both

fruiting bodies and mycelium.

Total phenols and flavonoid content

The total phenol and flavonoid contents of the aqueous extract of V. volvacea

was determined.

Many studies have found that edible mushrooms possess potent antioxidants,

such as phenolics, flavonoids and α-tocopherol (Lo and Cheung, 2005; Lee

et al., 2007; Jayakumar et al., 2009). Phenols are important plant constituents known

to be powerful chain-breaking antioxidants (Duh et al., 1999) and may directly relate

to the antioxidant action by their ability to scavenging free radicals by single-electron

transfer (Ribeiro et al., 2006; Lee et al., 2007; Kim et al., 2008) and play an important

role in stabilizing lipid peroxidation (Yen et al., 1993).

The total phenolic content expressed as mg of gallic acid equivalents (GAE),

catechol equivalents (CE), catechin equivalents (CAE) and flavonoid content as

catechol equivalents (CE), rutin equivalents (RE) and quercetin equivalents (QE) per

gram in fruiting bodies and mycelia of V. volvacea are depicted in Table 6.

The phenolic content of fruiting bodies and mycelium extracts of V. volvacea

were reported to be 28.42 ± 0.73 and 19.08 ± 0.45 mg/g GAE, 31.39 ± 1.56 and 11.90

± 0.52 mg/g CAE, 26.17 ± 1.12 and 13.28 ± 0.66 mg/g CE respectively.

The total flavonoid content of fruiting bodies and mycelium extracts of

V. volvacea were reported to be 8.75 ± 0.39 and 6.86 ± 0.31 mg/g CAE; 9.71± 0.51

and 8.23 ± 0.27 mg/g RE; 6.78 ± 0.34 and 6.75 ± 0.48 mg/g QE respectively.

Flavonoids are polyphenolic compounds widely distributed in fruits,

vegetables, plant extracts as well as in plant-derived beverages such as tea and red

wine (Hertog et al., 1993; Robards and Antolovich, 1997). Flavonoid antioxidants

function as scavengers of free radicals by rapid donation of hydrogen atom radicals.

Many of the pharmacological effects of flavonoids are related to their interaction with

several enzymes (Di-Carlo et al., 1990; Chang et al., 1993) and to their antioxidant

activity, which can be due to their ability to scavenge free radicals.

The antioxidant activity of putative antioxidants have been attributed to various

mechanisms, among which are prevention of chain initiation, binding of transition metal

ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction,

reductive capacity and radical scavenging (Diplock, 1997; Gulcin et al., 2002 a,b).

Therefore, the results of the present study suggest that the extracts of

V. volvacea as well as other edible mushrooms, might reduce oxidative damage in the

human body and provide health protection.

4.3 In vitro antioxidant activity of V. volvacea

The aqueous extracts of the fruiting bodies and mycelia were investigated for

antioxidant activity by several in vitro methods, such as DPPH, ABTS, DMPD and

hydroxyl radical scavenging assays, reducing power assay, ferric antioxidant reducing

power (FRAP) assay, cupric ion reducing antioxidant capacity (CUPRAC) assay, lipid

peroxidation inhibition assay, -carotene bleaching assay, erythrocyte hemolysis

inhibition assay, phosphomolydenum assay and ferrous ion chelating assay.

4.3.1 DPPH radical scavenging activity

DPPH is a free radical compound that has been widely used to determine the

free radical scavenging ability of various samples. This method is based on the

reduction of DPPH in solution, in the presence of a hydrogen donating antioxidant and

is dependent on the formation of the non-radical form, DPPH-H in the reaction. The

colour of the DPPH radical solution becomes lighter and its absorbance goes down in

the presence of an antioxidant compound (Gulcin et al., 2003).

At concentrations of 0.2 - 1.0 mg/mL, the scavenging abilities of FBAE, FBPS,

MAE, IPS and CFAE of V. volvacea on DPPH radicals were between 28.60 - 75.36 %,

19.41 - 70.33 %, 19.79 - 69.12 %, 12.36 - 64.02 % and 9.37 - 57.31 % (Table 7).

At 0.02 - 0.10 mg/mL, the radical scavenging ability of positive controls

ascorbic acid, trolox, BHA and quercetin were between 89.69 - 94.77 %, 94.34 - 97.09

%, 89.76 - 95.72 % and 92.08 - 96.37 % respectively.

EC50 values of the mushroom extracts ranged from 0.65 to 0.88 mg/mL. The

hierarchy of activity decreased in the order: quercetin (0.03) ~ BHA (0.03) ~ trolox

(0.03) ~ ascorbic acid (0.03) > FBAE (0.65) > MAE (0.69) > FBPS (0.71) > IPS (0.78)

> CFAE (0.88) mg/mL respectively.

A significant difference (P

All the tested compounds exhibited effective radical cation scavenging activity.

As seen in Table 8 various extracts from the mushroom, V. volvacea is an effective

ABTS•+

radical scavenger in a concentration dependent manner (0.2 - 1.0 mg/mL).

The scavenging abilities of FBAE, FBPS, MAE, IPS and CFAE of

V. volvacea on ABTS radicals varied from 21.12 - 86.84 %, 17.19 - 68.01 %, 21.41

- 74.46 %, 18.04 - 62.04 % and 10.10 - 52.12 % respectively at 0.2 - 1.0 mg/mL.

The ABTS radical scavenging ability of positive controls ascorbic acid, trolox,

BHA and quercetin were between 21.92 - 95.32 %, 23.53 - 97.07 %, 61.25 - 96.78 %

and 56.72 - 92.54 % at 0.1 - 0.5 mg/mL respectively.

EC50 values of the extracts of fruiting bodies and mycelia ranged from

0.56 - 1.00 mg/mL. EC50 values of ABTS radical scavenging activity of fruiting

bodies, mycelia and standard antioxidants was found to be in the order: BHA (0.08) >

quercetin (0.09) > trolox (0.20) ~ ascorbic acid (0.20) > FBAE (0.56) > MAE (0.68) >

FBPS (0.70) > IPS (0.81) > CFAE (1.0) mg/mL respectively.

A significant difference (P

EC50 values of the fruiting bodies and mycelial extracts on DMPD radicals

ranged from 0.55 to 0.75 mg/mL. The scavenging effect of fruiting bodies, mycelia

and standard antioxidants on DMPD radicals were ranked in the order: trolox (0.02) >

ascorbic acid (0.03) > BHA (0.10) > quercetin (0.14) > FBAE (0.55) > MAE (0.61) >

IPS (0.62) > CFAE (0.69) > FBPS (0.75) mg/mL respectively.

A significant difference (P trolox (0.20) > ascorbic acid (0.37) ~ quercetin (0.37)

> FBAE (0.62) > MAE (0.68) > FBPS (0.82) > IPS (0.91) > CFAE (1.01) mg/mL

respectively.

A significant difference (P ascorbic acid (0.40) > BHA

(0.44) > FBAE (0.50) > MAE (0.51) > trolox (0.76) > FBPS (0.78) > IPS (0.87) >

CFAE (1.05) mg/mL respectively. All the tested samples presented much lower

reducing powers than those of positive controls, which indicate that synthetic

antioxidants have better reducing ability than the antioxidants from mushroom fruiting

bodies and mycelium.

A significant difference (P ascorbic

acid (0.03) > trolox (0.04) > BHA (0.05) > FBAE (0.75) > MAE (0.77) > FBPS (0.96)

> IPS (1.01) > CFAE (1.06) mg/mL based on the EC50 values (Table 12).

4.3.7 Cupric ion reducing antioxidant capacity (CUPRAC) assay

The CUPRAC method has also been used to determine the reducing power of

antioxidant compounds (Apak et al., 2004). In this assay, a higher absorbance

indicates higher cupric ion (Cu2+

) reducing ability. Cu2+

reducing capability of

V. volvacea extracts was found to be concentration dependent (0.2 - 1 mg/mL).

At 0.2 to 1mg mL, cupric ion (Cu2+

) reducing ability of FBAE, FBPS, MAE,

IPS and CFAE of V. volvacea ranged between 0.166 - 0.403, 0.179 - 0.398, 0.135 -

0.515, 0.129 - 0.531 and 0.136 - 0.496 respectively.

At 0.02 - 0.10 mg/mL, cupric ion (Cu2+

) reducing ability of positive controls of

ascorbic acid, trolox, BHA and quercetin were between 0.475 - 1.632, 0.344 - 0.824,

0.582 - 1.928 and 0.776 - 1.991 respectively.

The EC50 values of the mushroom fruiting bodies and mycelial extracts by the

cupric ion (Cu2+

) reducing antioxidant power assay ranged from 0.84 to 1.20 mg/mL.

Cupric ions (Cu2+

) reducing ability of mushroom fruiting bodies, mycelium and

standard compounds based on EC50 values exhibited the following order: BHA (0.01)

~ quercetin (0.01) > trolox (0.03) > ascorbic acid (0.05) > MAE (0.84) > IPS (0.92) >

CFAE (1.03) > FBAE (1.10) > MCPF (1.20) mg/mL (Table 13).

A statistically significant difference (P

84.62%, 9.31 - 51.74 % and 5.44 - 46.15 % respectively. At 0.2 - 1.0 mg/mL, the

β-carotene bleaching inhibition of positive controls ascorbic acid, trolox, BHA and

quercetin were between 61.16 - 85.99 %, 71.67 - 94.59 %, 87.22 - 94.47 % and 84.52 -

89.64 % respectively.

EC50 values of the β-carotene bleaching inhibition by the aqueous extracts of

fruiting bodies and mycelia ranged from 6.50 to 12.41 mg/mL. The 50 % inhibitory

potential of β-carotene bleaching of various extracts from mushroom fruiting bodies

and mycelium of V. volvacea were found to be in the order of: BHA (0.12) > trolox

(0.16) > ascorbic acid (0.18) > quercetin (0.19) > MAE (6.50) > FBAE (6.70) > FBPS

(7.30) > IPS (8.87) > CFAE (12.41) mg/mL respectively.

A statistically significant difference (P FBAE (0.68) > FBPS

(0.88) ~ MAE (0.88) > CFAE (1.18) > IPS (1.33) mg/mL.

A statistically significant difference (P

mushroom and mycelium. The various extracts exhibited good chelating activity on

ferrous ions.

4.3.10 Inhibition of lipid peroxidation in rat liver homogenate

Lipid peroxidation, a process induced by free radicals, leads to oxidative

deterioration of polyunsaturated lipids. LPO inactivates cellular components and there

in plays a key role in oxidative stress in biological systems. Several toxic byproducts

of LPO can damage other biomolecules, including DNA, although these biomolecules

are distant to the site of their generation (Box and Maccubbin, 1997). Transition metal

ions, such as iron and copper, are known to stimulate LPO through various

mechanisms (Halliwell and Gutteridge, 1984). These metal ions may generate OH. to

initiate the LPO process and/or propagate the chain process via decomposition of lipid

hydroperoxide (Braughler et al.,1987). Hence, the inhibitory activity of the mushroom

extracts on LPO was evaluated.

The LPO of rat liver was triggered by FeCl2-H2O2 and the end products of the

process were measured in terms of thiobarbituric acid reactive substances (TBARS)

formed. The various extracts of mushroom and mycelium inhibited lipid peroxidation

in a concentration dependent manner. The lipid peroxidation inhibition of rat liver

homogenate induced by Fe2+

/ascorbate, by the mushroom extracts at different

concentration are presented in Table 16.

The LPO inhibition by FBAE, FBPS, MAE, IPS and CFAE of V. volvacea

were between 23.71 - 80.60 %, 19.25 - 64.34 %, 15.30 - 67.24 %, 13.16 - 62.80

%, 10.22 - 50.03 % at 1 - 5 mg/mL respectively.

At 0.02 - 0.10 mg/mL, LPO inhibition of positive controls ascorbic acid,

trolox, BHA and quercetin were between 51.42 - 71.43 %, 52.27 - 63.42 %, 48.57 -

71.43 % and 41.71 - 86.57 % respectively.

EC50 values ranged from 2.93 to 4.46 mg/mL. Based on the EC50 values, the

lipid peroxide inhibition increased in the order: ascorbic acid (0.02) ~ trolox (0.02) >

BHA (0.03) > quercetin (0.05) > FBAE (2.93) > FBPS (3.52) > MAE (3.59) > IPS

(3.95) > CFAE (4.46) mg/mL respectively.

The various mushroom extracts could inhibit lipid peroxidation by scavenging

the OH- or O2

- radicals or by chelating the iron itself. The mechanism of lipid

peroxidation inhibiting effect may be relative to the membrane stabilization of

polysaccharide.

4.3.11 Inhibition of lipid peroxidation in rat liver mitochondria

Peroxidation is important in food deterioration and in the oxidative

modification of biological molecules particularly lipids. Inhibition of lipid

peroxidation by any external agent is often used to evaluate its antioxidant capacity. In

the present study the inhibition of lipid peroxidation induced by FeSO4-ascorbate by

the mushroom extracts was carried out in rat liver mitochondria.

The mushroom extracts inhibited lipid peroxidation in rat liver mitochondria in

a concentration dependent manner (Table 17).

The LPO inhibition by FBAE, FBPS, MAE, IPS and CFAE of V. volvacea was

found to be 32.88 - 85.18 %, 14.20 - 54.20 %, 35.09 - 82.28 %, 20.10 - 55.83 %

and 11.99 - 47.15 % respectively at a concentration of 1 - 5 mg/mL.

Positive controls ascorbic acid, gallic acid and BHT at 0.05 - 0.25 mg/mL

showed an inhibition between 50.50 - 74.00 %, 42.00 - 67.00 % and 55.50 - 66.06 %

respectively.

EC50 values of the LPO inhibition in rat liver mitochondria of mushroom

fruiting bodies and mycelium extracts ranged from 2.71 to 4.94 mg/mL and 50 %

inhibitory potential of various extracts and standard antioxidants follows the order:

BHT (0.04) > ascorbic acid (0.05) > gallic acid (0.07) > FBAE (2.71) > MAE (2.79) >

IPS (4.27) > FBPS (4.42) > CFAE (4.94) mg/mL respectively.

4.3.12 Lipid peroxidation inhibition assay in egg yolk

Lipid peroxidation involves the formation and propagation of lipid radicals

with numerous deleterious effects, including destruction of membrane lipids,

metabolic disorders and inflammation and production of malondialdehyde (MDA) is a

hallmark of this process. Inhibition of lipid peroxidation was assessed by the amount

of MDA produced. Lipids in egg yolk undergo rapid non-enzymatic peroxidation in

the presence of ferrous sulphate. Various extracts from the mushroom showed a dose

dependent lipid peroxidation inhibition.

The LPO inhibition in egg homogenate by the FBAE, FBPS, MAE, IPS and

CFAE of V. volvacea were 21.14 %, 18.37 %, 20.93 %, 11.76 % and 10.06 % at 1

mg/mL and 75.32 %, 61.29 %, 61.18 %, 45.16 % and 40.08 % at 5 mg/mL respectively.

The egg yolk LPO inhibition by the positive controls ascorbic acid, trolox,

BHT and quercetin were 21.05, 35.89, 23.08 and 53.24 at 0.02 mg/mL and 68.21,

67.18, 55.90 and 92.46 at 0.10 mg/mL respectively.

EC50 values of the mushroom extracts ranged from 3.21 to 5.83 mg/mL. Based

on the EC50 values, the lipid peroxide in egg yolk inhibition increased in the order:

trolox (0.06) > BHT (0.07) ~ ascorbic acid (0.07) > quercetin (0.09) > FBAE (3.21) >

FBPS (3.79) > MAE (4.13) > IPS (5.28) > CFAE (5.83) mg/mL (Table 18).

A statistically significant difference (P rutin (0.21) > FBAE (0.62) >FBPS (0.67) > MAE (0.68)

> CFAE (0.77) > IPS (0.83) mg/mL respectively.

A statistically significant difference (P

4.3.14 Phosphomolybdenum assay

The phosphomolybdenum method is based on the reduction of Mo (VI) to

Mo (V) by the antioxidant compounds and the formation of green phosphate/Mo (V)

complex with the maximal absorption at 695 nm. The assay being simple and

independent of other antioxidant measurements commonly employed, its application

was extended to plant polyphenols (Prieto et al., 1999). Higher absorbance indicates a

higher antioxidative activity. The total antioxidant capacity was expressed as number

of equivalents of gallic acid (GAE) and ascorbic acid (AAE)/g extract.

The total antioxidant activity of mushroom fruiting bodies and mycelium extracts

is depicted in Table 20. Different extracts of mushroom and mycelium exhibited

various degrees of antioxidant capacity.

The total antioxidant capacity was 171.87 and 102.90 mg/g GAE and 93.66 and

56.08 mg/g AAE for FBAE and FBPS from V. volvacea fruiting bodies respectively.

The total antioxidant capacity was 62.75, 30.50 and 24.00 mg/g GAE and 35.48,

20.54 and 22.08 mg/g AAE for MAE, IPS and CFAE from V. volvacea mycelium

respectively.

There are many different methods for determining antioxidant function each of

which depends on a particular generator of free radicals, acting by different mechanisms

(Huang et al., 2005). Antioxidants may act in various ways such as scavenging the

radicals, decomposing the peroxides and chelating the metal ions (Cam et al., 2009).

Four mechanisms have been proposed to explain how phenolic antioxidants can

play their role. The first one involves a direct hydrogen atom transfer (HAT) (Mayer

and Rhile, 2004) from the antioxidant to the radical. The second mechanism, involves

single electron transfer (SET) (Rojano et al., 2008) from the antioxidant to the radical,

leading to indirect H-abstraction. The third has been termed sequential proton loss

electron transfer (SPLET) (Klein and Lukes, 2007) and takes place once the anion has

been formed. The fourth mechanism is metal chelating activity. Metals chelation may

provide important antioxidative effects by retarding metal catalyzed oxidation

(Gulcin et al., 2010). All four mechanisms may occur in parallel, but at different rates.

It is very difficult to assess the antioxidant activity of a product on the basis of a single

method because the antioxidant mechanism in biological matrices is quite complex and

several different factors play a role in these mechanisms (Huang et al., 2005).

According to the results of this study, it is clearly indicated that the aqueous

extract of V. volvacea has significant antioxidant activity against various antioxidant

systems with the fruiting bodies showing higher activity than the mycelium in vitro;

moreover, the mushroom species can be used as an easily accessible source of natural

antioxidants and as a possible food supplement or in pharmaceutical industry. The

various antioxidant mechanisms of the mushroom extract may be attributed to strong

hydrogen-donating ability, a metal chelating ability and their effectiveness as good

scavengers of superoxide and free radical.

4.4 Determination of the acute oral toxicity of the aqueous extract from the

fruiting bodies of V. volvacea.

In short-term toxicity studies, the effect of oral administration of aqueous

extract from the fruiting bodies of V. volvacea (VVAE) was analyzed. The effect of

VVAE at doses of 1000, 2000 and 3000 mg/kg b.wt were assessed. The doses were

administered for a period of 30 consecutive days. During the study period, the animals

were observed for any clinical signs and body weight changes. The activities of the

marker enzymes, antioxidant status and serum metabolites were analyzed.

4.4.1 Effect of VVAE on body and organ weights of experimental rats

Changes in body weight have been used as an indicator of adverse effects of

drugs and chemicals (El-Hilaly et al., 2004). The body weights of control and VVAE

treated rats are presented in Table 21. Absolute and relative organ weights of rats were

measured at the end of acute toxicity test period (30 days). No significant difference

(P

that VVAE had no disturbing effects on the circulating blood cells or on

hematopoiesis.

The transaminases (AST and ALT) are well-known enzymes used as good

indicators of liver function predicting possible toxicity (El-Hilaly et al., 2004).

Generally, any damage to the parenchymal liver cells results in elevations of both

transaminases in the blood and AST found in the serum is of both mitochondrial and

cytoplasmic origin and any rise can be taken as a first sign of cell damage that leads to

the outflow of the enzymes into the serum. Bilirubin is one of the most useful clinical

clues to the severity of necrosis and its accumulation is a measure of binding,

conjugation and excretory capacity of hepatocyte (Manokaran et al., 2008). Serum total

protein levels reflects the functional status of the liver. Therefore, no significant changes

in total bilirubin, protein, ALT and AST activities observed, suggest that the subacute

administration of VVAE did not alter the hepatocyte function and metabolism.

As a measure of renal function status, serum urea and creatinine are often

regarded as reliable markers. Any elevation in the serum concentrations of these markers

is indicative of renal injury (Adelman et al., 1981; Lameire et al., 2005). Thus, the

results recorded in this study suggest that VVAE did not affect the renal function.

The liver is the site of cholesterol disposal or degradation and the major site of

cholesterol synthesis. No significant (P

levels on VVAE treatment. The results observed suggest that VVAE has no toxic

effect on the antioxidant system and did not cause oxidative stress.

4.4.4 Histopathological examination of liver and kidney of the experimental rats

Microscopic observations of liver and kidney tissues are presented in

photomicrograph 1 and 2 (a-d). The sectioning revealed normal liver histomorphology

and renal architecture and absence of any gross pathological lesions in kidney and liver.

In conclusion, the present investigation provides valuable information on the

acute toxicity profiles of oral administration of aqueous extract of V. volvacea. At the

oral doses tested, VVAE can be considered safe as it did not cause either any lethality or

adverse changes in the general behavior or hematological and biochemical parameters.

4.5 Antiproliferative study

Mushrooms are nutritional foods and contain nontoxic compounds that have

medicinal benefits. Mushrooms have a variety of accumulated secondary metabolites

such as phenolic compounds, polypetides, terpenes and steroids. Mushrooms also have

lectins, polysaccharides, polysaccharide-peptides and polysaccharide-protein

complexes which are known to have immunomodulatory and anticancer activities

(Zhang et al., 2007; Sun and Liu, 2009).

Kim et al. (2009) reported the antiproliferative properties of Pleurotus ostreatus,

Pleurotus cornucopiae and Pleurotus salmoneostramineus on HT-29 cell lines.

The antiproliferative activity of VVAE was analysed in cancer cell lines

namely, HEp- 2, Hep G2, T47D and A549. The viability of the tumour cells was found

to decrease with the increase in the concentration of the extract. The cytotoxicity of the

extract was also assessed in vero cell line. VVAE showed inhibitory effects on the

proliferation of cancer cells but had less effect on normal cells indicating a degree of

specificity for cancer cell lines.

The results obtained are presented in Table 24, 25 and Photomicrograph 3 (a-j).

VVAE at a concentration of 31.25 to1000 μg/mL was assessed for ctytotoxicity

over cancer cell lines. The cell viability was found to be 98.90 ± 2.12 % at

31.25 μg/mL for vero cell lines and 84.30 ± 1.09 % at 1000 μg/mL by MTT assay. At

the concentration of 1000 μg/mL the cell viability percentage was found to be 33.30 ±

1.21 % on A549 cells, 11.14 ± 0.92 % on HEp-2 cells, 17.20 ± 0.63 % on Hep G2 cells

and 24.23 ± 1.19 % on T47D cells. The CTC50 was found to be 201.10 ± 5.20 μg/mL,

190.50 ± 7.20 μg/mL, 200.00 ± 10.10 μg/mL and 380.00 ± 12.25 μg/mL on A549,

HEp-2, Hep G2 and in T47D respectively as assessed by MTT method. The CTC50

value was observed to be > 1000 μg/mL on vero cell lines (Table 26).

The effect of VVAE on cancer cell lines was also assessed by SRB method and

the results are depicted in Table 25. The cell viability on vero cells was observed to be

93.25 ± 3.11 % at 31.25 μg/mL and the viability percentage was found to increase with

an increase in concentration and was found to be 83.80 ± 3.03 % at 1000 μg/mL. The

survival percentage was observed to be 88.72 ± 4.12 %, 83.20 ± 2.94 %,

90.19 ± 3.56 % and 86.03 ± 4.10 % at 31.25 μg/mL on A549, HEp-2, Hep G2 and in

T47D respectively. The cell survival percentage was found to be 19.12 ± 0.77 %, 16.79

± 1.02 %, 17.09 ± 0.71 % and 18.04 ± 0.63 % at the concentration of 1000 μg/mL on

A549, HEp- 2, Hep G2 and in T47D respectively as assessed by SRB method.

The CTC50 of VVAE was found to be 180.00 ± 6.25 μg/mL,

215.10 ± 10.00 μg/mL, 195.00 ± 5.15 μg/mL and 191.15 ± 6.10 μg/mL on A549, HEp-

2, Hep G2 and in T47D respectively by SRB method. The CTC50 value was observed

to be > 1000 μg/mL on vero cell lines (Table 26).

Lavi et al. (2006) reported that the methanol extract of P. ostreatus at

concentration of 1000 μg/mL inhibited the growth of MCF-7 cells and HT-29 cells

with the proliferation index percent of 70 % and 17 %, respectively.

Ghazanfari et al. (2011) observed the aqueous extract of the fruiting bodies of

P. florida to have inhibited the growth of Hela, Hep-G2, MCF-7 and PC-12 cells

with the growth inhibition percent of 65. 44 ± 0.28 %, 69.79 ± 1.62 %, 75.14 ± 1.2 %

and 39.91± 2.86 % respectively.

The methanolic extract from Erycibe elliptilimba had an antiproliferative

effect on SKBR3 and MDA-MB435 human breast cancer cells (Kummalue et al.,

2007). It has been reported that the aqueous extract of Inonotus obliquus

significantly inhibited the growth of Sarcoma 180 cells (Chen et al., 2007) and Hep

G2 cells (Youn et al., 2009).

The obtained results have indicated the efficacy of VVAE as a potent

antiproliferative agent and the activity could be attributed due to the bioactive

substances present in the extract.

4.6 Protective effect of VVAE against EAC induced ascites carcinoma in mice

Ehrlich ascites tumour is a transplantable, poorly differentiated malignant

tumour which originally appeared as a spontaneous breast carcinoma in a mouse. It

grows in both solid and ascitic forms and is able to grow in almost all strains of mice.

Ehrlich tumour is a rapidly growing carcinoma with very aggressive behavior

(Segura et al., 2000). The Ehrlich ascites tumour is used as a transplantable tumour

model to investigate the antineoplastic effect of the compounds.

The EAC model was used for evaluating the antitumour efficacy of the

aqueous extract of V. volvacea at 500 and 1000 mg/kg.

4.6.1 Effect of VVAE on hematological parameters and tumour growth response

of EAC bearing mice

Antitumour activity of VVAE against EAC tumour bearing mice was assessed

by measuring tumour volume, tumour weight, cell count (viable and nonviable), mean

survival time and percentage increase in life span (Table 27). Oral administration of

the VVAE at the dose of 500 and 1000 mg/kg markedly (P

results could be due to the direct cytotoxic effect of the extract or indirectly preventing

the local inflammatory responses. It could be suggested that the mushroom extract by

decreasing the ascitic volume and by arresting the tumour growth, increases the life

span of EAC bearing mice.

Usually, in cancer chemotherapy the major problems that are being

encountered are of myelosuppression and anemia (Hogland, 1982). The anemia

encountered in tumour bearing mice is mainly due to reduction in RBC or hemoglobin

percentage and this may occur either due to iron deficiency or due to hemolytic or

myelopathic conditions (Clarkson and Burchenal,1965). Previous studies have

reported an increase in WBC count and decrease in RBC, PLT and Hb % in EAC

bearing mice (Muthuraman et al., 2008; Sangameswaran et al., 2012).

Treatment with VVAE replenishes the hemoglobin (Hb) content, RBC and

WBC count to the normal levels. It is evident from the result that VVAE possess

protective action on hemopoietic system.

The ethanolic extracts of Phellinus linteus (Song et al., 2003) exhibited

antitumour activity against EAC which was in agreement with our findings.

Joseph et al. (2011) reported the antitumour activity of the polysaccharide isolated

from G. lucidum. The polysaccharide was able to effectively reduce the tumour

volume. Ethyl actetate extracts of Phellinus rimosus was reported to possess

antitumour activity against EAC cells. The extract markedly reduced the tumour

volume (Ajith and Janardhanan, 2003). Nitha et al. (2007) reported that treatment with

aqueous-ethanolic extract of Morchella esculenta mycelium increased the life span and

decreased the tumour volume and viable cell counts in EAC bearing mice.

Treatment with VVAE at both the doses (500 and 1000 mg/kg b.wt) significantly

restored the hematological parameters and altered the tumour growth reponse in tumour

bearing mice in a dose dependent manner in line with the anticancer drug, CP.

4.6.2 Effect of VVAE on the liver marker enzymes in EAC bearing mice

The enzyme (AST, ALT, LDH and ALP) activities was observed to be

decreased significantly (P

restored the altered enzyme activity in serum and liver tissue to near normal levels

(Table 28).

Enzymes in serum have been studied for many years as possible early

indicators of neoplasia and as aids in following the progression and regression of

disease (Kathiriya et al., 2010). Hepatotoxicity may occur due to cytotoxic agent itself

or due to its toxic metabolites and in certain circumstances they can be carcinogenic.

The activities of the liver marker enzymes are proportional to the extent of

malignancy and can thus be used as indicators for the diagnosis and prognosis of

disease. LDH is increased in acute necrosis of the liver and is a sensitive indicator

for hepatic damage (Kim et al., 2001). The abnormal variation in the marker

enzymes reflect the overall change in the metabolism that occurs during malignancy

(Rajkapoor et al., 2005).

-GT is a broad specificity transferase that catalyses the transfer of gamma

glutamyl groups from a wide variety of peptide donors to a wide range of aminoacids

and peptide receptors (Yildrim et al., 1999). -GT cleaves extracellular GSH thereby

providing the increased intracellular glutathione synthesis (Valentich et al., 1992).

-GT activity serves as a marker for the progress of carcinogenic events.

Tissue damage is a sensitive feature in cancerous condition, therefore such

deterioration or destruction of the membrane can lead to leakage of these enzymes

from the tissues. Hence elevation of these liver specific enzymes observed a possible

indicator of the progression of tumour growth (El-Beshbishy, 2005).

Marappan and Subramaniyan (2012) have also reported a significant increase

in the levels of AST, ALT and ALP in the serum of EAC induced mice. Saha et al.

(2008) reported that the treatment of Ipomoea reptens, restored the altered enzyme

activity in the EAC bearing mice. Similar results were obtained by Dolai et al. (2012).

The restoration of the activities of the marker enzymes in the liver and serum

observed in the present study on treatment with VVAE, suggests the role of the

extract in defending the hepatic membrane against ROS produced in cancer

conditions. Thus, indicating the antitumour efficiency of the extract as with the

standard antitumour drug, CP.

4.6.3 Effect of VVAE on hepatic antioxidants in EAC bearing mice

The effect of VVAE on activities of the antioxidant enzymes and levels of non-

enzymic antioxidants are presented Table 29.

EAC control group mice showed a significant decline (P

initiate lipid peroxidation and DNA damage leading to mutagenesis, carcinogenesis

and cell death if the antioxidant potential is insufficient.

The antitumerogenic effect of aqueous extract of V.volvaceae may be due to

the antioxidant and the free radical quenching property of the phytoconstituents in

V. volvaceae.

Pleurotus florida extract was able to reduce lipid peroxidation in EAC

induced tumour in mice (Ajith and Janardanan, 2007). The results obtained in the

present study correlated with the findings reported by Gupta et al. (2004) and

Loganayaki and Manian (2012).

4.6.5 Effect of VVAE on serum biochemical parameters in EAC bearing mice

In the present study, a significant (P

4.6.6 Effect of VVAE on serum lipid profile of EAC bearing mice

A significant (P

vein was observed. Photomicrograph 5b shows the liver tissue section of EAC induced

tumour control mice presenting marked dilatation of central vein, congestion of

perivenular hepatocytes and haemorrhage into the interstitium. Photomicrograph 5c

presents the hepatic tissue of mice treated with VVAE at 500 mg/kg. The section

reveals mild congestion of centrilobular zone, glycogenation, focal endothelial

proliferation and mild histocytic aggregation. Photomicrograph 5d reveals the hepatic

tissue of mice treated with VVAE at 1000 mg/kg. The tissue section presents mild

dilattion of central vein and almost normal hepatocyte architecture. Photomicrograph

5e shows the liver tissue of the mice treated with the standard drug, CP. This tissue

section indicates near normal hepatocyte architecture with negligible dilatation of

central vein and sinusoids.

The aqueous extract of the paddy straw mushroom, VVAE was found to

potentially reduce the tumour volume, viable cells, reverse biochemical alterations in

the serum and ameliorate the oxidative stress posed by EAC cells. Thus from the

results of the biochemical analysis and histopathological studies, it could be suggested

the extract possessed antitumour activity.

4.7 Chemopreventive effect of VVAE in DMBA induced mammary carcinoma

Breast cancer is one of the most common cancers in women in both developed

and developing countries (Parkin and Fernandez, 2006). Increased lifetime exposure to

endogenous or exogenous estrogen is recognized as the single most important risk

factor in the development of breast cancer (Yager and Davidson, 2006).

The rat mammary carcinogenesis model is the best known animal system for

investigating the efficacy of chemopreventive agents. DMBA, a polycyclic aromatic

hydrocarbon (PAH) is one of the reference compounds used as a mammary carcinogen

in rodents (Costa et al., 2002). Mammary tumours induced by the administration of

DMBA are morphologically and histologically similar to human mammary tumours

(Malejka-Giganti et al., 2000).

The present study was undertaken to evaluate the chemopreventive potency of

the aqueous extract of Volvariella volvacea and the results are discussed as follows:

4.7.1 Effect of VVAE on body weight and tumour weight of DMBA induced rats

A marked (P

resulted in a significant improvement in body weight and reduction in tumour weight

in a dose dependent manner as compared with DMBA control rats.

Fig. 1: Effect of VVAE on body weight of DMBA induced rats

Fig. 2: Effect of VVAE on tumour weight of DMBA induced rats

In mammary carcinoma bearing animals, the sharp drop in the body weight

observed may be due to cancer cachexia. Cancer cachexia results in progressive loss of

body weight, which is mainly accounted by wasting of host body compartments such

as skeletal muscle and adipose tissue. Weight loss and tissue wasting are observed in

cancer patients. The loss of body weight implies poor prognosis and shorter survival

time for cancer patients (Khan et al., 1999).

Semecarpus anacardium Linn extract was found to prevent the significant

weight reduction in the DMBA induced rats (Mathivadhani et al., 2007). Cordeiro and

Kaliwal (2011) reported the treatment of Bridelia retusa Spreng increased the body

weight of the DMBA induced animals. Lakshmi et al. (2009) reported that the

methanolic extract of Ganoderma lucidum inhibited the tumour weight in DMBA

induced rats.

The reduction in tumour burden and improved body weight in DMBA induced

rats on treatment with VVAE is indicative of health improvement in cancer and results

of the study suggest the chemoprotective potential of VVAE.

4.7.2 Effect of VVAE on the activities of carbohydrate metabolizing enzymes in

DMBA induced rats

Table 33 represents the activities of carbohydrate metabolizing enzymes in

DMBA induced animals. A marked (P

the host to depend on gluconeogenesis from non-carbohydrate compounds to maintain

its blood glucose level.

The animals that were treated with VVAE at both doses (500 mg and 1000

mg/kg) showed a significant drop in the activity of glycolytic enzymes and a

concomitant elevation in the gluconeogenic enzymes. The observed changes may be

due to the antitumour potential of the extract that is manifested by inhibiting the

glycolytic enzyme activity or by the suppression of tumour progression. This activity

may be attributed to the phytochemicals present in the extract.

Arathi and Sachadanandam (2003) reported Semicarpus anacardium Linn nut

milk extract significantly restored the alterations in the activity of glycolytic enzymes

and gluconeogenic enzymes in the liver and kidney of DMBA induced animals.

4.7.3 Effect of VVAE on the activities of marker enzymes in serum of DMBA

induced rats

The effect of VVAE on the levels of AST, ALT, ALP and LDH in the liver of

control and experimental animals are presented in Table 34. A significant (P

as diagnostic tool in malignant diseases (Ozdemir et al., 2007). Activity of Alkaline

phosphatase (ALP) enzyme is used as a specific tumour marker during diagnosis and

in the early detection of cancer (Bedi and Priyanka, 2012). LDH is a tetrameric

enzyme and is recognized as potential tumour marker especially for solid tumours in

assessing the progression of proliferating malignant cells (Helmes et al., 1998). LDH

also serves as a marker for membrane integrity and is a regulator of many biochemical

reactions and was found to be increased in serum of the cancer bearing animals. The

elevated activity of LDH may be due to over production by tumour cells, or it may be

due to the release of isoenzymes from destroyed tissues.Conversely, administration of

VVAE, controls ALP and LDH levels by decreasing permeability of the membrane

and renders protection to membrane integrity.

Khataibeh et al. (2007) reported the increased serum levels of AST, ALT and

LDH in DMBA induced mammary carcinoma was reversed back to near normal after

treatment with soy and garlic extract. The results suggest the capacity of the extract in

maintaining membrane integrity and permeability.

4.7.4 Effect of VVAE on antioxidant status in DMBA induced rats

Table 35 depicts the status of enzymatic (SOD, CAT, GPx, GR, GST) and non-

enzymatic (GSH and vitamin C) antioxidants in liver, kidney and mammary tissue of

experimental rats. Marked (P

neutralize ROS (Deneke, 2006). GPx, a selenium enzyme, plays a major role in

regulating the concentration of H2O2 (Sies, 1991). GSTs are a family of enzymes that

catalyze the conjugation of reactive chemicals with GSH thereby protecting cells

(Yoshimasa et al., 2000). GR catalyzes the NADPH-dependent reduction of GSSG to

GSH, thus maintaining GSH levels in the cell (Yeh et al., 2005).

The depletion of GSH status in cancer induced animals and reverting back to

near normal in drug treatment is evident to prove the protective nature of the

mushroom extract against cell proliferation by directly reacting with ROS. Vitamin C

may reduce carcinogenesis through stimulation of the immune system (Bendich,

1997).The decreased vitamin C in cancer bearing rats might be due to increased free

radical generation. Medicinal plants rich in radical scavenging antioxidants have been

demonstrated to exhibit antiproliferative and apoptotic effects against human breast

cancer cells as well as DMBA-induced mammary carcinomas (Anbuselvam et al.,

2007; Mathivadhani et al., 2007).

Thus ROS induced lipid and protein oxidation with compromised antioxidant

defenses in the present study is in line with Padmavathi et al. (2006) and

Kumaraguruparan et al. (2007). The results of the present study are in accordance with

Vinothini et al. (2009) who reported that treatment with Azadirachta indica resulted in

increase in the levels of antioxidant enzymes in DMBA induced rats.

The observed increase in the antioxidants after the treatment of VVAE suggests

the protective nature of the extract.

4.7.5 Effect of VVAE on lipid peroxides and hydroperoxides in DMBA induced

rats.

A significant (P

rats could be attributed to the overproduction of ROS (Bhuvaneshwari et al., 2004).

However, the administration of VVAE decreased the LPO and HPO levels which may

be due to the free radical scavenging activity of VVAE.

Cordeiro and Kaliwal (2011) observed significant decrease in LPO upon

treatment with the extract of Bridelia retusa Spreng in DMBA induced rats.

The significant decrease in the levels of LPO and HPO in the animals treated

with VVAE suggests the protective nature of the extract in countering ROS. The

antitumerogenic effect of aqueous extract of V. volvaceae may be due to the

antioxidant and the free radical quenching property of the phytoconstituents of

aqueous extract of V. volvacea.

4.7.6 Effect of VVAE on the mitochondrial marker enzymes in DMBA induced

rats

A significant (P

Administration of VVAE (500 and 1000 mg/kg) showed significant (P

reported in tumour (Damen et al., 1984). Defects in the catabolism of triacylglycerol

rich lipoproteins by lipoprotein lipase may give rise to hypertriglyceridemia

(Fredrickson et al., 1978). Significantly higher levels of triglycerides were observed in

breast cancer patients when compared with their controls (Zlelinski et al., 1988).

Veena et al. (2006) reported that the increased levels of lipids in plasma and

liver in DMBA induced rats, was restored to near normal indices upon administration

of the extract of Semicarpus anacardium and Kalpaamruthaa.

The data obtained in the present study shows that the lipid levels were reverted

back to the near normal levels on treatment with aqueous extract of V. volvaceae. This

pharmacological property of the extract may be due to hypolipidemic property and

cytoprotective effect of the drug on deteriorated cell membrane, which is a crucial

condition in the cancerous condition.

4.7.9 Effect of VVAE on the hematological parameters in DMBA induced rats

Table 40 presents the effect of VVAE on the hematological parameters. A

significant decrease (P

4.7.10 Effect of VVAE on the histology of mammary tissue in DMBA induced rats

Photomicrograph 6 (a-d) presents the histopathological study of the mammary

tissue of the control and DMBA induced experimental rats.

Photomicrograph 6a reveals the normal architecture of the mammary tissues of

normal control animals presenting absence of any pathological condition.

Photomicrograph 6b presents the mammary tissue sectioning of DMBA-tumour

control group revealing hyperplasia of the lobules. The cellular architecture was found

to be altered and enlargement of the alveolus was seen. Inflammatory cell infiltration

was observed. Photomicrograph 6c shows the mammary tissue section of rats treated

with 500 mg dose of VVAE. Mild ductular proliferation with focal epithelial

hyperplasia observed in Periductular adipose tissue was present with mild

inflammatory cellular infiltration. Photomicrograph 6d represents the mammary tissue

of rats administered with 1000 mg dose of VVAE presented a histological profile

similar to the normal mammary tissue. Epithelial cells were uniform in size with

neglible infiltration of inflammatory cells.

This observation of VVAE treatment is supported by previous reports on

mammary carcinoma. Semicardium annardicum extract treatment was found to

improve the derangements caused by DMBA (Arathi and Sachdanandam, 2003).

Arulkumaran et al. (2007) reported that treatment with Kalpaamrutha increased the

activities of the MDH and SDH in DMBA induced rats. Jagatheesh et al. (2010)

reported that the A. paeonifolius extract restored the the hematological alterations in

the DMBA induced rats.

Hence, it is concluded from the present investigation that VVAE improves the

body weight of animals, attenuates LPO, normalizes the status of antioxidants,

hematological parameters, activity of carbohydrate metabolizing enzymes, cytosolic,

lysosomal and mitochondrial marker enzymes. The extract also presents hypolipidemic

activity. Thus the results of present investigation have confirmed the usefulness of

VVAE as an effective anticancer agent in experimental mammary carcinoma.

4.8 Cardioprotective effect of VVAE against isoproterenol induced myocardial

infarction

MI is the acute condition of necrosis of the myocardium that occurs as a result of

imbalance between coronary blood supply and myocardial demand. Experimental

induction of MI by ISO in animals is a well established model to study the protective

role of various cardioprotective agents. The present study was undertaken to investigate

the protective efficacy of aqueous extract of Volvariella volvacea in ISO induced rats.

4.8.1 Effect of VVAE on the heart weight of the ISO induced myocardial

infarcted rats

Fig. 3 presents the heart weight of the experimental animals. In the present

study, cardiac hypertrophy, i.e., enlargement of the heart has been observed in ISO-

administered rats. A significant increase (P

compared to normal control rats. Pretreatment with VVAE (500 and 1000 mg/kg) daily

for a period of 21 days prior to ISO-induction resulted in a marked (P

4.8.3 Effect of VVAE on the activities of CK and CK-MB in serum of ISO

induced myocardial infarcted rats

Activities of CK and CK-MB in serum of normal and ISO induced myocardial

infarcted rats are given in Fig. 5.

Fig. 5: Effect of VVAE on the activities of CK and CK-MB in serum of ISO induced myocardial

infarcted rats

The rats induced with ISO showed a significant (P

4.8.4 Effect of VVAE on the activities of cytosolic marker enzymes of ISO

induced myocardial infarcted rats

The effect of VVAE on the activities of marker enzymes (AST, ALT and

LDH) in serum and heart of normal and ISO induced myocardial infarcted rats are

presented in Table 41. ISO induction caused a significant (P

Fig. 6: Effect of VVAE on the LDH isoenzymes of ISO induced myocardial infarcted rats

L1 - Control; L2 – ISO induced; L3 - ISO + VVAE (500 mg/kg); L4 - ISO + VVAE (1000 mg/kg)

In cardiac tissue LDH-1 and LDH-2 are predominant hence, detection of

elevated concentration of this enzyme released into the blood stream from the

damaged tissue has become a definitive diagnostic and prognostic criterion for various

diseases and disorders and a study of its isoenzymes has found importance in the

location of tissue damage (Plaa and Zimmerson, 1997).

The increased intensity of LDH-1 and LDH-2 bands in serum observed in the

study could be due to the ISO induced necrosis of the myocardium.

Pretreatment with VVAE significantly decreased the intensity of LDH-1 and

LDH-2 bands in ISO-induced rats which could be due to the reduction in the degree of

damage in the myocardium by VVAE thereby preventing their leakage.

4.8.5 Effect of VVAE on the antioxidants in cardiac tissue of ISO induced

myocardial infracted rats

The effect of aqueous extract of Volvariella volvacea on the antioxidant

enzymes in the heart of the experimental animals are presented in Table 42.

In the present study, the ISO-induced rats showed a significant (P

anion, hydrogen peroxide causes myocardial cell damage mediated by ISO (Searle and

Wilson, 1980; Guarnieri et al., 1980).

Prior treatment with VVAE was found to improve the activities of SOD and

CAT by scavenging superoxide and hydrogen peroxides produced by ISO, in a dose

dependent manner.GSH is important in protecting the myocardium against oxygen free

radical injury, the observed decrease in reduced glutathione levels might be due to

increased utilization in protecting thiol containing proteins from lipid peroxides and

from other reactive oxygen species which causes the reduction in the activities of GPx,

GRx and GST (Priscilla and Prince, 2009).

Vitamin C reduces the risk of CVD by reducing blood pressure, blood

cholesterol and the formation of oxidized LDL cholesterol (Ondrejickova et al., 1991).

Kumaran and Prince (2010) reported decrease levels of vitamin C and vitamin E in

ISO induced rats. The decrease in the vitamin C levels observed in the present study

could be due to the increase utilization due to oxidative stress caused by free radicals.

The consequent improvement in the levels of GSH and vitamin C upon prior treatment

with VVAE could be due to the antioxidant capacity of the extract.

Priscilla and Prince (2009) reported an increase in the activities of GPx, GR

and GST on pretreatment with gallic acid in the ISO-induced rats. The results obtained

were in accordance with Sudheesh et al. (2013).

4.8.6 Effect of VVAE on levels of cardiac lipid peroxidation in ISO induced

myocardial infarcted rats

Table 43 depicts the levels of LPO and HPO in heart of normal and

experimental rats. Rats induced with ISO, showed a significant (P

Elevation of lipid peroxides in ISO treated rats could be attributed to the

accumulation of lipids in the heart and damage to the myocardial membranes. Oral

treatment with VVAE decreased the levels of lipid peroxidation products in ISO

induced rats. Thus, it could be suggested that VVAE scavenges the ROS produced

excessively by ISO, protected the cardiac tissue because of its antioxidant effect. In

this context, the results of the previous in vitro radical scavenging studies suggest the

scavenging action of VVAE. The observed decrease in peroxidation products implies

the protective nature of the extract against ISO induction.

Kumaran and Prince (2010) had reproted that prior treatment with caffeic acid

decreased the lipid peroxidation levels.

4.8.7 Effect of VVAE on the activities of lysosomal enzymes in ISO induced

myocardial infarcted rats

The activity of the lysosomal enzymes is presented in Table 44. A significant

reduction (P

4.8.8 Protective effect of VVAE on the activities of mitochondrial dehydrogenases

in cardiac tissue of ISO induced myocardial infarcted rats

The ISO treatment significantly (P

K+, Mg

2+ (Mourelle and Franco, 1991). Changes in the properties of these ion pumps

affect the cardiac function.

The decreased activity of Na+/K

+ ATPase could be due to enhanced lipid

peroxidation due to free radicals on ISO induction, since Na+/K

+ ATPase is a thiol

group containing enzyme and is lipid dependent (Ithayarasi and Devi 1997; Paritha

and Devi, 1997). Decreased activity of Na+/K

+ ATPase can lead to a decrease in

sodium efflux, thereby altering membrane permeability (Finotti and Palatini 1986).

Ca2+

ATPase regulates the calcium pump activity. Enhanced Ca2+

ATPase

activity observed during β-adrenergic stimulation could be due to elevated activity of

cyclic AMP that posphorylates at several sites on C-terminal chains of the Ca2+

channel and increases the probability of opening of the Ca2+

channel (Varadi et al.,

1995). The enahanced activity of Ca2+-

ATPase could cause the depletion of high

energy phosphate stores, thereby indirectly inhibiting Na+ and K

+ transport and

inactivation of Na+/K

+ATPase (Ithayarasi and Devi 1997). Mg

2+ ATPase activity is

involved in other energy requiring process in the cell and its activity is sensitive to

lipid peroxidation. Our study shows that pretreatment with VVAE (500 and

1000 mg/kg) maintains the activities of ATPases to near normal in ISO-treated rats.

4.8.10 Effect of VVAE on the levels of glycoproteins in heart and plasma of ISO

induced myocardial infarcted rats

Table 47 presents the levels of glycoproteins in the experimental animals. In

this study, a significant (P

Wexler (1970) suggested that glycoproteins are involved in the myocardial necrosis

and repair. Mathew et al. (1982) also reported similar changes in serum and the heart

glycoproteins in ISO induced MI in rats.

The results indicate that VVAE protected the myocardium against ISO induced

toxicity and maintained the levels of glycoprotein components by its antiglycative effect.

4.8.11 Effect of VVAE on serum and tissue lipids of ISO induced myocardial

infracted rats

ISO induced myocardial infarcted rats showed significant (P

with VVAE lowered the levels of free fatty acids in the serum and heart of myocardial

infarcted rats.

Degradation of PL by phospholipases is considered to be a vital factor in the

genesis of ischemic cellular injury. Increased peroxidation of membrane PL released

via phospholipase A2 resulted in decreased content of heart PL (Chien et al., 1984).

VVAE pretreatment increased the levels of PL in the heart of ISO induced myocardial

infarcted rats. This effect is due to the ability of VVAE to prevent peroxidation of

membrane PL.

Lipoproteins are closely associated with MI. Increased levels of LDL-C reveal

a positive correlation with MI, whereas HDL-C levels reveal a negative correlation.

HDL-C inhibits the uptake of LDL-C from arterial wall and facilitates the transport of

cholesterol from peripheral tissues to the liver where it is catabolised and excreted

from the body. Pretreatment with VVAE minimized the alterations in the levels of

serum lipoproteins by increasing HDL-C and decreasing LDL-C and VLDL-C levels

in ISO induced myocardial infarcted rats.

The pretreatment with VVAE showed a well-stabilizing effect on lipids in the

treatment group (group III and group IV) rats when compared to ISO administered

rats. The results are in agreement with Punithavathi and Prince (2009).

4.8.12 Effect of VVAE on the levels of serum NO, CRP, uric acid and total

protein in ISO induced myocardial infarcted rats

Table 49 depicts the effect of aqueous extract of V. volvaceae on the levels of

nitric oxide, uric acid, protein and CRP in serum of normal and ISO induced

myocardial infarcted rats. The rats induced with ISO showed a significant (P

Serum uric acid is considered to be a risk factor in the development of MI

(Weir et al., 2003). Our results are in accordance with Priscilla and Prince (2009).

Supplementation of mangiferin to ISO induced rats significantly reduced serum uric

acid level compared to ISO administered rats (Prabhu et al., 2006).

CRP is observed to be deposited within all acute myocardial infarcts (Lagrand

et al., 1997) and is an exquisitely sensitive systemic marker of a pathological condition.

The results of various studies indicate that involvement of CRP not only reflects tissue

damage but has a role in ischemic myocardial damage (Griselli et al., 1999).

The results of our study indicate that the elevated levels of serum CRP in the

ISO induced animals is due to the myocardial necrosis and infarction in rats. The prior

treatment of VVAE at both the doses significantly decreased the serum CRP levels,

revealing the protective nature of the extract. This could be due to the capacity of the

extract to reduce inflammation and thereby reducing the effects of ISO.

A decrease in the levels of serum total proteins observed in ISO-induced rats

could be due to increased free radical production by ISO that hinders protein synthesis.

Pretreatment with VVAE significantly increased the levels of total proteins. The effect

could be attributed to the ability of VVAE to scavenge free radicals and to inhibit lipid

peroxidation.

4.8.13 Effect of VVAE on the histopathology of the cardiac tissue in ISO induced

myocardial infarcted rats

Photomicrograph 7 (a-d) reveal the histopathological sectioning of the cardiac

tissue of the experimental rats. Photomicrograph 7a presents the tissue sectioning of

normal rats. The section reveals the normal architecture of the myocardium.

Photomicrograph 7b represents the cardiac cell architecture of ISO induced MI

control rats. The myocardial architecture of the ISO induced rats reveals focal

confluent necrosis of the muscle fiber with inflammatory cell infiltration and edema.

Marked vacuolar changes were also observed. Photomicrograph 7c and d presents

the histological sectioning of the VVAE pretreated groups (500 and 1000 mg/kg)

repectively. The sectioning demonstrated the reversal of myonecrosis seen with ISO

alone group. Pretreatment with VVAE demonstrated marked improvement in ISO

induced alterations such as vacuolar changes, edema, capillary dilatation and

leukocyte infiltration compared to ISO administered group. The reversal to normal

architecture was found to be seen more in the 1000 mg/kg dose when compared to

500 mg/kg dose of VVAE. Both the doses were effective in reversing the necrosis

and inflammation induced by ISO.

Thus our data indicate that VVAE may provide potential therapeutic value in

the treatment of MI. Present study demonstrates the cardioprotective potential of the

aqueous extract of the paddy straw mushroom, Volvariella volvacea in ISO-induced

model of myocardial necrosis, as evidenced by amelioration of cardiac dysfunction,

improvement in endogenous antioxidant defense system, improvement in the activities

of TCA cycle enzymes, increased the activities of the membrane bound ATPases and

decreased lipid peroxidation. VVAE also protected the heart from the accumulation of

lipids and glycoprotein components.

4.9 Antihyperlipidemic effect of VVAE on high fat diet fed rats

Cardiovascular disease (CVD) is the major cause of death in developed

countries and hyperlipidemia is regarded as one of the important risks in the

initiation and progression of atherosclerotic impasse and development of CVD. The

common epidemic reason for hyperlipidemia is an excessive or improper lipid intake

(Harrison et al., 2003). Dietary fat intake has been shown to be important in the

development of human obesity and can be associated with increased oxidative stress

in mammals (Ibrahim et al., 1997). Research data have indicated that high fat diet

(HFD) increases the incidence of diabetes, hypertension and other degenerative

diseases (Huang, et al., 2007).

The present study was undertaken to investigate the hypolipidemic effect of

VVAE in high fat diet induced rats.

4.9.1 Effect of VVAE on body weight gain and organ weights of HFD fed

experimental animals

A significant (P

Makhni et al. (2008) reported that hyperlipidemia, including hypercholesterolemia and

hypertriglyceridemia, is a major risk factor for the development of CVD.

The increase in the body weight of the rats of HFD alone group reflects the

high calorie intake of the rats. The observed increase in weights of the visceral organs

(liver and heart), aorta and adipose tissue is indicative of the accumulation of the fat

due to HFD. Previous reports suggest weight gain in the HFD fed rats (Li et al., 2009;

Lee et al., 2010).

The accumulation of visceral fat is the most important cause of the metabolic

syndrome and excess adipose tissue increases the mortality and risk for disorders such

as hyperlipidemia, hypertension, diabetes and atherosclerosis (Bernardo, 2000). These

changes are frequently associated with a cluster of dyslipidemia, endothelial

dysfunction, insulin resistance and inflammation (Lyon et al., 2003; Goldstein and

Scalia, 2004).

The observed results were suggestive of the antihyperipidemic activity of

VVAE. This could be due to the phytochemicals present in the extract.

Phytochemicals, as phenolic compounds have an anti-obesity effect through the

suppression of dyslipidemia, hepatosteatosis and oxidative stress in obese rats

(Manach et al., 1996; Niho et al., 2001; Hasumura et al., 2004).

Various edible mushrooms have already proven to be an important natural

regimen for controlling hyperlipidemia (Sugiyama et al., 1992; Bobek et al., 1998)

Li et al. (2009) reported the polysaccharide from the mushroom Pholiota

nameko has anti-obesity effect and reduced the relative body weight gain, decreased

adipose tissue and reduced the liver and heart weight in animals that were fed with HFD.

4.9.2 Lipid lowering effect of VVAE in HFD fed experimental animals

Table 51 presents the serum and tissue lipid status in HFD fed rats.

Administration of HFD to the rats resulted in a marked elevation (P

markedly (P

The observed increase in serum HDL-C levels in animals treated with VVAE

suggests the capacity of the mushroom in preventing the accumulation of atherogenic

plaques.

Atherogenic index, (AI) is an important prognostic marker for CVD

(Boers et al., 2003; Parthasarathy et al., 1990). Some healthcare professionals often

use this ratio to assess the risk for developing heart disease. Lower ratio of TC to

HDL-C means lower risk. An elevated TC/HDL-C ratio is usually associated with a

low HDL-C and/or elevated TG.

The increase in the AI in the HFD fed rats is suggestive of lipid accumulation

and hyperlipidemic condition that has resulted in increased AI. The observed reduction

in the AI levels upon administration of VVAE indicates the lipid lowering capacity of

the extract that could be attributed to the phytochemicals present in the extract. Extracts

rich in phytochemicals as phenols, flavonoids were reported to decrease the

hypercholesterolemia and lower the serum lipid levels (Anila and Vijayalakshmi, 2003).

Polysaccharides are well known to be the major structural components of

mushrooms and polysaccharides isolated from different mushrooms have shown good

cholesterol- lowering activity (Rajewska and Bałasinska, 2004). Polysaccharides from

Pholiota nameko were observed to reduce the AI in HFD fed experimental animals

(Li et al., 2009).

TC /HDL-C and LDL-C/ HDL-C ratios are also predictors of coronary risk. In

this study these ratios were markedly reduced by treatement with VVAE (Table 51).

High levels of TC and more importantly, LDL-C are major coronary risk factors

(Temme et al., 2002). It is desirable to have higher plasma HDL-C and lower LDL-C

to prevent atherogenesis, since there is a positive correlation between an increased

LDL-C/HDL-C ratio and the development of atherosclerosis. Administration of VVAE

significantly suppressed the higher values of LDL-C/HDL-C ratio showing the

beneficial effect of the mushroom in preventing atherosclerosis incidence. Li et al.

(2009) reported a decrease in the levels of serum and liver TG and on supplementation

of the extract of the mushroom, P. nameko in HFD fed rats.

Thus the results suggest the administration of the aqueous extract of

V. volvacea extract could lower the risk of cardiovascular diseases by restoring AI,

LDL-C/HDL-C and TC/HDL-C ratios. Thus VVAE has anti-obesity effects that are

reflected by reducing the body weight gain.

4.9.3 Effect of VVAE on antioxidant status of HFD fed experimental animals

In our study, the activities of the antioxidant enzymes (CAT, SOD, GPx, GR

and GST) and levels of non-enzymic antioxidants (GSH and vitamin C) in liver, heart

and adipose tissue were found to be significantly (P

4.9.4 Effect of VVAE on lipid peroxidation in HFD fed experimental animals

Significant (P

The observed increase in the blood glucose levels could be due to increase food

intake in the HFD fed rats. Ishii et al. (2010) reported hyperglycemia in rats fed with

HFD.

Increase in the levels of serum NO observed in the study could be due to the

ROS in the HFD fed rats. High level of NO is deleterious to cells. Increased levels of

serum NO in HFD fed rats was reported by El-Krish et al. (2011).

Observed decrease in the total protein levels in the HFD fed group II animals

could be due to alteration in metabolism in hyperlipidemic conditions. Chen et al.

(2009) reported a reduction in total protein levels in serum of HFD fed rats.

Thus the observed reduction in the serum NO and increase in protein levels

indicate the protective nature of the extract.

4.9.6 Effect of VVAE on the activity of CK and CK-MB in serum of HFD fed

experimental animals

The HFD fed obese rats showed a significant (P

4.9.7 Effect of VVAE on the histology of liver and aorta of HFD fed experimental

animals

Photomicrograph 8a-e presents the effect of VVAE on the hitstology of liver.

The histology appeared normal in control group (Photomicrograph 8a)

revealing the normal architecture of the hepatic tissue. Photomicrograph 8b presents

the hepatic tissue of the HFD fed group II animals. Livers of the HFD group were

larger compared with those in the normal group. In addition, the sizes of lipid droplets

in the VVAE group (500 and 1000 mg/kg b.wt) were remarkably smaller than those of

HFD group, suggesting that VVAE could reduce the accumulation of lipid droplets

and keep hepatocytes normal (Photomicrograph 8c and d) similar to atorvastatin

treatment (Photomicrograph 8e)

Photomicrograph 9 a-e presents the effect of VVAE on aorta morphology in

rats fed high-cholesterol diet.

Photomicrograph 9a presents the aorta of the control group animals. The

section reveals the normal architecture of the aorta. Photomicrograph 9b reveals the

aorta in HFD control rats with layered fat deposit. Photomicrograph 9c and d presents

the VVAE treatment group (500 and 1000 mg/kg b.wt) showing significantly

negligible fat deposits in line with atorvastatin (Photomicrograph 9e).

The histology of results suggests the capacity of the extract in normalizing the

architecture of liver and aorta, thereby reducing the fat deposits. The result implies the

lipid lowering efficiency of the extract similar to standard cholesterol lowering drug,

atorvastatin. Thus by reducing the body weight gain and restoring the organ indices,

AI, lipid profiles, antioxidant status, NO and protein levels and CK activity, VVAE

serves as a potent source for tapping efficient anti hyperlipidemic drugs.

4.10 Antidiabetic effect of VVAE on STZ induced diabetes

Diabetes mellitus is a serious, complex metabolic disorder of multiple

etiologies, characterized by chronic hyperglycemia with disturbances of carbohydrate,

fat and protein metabolism resulting from defects in insulin secretion (β-cell

dysfunction), insulin action (insulin resistance) or both (Kardesler et al,. 2008).

STZ, an antibiotic produced by Streptomyces achromogenes, is a selective

pancreatic β-cell genotoxicant used to induce experimental diabetes in model

organisms. Several evidences indicate that free radicals may play an essential role in

the mechanism of β-cell damage and diabetogenic effect of STZ (Takasu et al., 1991;

Ohkuwa et al., 1995). STZ induced diabetic animals exhibit many of the complications

observed in human diabetes.

The present investigation was undertaken to assess the antihyperglycemic

effect of aqueous extract of Volvariella volvacea against STZ- Nicotinamide induced

diabetic model. The results of the study are discussed below:

4.10.1 Effect of VVAE on the levels of glucose and insulin in serum and glycated

hemoglobin in STZ induced diabetic rats

STZ induction resulted in a marked (P

Hyperglycemia is the hallmark of diabetes. The STZ induced elevation of blood

glucose levels could be due to β-cell damage resulting in insulin deficiency. The marked

reduction in the serum insulin levels observed in the study could be due to the effect of

STZ owing to its ability to destroy pancreatic β-cells, possibly by a free radical

mechanism (Halliwell and Gutteridge, 1994; Simmons, 1984). A marked increase in

insulin levels observed on treatment with VVAE is implicative of the role of the

mushroom extract in combating the damage caused by STZ on pancreatic β cells.

Hypoglycemic effects of various mushrooms have been reported (Kim et al.,

2001; Yang et al., 2002). Oral treatment of VVAE was found to significantly reduce

the blood glucose levels, which is indicative of the hypoglycemic effect of the extract

in line with glibenclamide.

Advanced glycation occurs during normal ageing but to a greater degree in

diabetes in which it plays a major role in the development of diabetic complications

(Ahmed, 2005). The persistent supra-physiological level of glucose non-enzymatically

reacts with Hb to form increased glycosylated hemoglobin (HbA1c) which is a standard

biochemical marker for the diagnosis of ambient glycemia. There are several studies,

which report that serum advanced glycation end products (s-AGEs) increase in senile

diabetic patients. Increased non-enzymatic and autooxidative glycosylation is one of the

possible mechanisms linking hyperglycemia and the vascular complications of diabetes

(Hall et al., 1984). Diabetic rats showed higher levels of glycated hemoglobin indicating

their poor glycemic control. The significant reduction in HbA1c levels indicates the

potent antihyperglycemic efficiency of VVAE similar to glibenclamide.

Hwang et al. (2005) had reported the hypoglycemic effects of Phellinus baumii.

Kiho et al. (1994) stated that the hot water extract of the fruiting bodies of Agrocybe

Fig. 11: Effect of VVAE on HbA1c levels in STZ induced diabetic animals

cylindracea also has hypoglycemic effect. Yuan et al. (1998) reported the hypoglycemic

activity of a water-soluble polysaccharide from the fruiting bodies of Auricularia

auricula-judae. Polysaccharides from Ganoderma lucidum was reported to significantly

raise the insulin levels and decrease blood glucose levels (Jia et al., 2009).

4.10.2 Effect of VVAE on the activity of carbohydrate metabolizing enzymes in

STZ induced diabetic rats

Over-production (excessive hepatic glycogenolysis and gluconeogenesis) and

decreased utilization of glucose by the tissues are the fundamental basis of

hyperglycemia in diabetes mellitus (Shirwaikar et al., 2006).

Table 55 presents the effect of VVAE on glycolytic and gluconeogenic

enzymes in the liver and kidney of the experimental animals. STZ induction resulted in

a significant (P< 0.05) decrease in the activity of glycolytic enzyme, hexokinase (HX)

and glucose-6-phosphate dehydrogenase (G6PDH) with a marked elevation in the

activity of gluconeogenic enzymes glucose-6-phosphatase (G6P) and fructose-1,6-

bisphosphatase (F1,6bP). Oral administration of VVAE (500 and 1000 mg/ kg) was

found to significantly (P

VVAE up regulates the activities of these enzymes in tissues through insulin

release and thereby it enhances the utilization of glucose for cellular biosynthesis,

which is marked by the significant decrease in plasma glucose levels.

It has been demonstrated that in diabetes mellitus, the increased rate of

gluconeogenesis is related to the increased expression of key gluconeogenic enzymes

such as phosphoenol pyruvate carboxykinase (PEPCK), glucose6-phosphatase,

fructose-1,6-bisphosphatase in hepatic tissues (Van deWerve et al., 2000).

The results of the study indicates a significant surge in the activity of the

gluconeogenic enzymes G6P and F1,6 bP on STZ induction.

Glucose-6-phosphatase (G6P), a key enzyme in the homeostatic regulation of

blood glucose concentration, is expressed mainly in the liver and kidney and is critical

in providing glucose to other organs during diabetes, prolonged fasting or starvation

(Bouche et al., 2004). Insulin deficiency achieved by experimental diabetic rats

increases glucose-6-phosphatase activity. It is concluded that enhanced hepatic glucose

output in STZ -nicotinamide-induced diabetic rats probably involves dysregulation of

both the liver and kidney G6P activity. The activity is restored close to normal in the

diabetic rats treated with VVAE.

An increase in the activity of fructose-1,6-bisphosphatase (F1,6bP) has been

suggested as a possible mechanism for the production of increased endogenous

glucose. The decreased activity of fructose-1,6-bisphosphatase in the diabetic animals

treated with VVAE enables inhibition of gluconeogenesis from all gluconeogenic

substrates while avoiding direct effects on glycogenolysis, glycolysis and the

tricarboxylic acid cycle and thereby maintain the blood glucose homeostasis.

Pari and Satheesh (2009) reported a significant increase in the activity of G6P

and F1,6bP in STZ induced diabetic rats. The observed decrease in the activity of

gluconeogenic enzymes and elevation in the activity of glycolytic enzymes on

treatment with VVAE, indicated the utilization of glucose, thereby normalizing the

metabolism and blood glucose concentration.

4.10.3 Effect of VVAE on the activity of polyol pathway enzymes in STZ induced

diabetic rats

Fig 12 presents the activity of aldose reductase and sorbitol dehydrogenase in

the eye of STZ induced diabetic animals. A significant (P

dependent manner as compared with STZ induced diabetic rats. Oral administration

with glibenclamide also resulted in a reduction in the activity of the enzymes.

Fig. 12: Effect of VVAE on actividies of polyol pathway enzymes in STZ induced diabetic animals

One consequence of excessive intracellular glucose levels is an increased rate of

oxidative phosphorylation under hyperglycemia condition, as well as the activation of

the polyol pathway (Forbes et al., 2007). Aldose reductase (AR), a key enzyme in the

polyol pathway, catalyzes NADPH dependent reduction of glucose to sorbitol. Sorbitol

is subsequently converted to fructose by sorbitol dehydrogenase with NAD+

as cofactor

(Dunlop, 2000). Thus the marked reduction in the activity of the enzymes suggests the

activity of VVAE in reducing hyperglycemia thereby reducing the activation of the

polyol pathway. The results obtained were in accordance with Ishii et al. (2008).

4.10.4 Effect of VVAE on antioxidants and tissue peroxidation status in the liver

and kidney of STZ induced diabetic rats

Type 2 diabetes is associated with increased oxidative stress associated with

generation of ROS leading to oxidative damage particularly in liver and kidney

(Kakkar et al., 1998; Mohamed et al., 1999).

STZ induction presents a significant increase (P

Picton et al. (2001) reported that oxidative stress in diabetes coexists with a

decrease in the antioxidant status, which increases the deleterious effects of free

radicals. It has been proposed that S