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Antioxidant activity and toxicity prole of total triterpenes isolated from Ganoderma lucidum (Fr.) P. Karst occurring in South India
T.P. Smina a , J. Mathew a , K.K. Janardhanan a, , T.P.A. Devasagayam b
a b

Amala Cancer Research Centre, Amala Nagar, Thrissur, Kerala 680555, India Bhabha Atomic Research Centre, Trombay, Mumbai 400085, India

a r t i c l e
Article history:

i n f o

a b s t r a c t
The total triterpene fraction isolated from Ganoderma lucidum, a highly nutritional and popular medicinal mushroom occurring in South India, was evaluated for its antioxidant activity in vitro and in vivo. Total triterpenes successfully scavenged DPPH+ , ABTS+ and superoxide radicals, showed signicant ferric reducing activity and was highly effective in reducing the in vitro lipid peroxidation. Activities of the antioxidant enzymes in blood and tissue were increased by the administration of total triterpenes to Swiss albino mice in vivo. The ability of total triterpenes to scavenge the free radicals and to enhance bodys antioxidant defence systems indicates its potential use as an antioxidant. An attempt was also done to gauge the toxicity of total triterpenes using acute and sub acute study models in Swiss albino mice. The results showed that Ganoderma triterpenes did not possess signicant toxicity. The ndings thus reveal the possible therapeutic use of Ganoderma triterpenes. 2011 Elsevier B.V. All rights reserved.

Received 23 March 2011 Received in revised form 8 August 2011 Accepted 23 August 2011 Available online 30 August 2011 Keywords: Antioxidants Toxicity Triterpenes Ganoderma lucidum ORAC FRAP

1.

Introduction

The role of free radicals in the development of disease has attracted attention from both scientists and professionals involved in human health care (Halliwell and Gutteridge, 1999). Free radicals are constantly generated in biological systems endogenously as by-products of metabolism during a

large number of reactions. Although human and other organisms possess antioxidant defence and repair mechanisms that have evolved to protect them against oxidative damage, these systems are often insufcient to prevent the damage totally. Deciency of antioxidant defence may lead to oxidative stress, which might be associated with a variety of disorders including coronary heart disease, neural disorders, diabetes, arthritis and cancer (Yoshikawa et al., 2000). When natural defences

Abbreviations: TT, total triterpenes from Ganoderma lucidum; ORAC, oxygen radical absorbance capacity; FRAP, ferric reducing antioxidant power; TLC, thin layer chromatography; HPTLC, High Performance Thin Layer Chromatography; ROS, reactive oxygen species; RNS, reactive nitrogen species; DPPH, 2,2-diphenyl-1-picryl hydrazyl; ABTS, 2,2 -azinobis (3-ethylbenzothiazolin-6-sulphonic acid); NBT, nitoblue tetrazolium; EDTA, ethylene diamine tetra acetic acid; DTNB, 5,5-dithiobis-(2-nitrobenzoic acid); TPTZ, 2,4,6-tripyridyl-s-triazine; AAPH, 2,2 -azobis (2-amidinopropane) dihydrochloride; PE, -phycoerythrin; TBA, thiobarbituric acid; SGOT, serum glutamic oxaloacetic transaminase; SGPT, serum glutamic pyruvic transaminase; ALP, alkaline phosphatase. Corresponding author. Tel.: +91 487 2304190; fax: +91 487 2307698. E-mail address: drkkjanardhanan@gmail.com (K.K. Janardhanan). 1382-6689/$ see front matter 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2011.08.011

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are overwhelmed by excessive generation of prooxidants, the use of antioxidants, both conventional as well as derived from some novel sources, has become important for the proper management of oxidative stress. Antioxidants are widely used ingredients in dietary supplements for maintaining health, as well as the prevention and cure of various diseases. The major food derived antioxidants include -carotene, leutin, lycopene, selenium, vitamin A, vitamin C and vitamin E. Owing to the increased demand and importance of antioxidants in day to day life, the search for effective, non toxic natural compounds with antioxidant activity has increasingly become a matter of interest. A large number of medicinal mushrooms have recently been reported to possess signicant antioxidant activity (Jones and Janardhanan, 2000; Mathew et al., 2008; Nitha et al., 2010). Ganoderma lucidum (Fr.) P. Karst is known as mushroom of immortality and considered to be a panacea to cure all kinds of diseases in the Chinese folklore. In clinical studies, G. lucidum products have been widely used as a single agent or in combination with other herbal medicines or chemotherapeutic drugs for many years, mainly in Asian countries. Our previous investigations have demonstrated signicant antioxidant and antitumour, antiinammatory, antinociceptive, antimutagenic, anti-carcinogenic, cardio protective and nephroprotective effects of various extracts of G. lucidum (Jones and Janardhanan, 2000; Sheena et al., 2005). Identifying the active fractions or ingredients responsible for the biological activities and their mechanism of action is of great importance for developing pharmaceutical products from this mushroom. At least 140 different triterpenes have been identied in G. lucidum (Wasser and Weis, 1997). Major triterpenes isolated from G. lucidum include ganoderic (highly oxygenated C30 lanostane-type triterpenoids), lucidenic, ganodermic, ganoderenic, ganolucidic and applanoxidic acids, lucidones, ganoderals and ganoderols (Boh et al., 2007; Nishitoba et al., 1986). Investigations have demonstrated that triterpenes are responsible for the major medicinal properties and demonstrated therapeutic efciency of G. lucidum (Paterson, 2006; Wasser and Weis, 1997; Hattori, 2001; Huie and Di, 2004; Kim and Kim, 1999; Lin et al., 2003; Min et al., 2000). It has been reported that some of the physiological effects and distinct properties of G. lucidum are depended upon the strains and the cultivating conditions (Nishitoba et al., 1986). Preliminary studies also indicated that triterpene composition of G. lucidum fruit body varies according to the area in which it is grown (Min et al., 2000). Hence we extended our studies to evaluate the in vitro and in vivo antioxidant activity of total triterpenes isolated from G. lucidum occurring in South India, which is believed to be the key mechanism for major therapeutical properties of mushroom derived components. G. lucidum and its extracts have been reported to show no recognisable toxicity in animal experiments (Wasser and Weis, 1997). However, the possibility of adverse effect of its derivatives especially triterpenes upon long term use may be suspected owing to its remarkable cytotoxicity in various tumour and cancer cell lines in vitro (Lin et al., 2003; Min et al., 2000; Yuen and Gohel, 2005). Thus far, there has been no report regarding this concern in vivo. Hence, we extended our studies to evaluate the toxicity of the total triterpenes using acute and sub acute toxicity models in Swiss albino mice.

2.
2.1.

Materials and methods

Collection of mushroom

Ninety day-old fresh fruiting bodies of G. lucidum, growing on Caesalpinia coriaria Wild. tree, were collected from Thrissur district, Kerala, South India. The specimen was identied with the help of the literature and the identication was conrmed by comparison with type specimens. A voucher specimen was deposited in the herbarium of Centre for Advanced Studies in Botany, University of Madras, Chennai, India (HERB MUBL3172).

2.2.

Isolation of total triterpenes (TT)

Dried and powdered fruiting bodies of G. lucidum (100 g) were extracted with ethanol. The extract was concentrated (9 g) and dissolved in chloroform. The chloroform soluble fraction was separated and the solvent completely recovered, the residue (3 g) was loaded on silica gel column (3 cm 60 cm) and eluted with petroleum ether, chloroform, methanol and various combinations of these solvents [petroleum ether (Fr. 126), chloroform:petroleum ether 1:1 (Fr. 2750), chloroform (Fr. 51150), chloroform:methanol 9:1 (Fr. 151165), methanol (Fr. 165180)]. Each fraction was analysed for the presence of triterpenes using LiebermannBurchard reagent (acetic anhydridecon. H2 SO4 ). The fractions were also spotted on TLC plate and analysed for the presence of triterpenes using the spray reagent anisaldehydeH2 SO4 . The fractions that answered the tests for triterpenes (Harborne, 1973) were combined together and concentrated to get the total triterpene fraction (TT) (1.5 g). The total triterpenes (TT) thus obtained were used for further studies.

2.2.1.

HPTLC analysis

The total triterpene fraction was also analysed by High Performance Thin Layer Chromatography (HPTLC) using CAMAG ADC2 system. 5 l of sample was applied on a precoated silicagel 60 F 254 TLC Plate (E. Merck) of uniform thickness of 0.2 mm using Camag Linomac 5 TLC sampler and developed in automatic developing chamber (CAMAG ADC2) to a distance of 85 mm. Plate was visualized (Camag TLC visualiser) under 254 nm, 366 nm, and in visible light after spraying with anisaldehydesulphuric acid reagent and heating at 110 for 10 min. Toluene:ethyl acetate (9:1) was used as solvent system. Camag TLC scanner 3 was used for scanning. HPTLC prole was obtained with Desaga Video Documentation Unit.

2.3.

Animals

Swiss albino mice (weighing 25 2 g) used in the study, were purchased from Small Animal Breeding Station, Mannuthy, Kerala, India and were housed in well ventilated cages under controlled conditions of light and humidity and provided with normal mouse chow (Sai Durga Food and Feeds, Bangalore, India) and water ad libitum. All the animal experiments were carried out as per the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Government

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of India, and by the approval of Institutional Animal Ethical Committee of the Research Centre (149/99/CPCSEA dated 2310-2009).

2.4. Determination of in vitro antioxidant activity of total triterpenes 2.4.1. DPPH radical scavenging assay

concentrations of total triterpenes (140 g/ml). This was incubated for 15 min at 37 C. An intense blue coloured complex was formed when Fe3+ TPTZ complex was reduced to the ferrous (Fe2+ ) form. The absorbance at 595 nm was recorded. The reducing power of the samples increased with the absorbance values.

DPPH (2,2-diphenyl-1-picryl hydrazyl) in its radical form has an absorption peak at 515 nm, which disappears on reduction by an antioxidant compound (Aquino et al., 2001). An aliquot (100 l) of different concentrations of the total triterpenes (10100 g/ml) were added to 1 ml of freshly prepared DPPH solution (0.25 g/l in methanol). Absorbance was measured at 515 nm, 20 min after the initiation of the reaction. The ability to scavenge DPPH radical was calculated by comparing the absorbance values of the control with that of treatment.

2.4.6.

Oxygen radical absorbance capacity (ORAC) assay

2.4.2.

ABTS+ radical scavenging assay

In ABTS assay, the total triterpenes was allowed to react with ABTS+ , a model stable-free radical derived from 2,2 azinobis (3-ethylbenzothiazolin-6-sulphonic acid) (ABTS) with the reaction of ammonium persulphate (Long and Halliwell, 2001). The ABTS+ radical solution was diluted to an absorbance of 0.75 at 734 nm in PBS. 10 l of different concentrations of the total triterpenes (10100 g/ml) were added to 1 ml of ABTS+ radical solution. Decrease in absorbance was measured by a spectrophotometer at 6 min after initial mixing, using PBS as reference.

The antioxidant activity of the total triterpenes was also estimated by ORAC assay (Ou et al., 2001). Reaction mixture (200 l) contained 175 l of 75 mM phosphate buffer (pH 7.0), 10 l of -phycoerythrin (0.5 mg in 7.3 ml buffer), 10 l total triterpene sample and 5 l of 160 mM AAPH. For standard assay, 50 M Trolox was added in place of sample. The uorescence was recorded every 5 min till the last reading becomes less than 5% of rst reading ( Exit 540 nm and Emis 565 nm) (Cao and Prior, 2002). The protective effect of an antioxidant is measured by assessing the area under the uorescence curve (AUC) of the sample as compared to that of the blank in which no antioxidant is present.

2.5. Determination of in vivo antioxidant activity of total triterpenes

Thirty Swiss albino mice (weighing 25 2 g) were divided into ve groups of six animals. Group I animals were kept as normal group without any drug treatment. Group II, III and IV animals were administered with total triterpenes (TT) at different doses 10, 50, and 100 mg/kg b.wt. orally for 30 days. Group V animals were given 250 l of sunower oil (which was used as vehicle to administer total triterpenes) orally for the same period. At the end of the experiment, animals were sacriced by cervical dislocation, and blood was collected by heart puncture. Liver was excised, washed and 10% homogenate was prepared in ice-cold TrisHCl buffer (0.1 M, pH 7.4). The liver homogenate was centrifuged at 3000 g for 20 min at 4 C to remove the cell debris, unbroken cells, nuclei, erythrocytes and mitochondria. The total protein was estimated by the method of Lowry et al. (1951). Haemoglobin was estimated by the cyanmethemoglobin solution using Drabkins method (Drabkin and Austin, 1932). The following assays were done in both blood and liver to assess the antioxidant status. Superoxide dismutase (SOD) activity was measured by the NBT reduction method of Mc Cord and Fridovich, 1969. Catalase (CAT) activity was estimated by the method of Aebi (1974) in blood and Beer and Sizer (1952) in tissue, by measuring the rate of decomposition of hydrogen peroxide at 240 nm. The assay of glutathione peroxidase (GPx) was done by the method of Hafeman et al. (1974) based on the degradation of H2 O2 in the presence of GSH. Reduced glutathione in the tissue was determined according to the method of Moron et al. (1979) based on the reaction with DTNB. Formation of lipid peroxides in liver was measured using the TBA method of Ohkawa et al. (1979).

2.4.3.

Assay for inhibition of lipid peroxidation

Lipid peroxidation in rat liver homogenate was estimated by TBA reaction method (Ohkawa et al., 1979). The reaction mixture containing 0.1 ml of mice liver homogenate (25%, w/v) in trisHCl buffer (20 mM, pH 7), KCl (30 mM), FeSO4 (NH4 )2 SO4 6H2 O (0.16 mM), ascorbate (0.06 mM) and various concentrations of the total triterpenes (1100 g/ml) were incubated for 1 h at 37 C and the resultant MDA and other aldehydes were allowed to react with TBA. Inhibition of lipid peroxidation was determined by comparing the optical density (532 nm) of the triterpene-treated tubes with that of the control.

2.4.4.

Superoxide radical scavenging activity

Superoxide radical (O2 ), generated from the photo reduction of riboavin was detected by NBT reduction method (Mc Cord and Fridovich, 1969). The reaction mixture contained EDTA (6 mM) containing NaCN (3 g), riboavin (2 g), NBT (50 g), KH2 PO4 Na2 HPO4 buffer (67 mM, pH 7.8) and various concentrations of the total triterpenes (1200 g/ml) in a nal volume of 3 ml. The tubes were illuminated under incandescent lamp for 15 min. The optical density at 530 nm was measured before and after illumination. The ability to scavenge superoxide radical was calculated by comparing the absorbance values of the control with that of treated.

2.4.5.

Ferric reducing antioxidant power (FRAP) assay

2.6.

Determination of toxicity of total triterpenes

The ferric reducing ability of the extract was measured at low pH (Benzie and Strain, 1996; Pulido et al., 2000). FRAP reagent (900 l) was mixed with an aliquot of 120 l of different

Acute and sub acute toxicity of total triterpenes (TT) was determined using Swiss albino mice (weighing 25 2 g).

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2.6.1.

Acute toxicity

Animals were divided into four groups of six animals each. A single dose of the total triterpenes was administered orally as follows:

3.
3.1.

Results
Isolation of total triterpenes (TT)

Group I: total triterpenes 1000 mg/kg b.wt. Group II: total triterpenes 2500 mg/kg b.wt. Group III: total triterpenes 5000 mg/kg b.wt. Group IV: vehicle sunower oil (250 l).

The animals were monitored for mortality, changes in body weight, clinical and behavioural changes such as diarrhoea, immobility, neuromuscular problems and adverse reactions for 14 days.

Total triterpene fraction (TT) was isolated from G. lucidum. TLC and HPTLC analysis followed by derivatisation with spray reagents conrmed the presence of a number of triterpenes in the fraction. In HPTLC analysis, seven green bands with Rf values 0.87, 0.78, 0.72, 0.49, 0.35, 0.30, and 0.19, four blue bands with Rf values 0.78, 0.44, 0.30, 0.09 and seven dark pinkish violet bands with Rf values 0.87, 0.78, 0.72, 0.63, 0.49, 0.30, and 0.19 were detected under 254 nm, 366 nm and visible light after spraying with anisaldehydesulphuric acid reagent respectively. HPTLC prole of the total triterpenes under 254 nm using solvent system toluene:ethyl acetate (9:1) is shown in Fig. 1.

2.6.2.

Sub acute toxicity studies

Animals were divided into ve groups of six animals in each group. The total triterpenes was administered orally once daily for 30 days as follows:

3.2. Determination of in vitro antioxidant activity of total triterpenes

The total triterpenes showed signicant DPPH scavenging activity (Fig. 2a). 100 g/ml of the total triterpenes showed a maximum of 81.81% activity. The IC50 value of triterpenes was 41.45 5.2 g/ml. The total triterpenes efciently scavenged ABTS+ radicals generated by the reaction of 2,2 -azinobis (3ethylbenzothiazolin-6-sulphonic acid) (ABTS) and ammonium per sulphate (IC50 30 5.4 g/ml) (Fig. 2b). The total triterpenes effectively inhibited the lipid peroxidation induced by Fe2+ ascorbate system in rat liver homogenate (Fig. 2c). The IC50 value was 84.62 4.1 g/ml. The total triterpenes were found to be very effective in scavenging the superoxide radicals generated from EDTANaCNriboavin system (Fig. 2d). IC50 value was 25 3 g/ml. The 167 g/ml of the total triterpenes could reduce 78.76% of superoxide radicals formed via photo reduction of riboavin. Total triterpenes showed signicant ferric reducing antioxidant power. It was able to scavenge all the ferric ions in the system even at very low concentrations (Fig. 2e). The IC50 value was 6 0.5 g/ml. This indicated the high reducing potential of the total triterpenes. ORAC value for the total triterpenes from G. lucidum was found to be 3515 23 micromoles of Trolox per gram, which point out its protective action against chemical damage and loss of PE uorescence, through its high radical scavenging ability.

Group I: normal (distilled water). Group II: total triterpenes 100 mg/kg b.wt. Group III: total triterpenes 250 mg/kg b.wt. Group IV: total triterpenes 500 mg/kg b.wt. Group V: vehicle sunower oil (250 l).

During the period the animals were monitored for clinical and behavioural changes such as diarrhoea, immobility, neuromuscular problems and after adverse reactions. The changes in body weights were recorded weekly with simultaneous observation of toxic manifestation and mortality. Twenty-four hours after the last dose of the extract the animals were sacriced by cervical dislocation. The blood was collected by direct heart puncture and one portion used for studying haematological parameters such as haemoglobin (Drabkin and Austin, 1932), total leukocyte count (Chaudhari, 2000) as well as differential count. Serum was used for determination of liver function enzymes such as GOT, GPT and alkaline phosphatase and also for renal function tests such as urea and creatinine. Activities of SGOT, SGPT and ALP were determined by kinetic method using the kits purchased from Agappae Diagnostic Ltd., India. Small portions of the selected tissues of liver and kidney of treated animals were xed in 10% neutral buffered formalin for histopathological analysis. The sections were stained with hematoxylineosin for evaluation.

3.3. Determination of in vivo antioxidant activity of total triterpenes

Administration of total triterpenes, to mice for a period of 30 days, enhanced the activities of blood antioxidants (Table 1). The SOD activity was elevated signicantly and the increase was 1.46 and 1.25 times more in 50 and 100 mg/kg b.wt. treated group respectively than that of normal. Activity of the major cellular non enzymatic antioxidant GSH was also increased signicantly in the entire treated group compared to the normal. Increase in GSH was 1.45 and 1.38 times higher than the normal in case of 50 and 100 mg/kg b.wt. treated groups respectively. Similarly elevations of CAT and GPx activity in 100 mg/kg b.wt. treated groups of animals were found 1.3 times as compared with normal.

2.7.

Statistical analysis

The values were expressed as mean standard deviation (S.D.). Statistical evaluation of the data was done by one way analysis of variance (ANOVA) followed by Bonferronis test using InStat Graphpad software. a P < 0.001, b P < 0.01, c P < 0.05 were considered as extremely signicant, signicant and moderately signicant respectively. d P > 0.05 were considered as non signicant with respect to normal group.

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Fig. 1 HPTLC prole of G. lucidum total triterpenes under 254 nm.

Table 1 Effect of total triterpenes administration on antioxidant enzymes in whole blood. Treatments (mg/kg b.wt.)
Normal TT 10 TT 50 TT 100 Vehicle
a

Catalase (K/g Hb)

313.48 340.14 376.14 418.87 319.62 51.38 96.96c 83.48c 83.25c 79.10c

Superoxide dismutase (U/g Hb)

265.27 292.48 333.92 389.76 289.91 15.6 55.18c 50.62b 1.84a 35.85

Glutathione peroxidase (U/ml of hemolysate)

1.83 1.89 2.04 2.41 1.76 0.62 0.40c 0.35c 0.58c 0.44c

Glutathione (nmol/ml)
10.67 14.00 14.83 15.50 11.50 2.99 2.69c 1.83c 0.84b 2.79c

TT, Ganoderma total triterpenes. Values are mean S.D. (n = 6). P < 0.001 with respect to normal. b P < 0.05 with respect to normal. c P > 0.05 with respect to normal.

Table 2 Effect of total triterpenes administration on antioxidant enzymes and MDA levels in liver. Treatments (mg/kg b.wt.) Catalase (K/mg protein)
32.32 54.05 59.04 66.50 34.29 14.43 13.88b 21.44b 15.68a 13.10b

Superoxide dismutase (U/mg protein)

17.65 22.87 24.59 27.41 18.06 4.29 5.92b 6.76b 6.20a 3.41b

Glutathione peroxidase (U/mg protein)

18.73 19.63 22.08 25.94 18.21 4.06 4.10b 0.99b 5.77a 3.10b

Glutathione (nmol/mg protein)

22.97 22.57 26.06 29.97 21.41 8.67 5.14b 5.11b 11.3b 4.20b

Lipid peroxidation (nmol of MDA formed/mg protein)

2.47 2.31 2.02 1.99 2.76 0.28 0.29b 0.27a 0.20a 0.12b

Normal TT 10 TT 50 TT 100 Vehicle

a

TT, Ganoderma total triterpenes. Values are mean S.D. (n = 6). P < 0.05 with respect to normal. b P > 0.05 with respect to normal.

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Fig. 2 In vitro antioxidant activity of total triterpenes from G. lucidum. (a) DPPH radical scavenging activity. (b) ABTS radical scavenging activity. (c) Inhibition of lipid peroxidation. (d) Superoxide radical scavenging activity. (e) Ferric reducing antioxidant power. Values are mean S.D. (n = 3), TT Ganoderma total triterpenes.

The effect of total triterpene administration on antioxidant system and lipid peroxidation levels in liver tissue is presented in Table 2. A marked increase in the activity of liver CAT was observed in all the groups after the administration of total triterpenes. The rise was 2.06, 1.8 and 1.6 times more than the normal. Increase in SOD and GPx was also high. SOD level increased 1.5 times in higher dosage group, where as GPx showed a 1.38 times increase. The increase in 1.3 times tissue GSH level was also observed after administration of total triterpenes. Lipid peroxidation in the tissue was found to decrease marginally. A slight increase in the level of antioxidant enzymes SOD and CAT were observed, in blood and liver of sunower oil alone treated group, compared to the normal group. However the values are not statistically signicant to support the antioxidant activity of sunower oil.

3.4.

Determination of toxicity of total triterpenes

Acute toxicity studies indicated that total triterpenes isolated from G. lucidum did not produce any symptoms of toxicity, behavioural change and mortality of animals in all the tested doses. Even at a high dosage of 5000 mg/kg b.wt., no toxic effect was observed. In sub acute model, three different concentrations of total triterpenes were given orally to the animals and no signicant change in the haematological and biochemical parameters were observed compared to the normal group of animals. There were no signicant changes in the haemoglobin content, total leukocyte count as well as differential count of the treated animals (data not given). There was no signicant variation in the body weight of the animals administered with total triterpenes, compared to the normal animals, in both acute and sub acute models. There was a

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Table 3 Effect of total triterpenes on the activity of liver function enzymes and renal markers in serum. Treatments (mg/kg b.wt.)
Normal TT 100 TT 250 TT 500 Vehicle

ALP (IU/l)
148.19 150.94 151.25 151.7 149.11 34.20 23.33 2.59 53.79 34.35

SGOT (IU/l)
47.15 43.22 42.43 45.97 42.04 7.03 3.60 1.12 3.28 2.90

SGPT (IU/l)
18.73 19.63 22.08 25.94 18.21 4.06 4.10 0.99 6.77 3.10

Urea (mg/dl)
25.38 27.15 24.24 27.40 27.39 3.10 1.16 3.30 3.44 4.19

Creatinine (mg/dl)
1.08 1.34 1.36 1.39 1.31 0.50 0.39 0.27 0.42 0.59

TT, Ganoderma total triterpenes. Values are mean S.D. (n = 6). Treatments are (P > 0.05) not signicantly different from normal.

slight increase in body weight of all the groups including normal group, in the sub acute study (data not given). However, the changes were not statistically signicant. The animals were absolutely healthy and devoid of any adverse reactions, throughout the treatment period. Serum transaminases (GOT and GPT) and ALP activities are good indices of liver damage. After 30 days of treatment with total triterpenes, non signicant increase in the activity of the liver marker enzymes such as GOT, GPT, ALP was observed compared to normal group of animals (Table 3). Kidney function test such as serum urea and creatinine did not show any signicant increase in the treated group (Table 3). Urea and creatinine levels were normal in the treated group. Histopathological examination of liver and kidney of treated animals did not show any pathological manifestations in the total triterpene treated animals as compared with the normal (Fig. 3).

4.

Discussion

The formation of excess amounts of free radicals and the subsequent oxidative stress is a key factor in many pathological conditions. The results of the present study revealed the potent antioxidant power of Ganoderma triterpenes, which were highly effective in scavenging most of the free radicals in vitro including superoxide, peroxyl, DPPH+ and ABTS+ . The ferric reducing ability of the total triterpene was also appreciable even at very low concentration, as Fe2+ ions, hydroxyl, superoxide and peroxyl radicals are oxidatively damaging in vivo. The superoxide scavenging ability of the total triterpene could prevent the further formation of HO2 and OH radicals which are highly reactive and toxic to cell organelles and membranes. The superoxide scavenging and ferric reducing ability of the triterpenes might be contributing to its effective prevention of lipid peroxidation caused by hydroxyl radical, produced in Fe2 + ascorbate system. In the ORAC assay, the chemical damage to -phycoerythrin (PE) and the consequent decrease in its uorescence emission, in the presence of reactive species, is considered as the index of oxidative damage. The uorescence of PE is highly sensitive to conformational and chemical integrity of protein. The inhibition of reactive species by the total triterpenes protects the PE from the chemical damage and loss of uorescence. The ORAC assay provides a unique and complete assessment in which the inhibition time and inhibition degree are measured as the reaction goes to completion. Unlike other popular antioxidant activity methods, the ORAC assay provides a direct measure of hydrophilic chain breaking antioxidant capacity against peroxyl radical.

The antioxidant enzymes operate as a balanced, coordinated system, play a major role in detoxication and coordinate the bodys antioxidant defence process. These innate antioxidant systems include enzymes such as SOD, CAT and GPx, micromolecules such as albumin, ceruloplasmin and an array of small molecules including ascorbic acid, -tocopherol, -carotene, ubiquinol-10, reduced glutathione (GSH), methionine, uric acid and bilirubin (Yu, 1994). The administration of total triterpenes for a period of one month was found to be capable of improving the activities of antioxidant enzymes such as SOD, CAT and GPx. The signicant increase in the activity of SOD, after the administration of triterpenes, promise protection against superoxides generated in vivo. Since superoxide is a major factor in oxygen toxicity, SOD is highly essential for life to sustain. The increased activity of CAT after the total triterpenes administration was helpful in removing hydrogen peroxide generated by several oxidase enzymes in vivo (Chance et al., 1979). Glutathione peroxidise also removes H2 O2 by coupling its reduction to H2 O with oxidation of reduced glutathione, GSH. They can also act on peroxides other than H2 O2 . The activity of GPx also increased moderately after total triterpene administration. An increase in these antioxidant enzymes is effective in protecting the body from many adverse conditions. Administration of total triterpenes also caused an increase in the level of GSH, a major non protein thiol containing tripeptide, is mainly involved in detoxication. Increased levels of GSH in biological systems account for its great potential to prevent free radical damage. GSH can react with OH , HOCl, peroxy nitrite, RO , RO2 carbon centred radicals and singlet oxygen. It can also chelate copper ions and diminish their ability to generate free radicals (Halliwell and Gutteridge, 1999). The ability of total triterpenes to scavenge the free radicals and enhance bodys defence systems against free radical attack indicates its high efciency as an antioxidant. Although it is generally believed that many mushrooms are safe because of their long history of usage, there remains some justication to fears of mushroom poisoning caused by inaccurate species identication. Before developing any pharmaceutical or dietary supplement it is essential to evaluate the toxicity of the compounds. The haemetotoxicological studies were performed to determine adverse effect of toxicants on mature blood cells in the haematopoietic tissue. The levels of renal and hepatic markers were also assessed to index the extent of toxicity. It is evident from our study that G. lucidum total triterpenes are devoid of apparent detectable toxicity and possess excellent in vivo and in vitro antioxidant property. Natural compounds, especially low toxicity mushroom derived components with potent antioxidant capacity, may be more easily

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Fig. 3 Histopathological analysis of organs from total triterpenes treated mice. (a) Liver from normal group. (b) Liver from TT 500 mg/kg b.wt. group. (c) Kidney from normal group. (d) Kidney from TT 500 mg/kg b.wt. group. TT Ganoderma total triterpenes.

and safely incorporated in human diets than other synthetic antioxidant chemicals. The ndings of the current studies suggest the potential therapeutic use of the Ganoderma triterpenes.

Conict of interest statement

The authors declare that there are no conicts of interest in publishing the manuscript. All the authors are associated and contributed to the work and there are no competing nancial interests.

Acknowledgement
The authors sincerely thank Dr. T.A. Ajith, Associate Professor Biochemistry, Amala Institute of Medical Sciences, Thrissur, Kerala, India for his valuable suggestions while preparing the manuscript.

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