e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 2 ( 2 0 1 1 ) 438–446

Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/etap

Antioxidant activity and toxicity profile 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 Amala Cancer Research Centre, Amala Nagar, Thrissur, Kerala 680555, India
b Bhabha Atomic Research Centre, Trombay, Mumbai 400085, India

a r t i c l e i n f o a b s t r a c t

Article history: The total triterpene fraction isolated from Ganoderma lucidum, a highly nutritional and popu-
Received 23 March 2011 lar medicinal mushroom occurring in South India, was evaluated for its antioxidant activity
Received in revised form in vitro and in vivo. Total triterpenes successfully scavenged DPPH+ , ABTS+ and superoxide
8 August 2011 radicals, showed significant ferric reducing activity and was highly effective in reducing the
Accepted 23 August 2011 in vitro lipid peroxidation. Activities of the antioxidant enzymes in blood and tissue were
Available online 30 August 2011 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 body’s antioxidant defence
Keywords: systems indicates its potential use as an antioxidant. An attempt was also done to gauge
Antioxidants the toxicity of total triterpenes using acute and sub acute study models in Swiss albino
Toxicity mice. The results showed that Ganoderma triterpenes did not possess significant toxicity.
Triterpenes The findings thus reveal the possible therapeutic use of Ganoderma triterpenes.
Ganoderma lucidum © 2011 Elsevier B.V. All rights reserved.
ORAC
FRAP

large number of reactions. Although human and other organ-

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 sys-
tems endogenously as by-products of metabolism during a
isms possess antioxidant defence and repair mechanisms that
have evolved to protect them against oxidative damage, these
systems are often insufficient to prevent the damage totally.
Deficiency of antioxidant defence may lead to oxidative stress,
which might be associated with a variety of disorders includ-
ing 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 antioxi-
dant 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, nito-
blue 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
 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 2 ( 2 0 1 1 ) 438–446
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 antiox-
idants 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 significant
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
significant antioxidant and antitumour, antiinflammatory,
antinociceptive, antimutagenic, anti-carcinogenic, cardio pro-
tective 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 identified in G. lucidum (Wasser and Weis, 1997). Major
triterpenes isolated from G. lucidum include ganoderic (highly
oxygenated C30 lanostane-type triterpenoids), lucidenic, gan-
odermic, 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 proper-
ties and demonstrated therapeutic efficiency 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 composi-
tion 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 activ-
ity 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 compo-
nents. 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.
439
2. Materials and methods
2.1. Collection of mushroom
Ninety day-old fresh fruiting bodies of G. lucidum, growing on
Caesalpinia coriaria Wild. tree, were collected from Thrissur dis-
trict, Kerala, South India. The specimen was identified with
the help of the literature and the identification was confirmed
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 MUBL-
3172).
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 frac-
tion 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.
1–26), chloroform:petroleum ether 1:1 (Fr. 27–50), chloroform
(Fr. 51–150), chloroform:methanol 9:1 (Fr. 151–165), methanol
(Fr. 165–180)]. Each fraction was analysed for the presence
of triterpenes using Liebermann–Burchard reagent (acetic
anhydride–con. H2 SO4 ). The fractions were also spotted on
TLC plate and analysed for the presence of triterpenes using
the spray reagent anisaldehyde–H2 SO4 . The fractions that
answered the tests for triterpenes (Harborne, 1973) were com-
bined 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 Per-
formance Thin Layer Chromatography (HPTLC) using CAMAG
ADC2 system. 5 ␮l of sample was applied on a precoated sil-
icagel 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 dis-
tance of 85 mm. Plate was visualized (Camag TLC visualiser)
under 254 nm, 366 nm, and in visible light after spraying with
anisaldehyde–sulphuric 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 profile
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 Pur-
pose of Control and Supervision of Experiments on Animals
(CPCSEA), Ministry of Environment and Forest, Government
440 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 2 ( 2 0 1 1 ) 438–446
of India, and by the approval of Institutional Animal Ethical
Committee of the Research Centre (149/99/CPCSEA dated 23-
10-2009).
2.4. Determination of in vitro antioxidant activity of
total triterpenes
2.4.1. DPPH radical scavenging assay
DPPH (2,2-diphenyl-1-picryl hydrazyl) in its radical form has
an absorption peak at 515 nm, which disappears on reduc-
tion by an antioxidant compound (Aquino et al., 2001). An
aliquot (100 ␮l) of different concentrations of the total triter-
penes (10–100 ␮g/ml) were added to 1 ml of freshly prepared
DPPH solution (0.25 g/l in methanol). Absorbance was mea-
sured 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.
2.4.2. 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 (10–100 ␮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.
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 reac-
tion mixture containing 0.1 ml of mice liver homogenate
(25%, w/v) in tris–HCl buffer (20 mM, pH 7), KCl (30 mM),
FeSO4 (NH4 )2 SO4 ·6H2 O (0.16 mM), ascorbate (0.06 mM) and var-
ious concentrations of the total triterpenes (1–100 ␮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 den-
sity (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 riboflavin was detected by NBT reduction method (Mc Cord
and Fridovich, 1969). The reaction mixture contained EDTA
(6 mM) containing NaCN (3 ␮g), riboflavin (2 ␮g), NBT (50 ␮g),
KH2 PO4 –Na2 HPO4 buffer (67 mM, pH 7.8) and various concen-
trations of the total triterpenes (1–200 ␮g/ml) in a final 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 rad-
ical was calculated by comparing the absorbance values of the
control with that of treated.
2.4.5. Ferric reducing antioxidant power (FRAP) assay
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
concentrations of total triterpenes (1–40 ␮g/ml). This was incu-
bated for 15 min at 37 ◦ C. An intense blue coloured complex
was formed when Fe3+ –TPTZ complex was reduced to the fer-
rous (Fe2+ ) form. The absorbance at 595 nm was recorded. The
reducing power of the samples increased with the absorbance
values.
2.4.6. Oxygen radical absorbance capacity (ORAC) assay
The antioxidant activity of the total triterpenes was also esti-
mated 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 fluores-
cence was recorded every 5 min till the last reading becomes
less than 5% of first 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 fluorescence 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
five groups of six animals. Group I animals were kept as nor-
mal group without any drug treatment. Group II, III and IV
animals were administered with total triterpenes (TT) at dif-
ferent doses 10, 50, and 100 mg/kg b.wt. orally for 30 days.
Group V animals were given 250 ␮l of sunflower oil (which was
used as vehicle to administer total triterpenes) orally for the
same period. At the end of the experiment, animals were sac-
rificed by cervical dislocation, and blood was collected by heart
puncture. Liver was excised, washed and 10% homogenate
was prepared in ice-cold Tris–HCl 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, ery-
throcytes and mitochondria. The total protein was estimated
by the method of Lowry et al. (1951). Haemoglobin was esti-
mated by the cyanmethemoglobin solution using Drabkin’s
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 deter-
mined 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).
2.6. Determination of toxicity of total triterpenes
Acute and sub acute toxicity of total triterpenes (TT) was deter-
mined using Swiss albino mice (weighing 25 ± 2 g).
 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 2 ( 2 0 1 1 ) 438–446
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:
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 – sunflower 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.
2.6.2. Sub acute toxicity studies
Animals were divided into five groups of six animals in each
group. The total triterpenes was administered orally once daily
for 30 days as follows:
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 – sunflower oil (250 ␮l).
During the period the animals were monitored for clini-
cal and behavioural changes such as diarrhoea, immobility,
neuromuscular problems and after adverse reactions. The
changes in body weights were recorded weekly with simul-
taneous observation of toxic manifestation and mortality.
Twenty-four hours after the last dose of the extract the ani-
mals were sacrificed 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 deter-
mination 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 fixed in 10%
neutral buffered formalin for histopathological analysis. The
sections were stained with hematoxylin–eosin for evaluation.
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 Bonferroni’s test
using InStat Graphpad software. a P < 0.001, b P < 0.01, c P < 0.05
were considered as extremely significant, significant and mod-
erately significant respectively. d P > 0.05 were considered as
non significant with respect to normal group.
441
3. Results
3.1. Isolation of total triterpenes (TT)
Total triterpene fraction (TT) was isolated from G. lucidum.
TLC and HPTLC analysis followed by derivatisation with spray
reagents confirmed 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 vio-
let 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 anisaldehyde–sulphuric acid reagent respec-
tively. HPTLC profile of the total triterpenes under 254 nm
using solvent system toluene:ethyl acetate (9:1) is shown in
Fig. 1.
3.2. Determination of in vitro antioxidant activity of
total triterpenes
The total triterpenes showed significant 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 efficiently scavenged
ABTS+ radicals generated by the reaction of 2,2 -azinobis (3-
ethylbenzothiazolin-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 gen-
erated from EDTA–NaCN–riboflavin 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 reduc-
tion of riboflavin. Total triterpenes showed significant 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 fluorescence,
through its high radical scavenging ability.
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 significantly 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
significantly in the entire treated group compared to the nor-
mal. 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.
442 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 2 ( 2 0 1 1 ) 438–446

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

Table 1 – Effect of total triterpenes administration on antioxidant enzymes in whole blood.

Treatments Catalase Superoxide Glutathione Glutathione
(mg/kg b.wt.) (K/g Hb) dismutase (U/g Hb) peroxidase (U/ml of (nmol/ml)
hemolysate)
Normal 313.48 ± 51.38 265.27 ± 15.6 1.83 ± 0.62 10.67 ± 2.99
TT 10 340.14 ± 96.96c 292.48 ± 55.18c 1.89 ± 0.40c 14.00 ± 2.69c
TT 50 376.14 ± 83.48c 333.92 ± 50.62b 2.04 ± 0.35c 14.83 ± 1.83c
TT 100 418.87 ± 83.25c 389.76 ± 1.84a 2.41 ± 0.58c 15.50 ± 0.84b
Vehicle 319.62 ± 79.10c 289.91 ± 35.85 1.76 ± 0.44c 11.50 ± 2.79c

TT, Ganoderma total triterpenes. Values are mean ± S.D. (n = 6).

a
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 Superoxide Glutathione Glutathione Lipid peroxidation
protein) dismutase peroxidase (nmol/mg (nmol of MDA
(U/mg protein) (U/mg protein) protein) formed/mg protein)
Normal 32.32 ± 14.43 17.65 ± 4.29 18.73 ± 4.06 22.97 ± 8.67 2.47 ± 0.28
TT 10 54.05 ± 13.88b 22.87 ± 5.92b 19.63 ± 4.10b 22.57 ± 5.14b 2.31 ± 0.29b
TT 50 59.04 ± 21.44b 24.59 ± 6.76b 22.08 ± 0.99b 26.06 ± 5.11b 2.02 ± 0.27a
TT 100 66.50 ± 15.68a 27.41 ± 6.20a 25.94 ± 5.77a 29.97 ± 11.3b 1.99 ± 0.20a
Vehicle 34.29 ± 13.10b 18.06 ± 3.41b 18.21 ± 3.10b 21.41 ± 4.20b 2.76 ± 0.12b

TT, Ganoderma total triterpenes. Values are mean ± S.D. (n = 6).

a
P < 0.05 with respect to normal.
b
P > 0.05 with respect to normal.
 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 2 ( 2 0 1 1 ) 438–446 443

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 antioxi-
dant 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 adminis-
tration 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 administra-
tion 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 sunflower oil alone treated group, compared to
the normal group. However the values are not statistically
significant to support the antioxidant activity of sunflower
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 concen-
trations of total triterpenes were given orally to the animals
and no significant change in the haematological and bio-
chemical parameters were observed compared to the normal
group of animals. There were no significant changes in the
haemoglobin content, total leukocyte count as well as differ-
ential count of the treated animals (data not given). There
was no significant 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
444 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 2 ( 2 0 1 1 ) 438–446
Table 3 – Effect of total triterpenes on the activity of liver function enzymes and renal markers in serum.
Treatments (mg/kg b.wt.) ALP (IU/l)
Normal 148.19 ± 34.20
TT 100 150.94 ± 23.33
TT 250 151.25 ± 2.59
TT 500 151.7 ± 53.79
Vehicle 149.11 ± 34.35
TT, Ganoderma total triterpenes. Values are mean ± S.D. (n = 6). Treatments are (P > 0.05) not significantly different from normal.
slight increase in body weight of all the groups including nor-
mal group, in the sub acute study (data not given). However,
the changes were not statistically significant. 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 significant increase in the activ-
ity of the liver marker enzymes such as GOT, GPT, ALP was
observed compared to normal group of animals (Table 3). Kid-
ney function test such as serum urea and creatinine did not
show any significant 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 patho-
logical 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 appre-
ciable 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 radi-
cals 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 fluorescence emission, in the presence of reac-
tive species, is considered as the index of oxidative damage.
The fluorescence of PE is highly sensitive to conformational
and chemical integrity of protein. The inhibition of reac-
tive species by the total triterpenes protects the PE from the
chemical damage and loss of fluorescence. The ORAC assay
provides a unique and complete assessment in which the inhi-
bition time and inhibition degree are measured as the reaction
goes to completion. Unlike other popular antioxidant activ-
ity methods, the ORAC assay provides a direct measure of
hydrophilic chain breaking antioxidant capacity against per-
oxyl radical.
SGOT (IU/l) SGPT (IU/l) Urea (mg/dl) Creatinine (mg/dl)
47.15 ± 7.03 18.73 ± 4.06 25.38 ± 3.10 1.08 ± 0.50
43.22 ± 3.60 19.63 ± 4.10 27.15 ± 1.16 1.34 ± 0.39
42.43 ± 1.12 22.08 ± 0.99 24.24 ± 3.30 1.36 ± 0.27
45.97 ± 3.28 25.94 ± 6.77 27.40 ± 3.44 1.39 ± 0.42
42.04 ± 2.90 18.21 ± 3.10 27.39 ± 4.19 1.31 ± 0.59
The antioxidant enzymes operate as a balanced, co-
ordinated system, play a major role in detoxification and
coordinate the body’s antioxidant defence process. These
innate antioxidant systems include enzymes such as SOD,
CAT and GPx, micromolecules such as albumin, ceruloplas-
min 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 antiox-
idant enzymes such as SOD, CAT and GPx. The significant
increase in the activity of SOD, after the administration of
triterpenes, promise protection against superoxides gener-
ated 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 adminis-
tration 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 adminis-
tration. An increase in these antioxidant enzymes is effective
in protecting the body from many adverse conditions. Admin-
istration of total triterpenes also caused an increase in the
level of GSH, a major non protein thiol containing tripeptide,
is mainly involved in detoxification. 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 gen-
erate free radicals (Halliwell and Gutteridge, 1999). The ability
of total triterpenes to scavenge the free radicals and enhance
body’s defence systems against free radical attack indicates its
high efficiency as an antioxidant.
Although it is generally believed that many mush-
rooms are safe because of their long history of usage,
there remains some justification to fears of mushroom poi-
soning caused by inaccurate species identification. 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 mark-
ers were also assessed to index the extent of toxicity.
It is evident from our study that G. lucidum total triter-
penes are devoid of apparent detectable toxicity and possess
excellent in vivo and in vitro antioxidant property. Natural
compounds, especially low toxicity mushroom derived com-
ponents with potent antioxidant capacity, may be more easily
 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 2 ( 2 0 1 1 ) 438–446
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 findings of the current studies
suggest the potential therapeutic use of the Ganoderma triter-
penes.
Conflict of interest statement
The authors declare that there are no conflicts of interest in
publishing the manuscript. All the authors are associated and
contributed to the work and there are no competing financial
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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