Antioxidant
activity and Folic acid content in indigenous isolates of Ganoderma lucidum
Krishna
Kondragunta. V, Karuppuraj.
V, Perumal. K*
Shri. A.M.M. Murugappa
Chettiar Research Centre, Taramani,
Chennai- 600113, Tamil Nadu, India
*Corresponding Author E-mail: perumalk@mcrc.murugappa.org
ABSTRACT:
Ganoderma lucidum, a medicinal
fungus called Linzhi in China and Reishi
in Japan, is an economically important fungal species and commonly utilized for
its nutraceutical properties. G. lucidum mushroom does not have cyto-toxicity and has been demonstrated to be safe due to
its’ long history of oral administration. G.
lucidum was subjected to an intensive scientific
research since 80s, demonstrating the multiplicity of its medicinal uses and it
is a major source for many bioactivities. Among many known and tested bioactive
compounds, particularly polysaccharides occupy a precious position as immuno modulators. Anti-oxidant activity of polysaccharides
has been identified from G. lucidum. In the present investigation, 4 different
indigenous isolates of G. lucidum (GL-01 to GL-04) were tested for its
antioxidant and folic acid content. The
DPPH radical scavenging activity of G. lucidum was reported and has the maximum antioxidant
activity (95.36±0.02%) recorded for GL-01 at 1mg/ml concentration and it is
followed by the same strain at 0.8mg/ml concentration (94.92±0.01%). GL-02,
GL-03 and GL-04 of methanol extract shown 93.18±0.17, 93.55±0.41 and 92.5±0.34%
scavenging activity respectively. The maximum activity with water extract
(62.18±1.96%) recorded by GL-03 at 0.8mg/ml concentration. Methanol extract has
shown excellent antioxidant potential compare to water extract. Maximum folic
acid content (77.7 µg/100g) was recorded in GL-03. The positive results of free
radical scavenging activity by fruit bodies of this is another alternative
platform to new finding in field of pharmaceutical and nutraceuticals.
KEYWORDS:
Anti-oxidant
activity, Free radical scavenging activity, DPPH, Folic acid and Ganoderma lucidum
INTRODUCTION:
Free radicals are molecules having an
unpaired electron in the outer orbit. They are not stable molecules and ready
to react. Free radicals play an important role in living systems. For example,
oxygen radicals wield critical actions such as signal transduction and gene
transcription. NO is one of the most widespread signalling
molecules and participate in every cellular and organic function1.
The beneficial effects of free radicals occur at low or moderate concentrations
and those levels are balanced by an efficient antioxidant network in the body.
The imbalance
between this oxidants and antioxidants causes oxidative damage of tissues and
bio molecules, eventually leading to disease conditions especially degenerative
diseases2. Some antioxidants are produced during metabolism (e.g.,
glutathione, ubiquinole and uric acid), while many
others are obtained from foods in the diet. However, this stockpile of defence mechanical system appeared to weaken
considerably with aging. Antioxidants are often reducing agents such as thiols, ascorbic acid, folic acid and polyphenols3.
Mushrooms have also been reported as organisms with antioxidant activity. Among
medicinal mushrooms, G. lucidum possessed profound antioxidant and anti tumour activities.
G. lucidum is a medicinal mushroom; the most famous
in this group is a legendry mushroom in china, with a long fascinating history
dating back over two thousand years. It produces several metabolites such as
polysaccharides, triterpinoids, steroids, alkaloids,
nucleotides and fatty acids with wide range of biological activity and
therapeutic use4. Scientific studies on the extracts and substances
from this mushroom demonstrated their anti-ageing, sedative. hepatoprotective, anti-allergic, hypoglycaemic,
cardiovascular, Immuno modulatory
and anti-viral effects.
The present
investigation aimed to evaluate the four indigenous strains of G. lucidum for
its antioxidant property and Folic acid content. Evaluation of antioxidant
activity of methanol and water extracts of different indigenous strains of G. lucidum
mushroom was done by DPPH radical scavenging activity. Folic acid is
antioxidant which used to prevent cancer during pregnancy. So that the content
of folic acid present in G. lucidum were also estimated.
MATERIALS AND METHODS:
Collection
of plant material and identification
Four fruiting bodies
of G. lucidum
collected from mushroom production unit, Shri. A.M.M.
Murugappa Chettiar research
center, Vadakkadampadi and authenticated as the G. lucidum by Karupuraj5.
Preparation of Mushroom extracts
The G. lucidum
extracts were made using methanol and water by following method as described by the method6. The fruit body
of mushroom was sun dried and powdered using mixer grinder to size 5mm. Five
grams of dried mushroom was homogenised with 100 ml
solvent using shaker at 150rpm and room temperature for 24hrs and filtered
through No. 4 Whatman filter paper. The residue was re-extracted twice. The
extract was kept for drying at room temperature until get dried completely. The
dried extract was re-dissolved at different concentrations in range of 0.2 to
1mg/ml and determined for antioxidant activity.
Quantitative
assay of DPPH free radical scavenging activity
The scavenging
activity for DPPH free radicals was determined according to the method
discovered by Zhao et al., 20067.
An aliquot of 2 ml of 0.1 mM DPPH solution in ethanol
and 1ml of two extracts in different concentration mentioned above were mixed.
The mixture was shaken vigorously and allowed to reach a steady state at room
temperature for 30 min. Decolourization of DPPH was
determined by measuring the decrease in absorbance at 517 nm, and the DPPH
radical scavenging effect was calculated according to the following equation:
Scavenging rate (%) = [(A0 –A1)/
A0] x 100
Where
A0 represents the absorbance of
DPPH solution.
A1 represents the absorbance of extract
with DPPH solution.
Quantification of Folic acid in powder of G. lucidum:
Fruit bodies of G. lucidum
(GL-01 to GL-04) were sun dried and powdered using mixer grinder in size 5mm.
Extraction procedure was followed by the method8. Three grams of
fruit body of G. lucidum
was mixed with 30ml of phosphate buffer (0.1M) were immersed in an ultrasonic
bath for 15 min. Trichloroacetic acid (TCA) (1ml) acetonitrile (10ml)
added to cleanup mixture and precipitate inferring substances. The total
volume was then made up to 50ml with 1M phosphate buffer. The extract was filtered through Whatman
filter paper No.1.
Folate content in G. lucidum mushroom is followed by the
method9. Fruit body extract (50ml) of each
mushroom were dissolved in 2ml of 0.1M sodium hydroxide solution. This was
reduced using zinc and 1ml of concentrated hydrochloric acid (HCl), filtered and diluted to 100ml using distilled water
in 250ml calibrated flask. Aliquots
of the working standard solution of reduced Folic acid extract of fruit body
were transferred into a series of 25-ml calibrated Flasks. 2ml of 5M HCl was added, cooled, followed by the addition of 2ml of
1% Sodium nitrite (NaNO2) and cooled, and 2 ml of 2% Sulfamic
acid was added. The solution was mixed with 2ml of 1% 3-aminophenol, heated in
a boiling water bath for 5 min, cooled, followed by the addition of 3 ml of 5M HCl, swirled, and diluted with water to the mark. After the
solutions were mixed thoroughly, the absorbance was measured at 460nm. Folic
acid was used as standard.
RESULTS
AND DISCUSSION:
DPPH radical which
possesses an unpaired electron and exhibits a strong violet colour
in solution (peak absorbance at 517nm) is used as a reagent for evaluation of
free radical scavenging activity of anti-oxidants. In present study Methanol
shows very good free radical scavenging activity. Free radical scavenging
activity of methanol extracts was shown in table1. The maximum activity (95.36±0.02%) recorded for GL-01 at 1mg/ml concentration
and it is followed by the same strain at 0.8mg/ml concentration (94.92±0.01%). Minimum activity (52.47%) traced by
GL-02 at the concentration 0.2mg/ml.
GL-03 and GL-04
noted 93.55±0.41 and 92.50±0.34 activity at the 1mg/ml concentration
respectively. The minimum activity of GL-03 observed at 0.2mg/ml concentration
is 69.68±0.21%. Table 2 shows results for free radical scavenging
activity of water extracts of G. lucidum mushroom powder.
Table 1. Antioxidant activity
of Methanol extracts of G. lucidum
|
Antioxidant activity (%) |
||||
|
Concentration (mg/ml) |
GL-01 |
GL-02 |
GL-03 |
GL-04 |
|
0.2 |
56.89±0.44 |
52.47±1.64 |
69.68±0.21 |
53.39±0.91 |
|
0.4 |
76.59±3.05 |
68.68±2.3 |
70.62±0.44 |
67.62±1.31 |
|
0.6 |
87.17±1.68 |
81.23±0.75 |
78.93±2.93 |
76.95±2.27 |
|
0.8 |
94.92±0.01 |
89.05±1.5 |
87.39±0.39 |
90.75±2.32 |
|
1.0 |
95.36±0.02 |
93.18±0.17 |
93.55±0.41 |
92.50±0.34 |
Table 2. Antioxidant activity
of water extracts of G. lucidum
|
Antioxidant activity (%) |
||||
|
Concentration (mg/ml) |
GL-01 |
GL-02 |
GL-03 |
GL-04 |
|
0.2 |
35.60±2.14 |
40.66±0.53 |
40.46±1.06 |
43.86±0.83 |
|
0.4 |
41.12±1.11 |
43.4±0.83 |
54.99±1.01 |
51.88±0.79 |
|
0.6 |
42.98±2.08 |
47.52±2.11 |
56.88±1.54 |
54.66±1.52 |
|
0.8 |
44.34±1.21 |
51.97±0.25 |
62.88±1.68 |
54.36±1.14 |
|
1.0 |
40.76±0.51 |
54.64±0.26 |
62.18±1.96 |
51.37±0.74 |
The maximum
activity (62.88±1.68%) recorded
by GL-03 in 0.8mg/ml concentration and it is almost equal (62.18±1.96%) to the concentration 1mg/ml which is
recorded by same strain GL-03.
GL-04 followed the
GL-03 which recorded 54.66±1.52% of activity at the low concentration 0.6mg/ml and it is followed by
GL-02 (54.64±0.26%) which is
almost same at the concentration 1mg/ml. Minimum activity against DPPH free
radical scavenging activity (35.60±2.14%) observed by GL-01 strain at the concentration of 0.2mg/ml.
Methanol extract
shown the better scavenging activity compares to the aqueous extracts in each
strain and it was observed roughly two times more in methanol extracts. In
previous studies it was recorded 93.1% for GL-PS at 0.3mg/ml concentration10,
20.32% at the concentration 1.25mg/ml11, 82.3% by EPS from G. lucidum
broth4, 96% by GL polysaccharides12 and it was reported
88.32% for G. lucidum
mushroom cultivated on brown rice13. Hence the activity differs from
strain to strain and solvent to solvent.
Maximum folic acid
content (77.7 µg) recorded in GL-03 and followed (54.2 µg) by GL-02. The
minimum content observed in the GL-04 strain
Table 3: Quantification of Folic acid from Fruit
body of G. lucidum
|
G. lucidum isolates |
Folic acid
µg/100g |
|
GL-01 |
48.8 |
|
GL-02 |
54.2 |
|
GL-03 |
77.7 |
|
GL-04 |
41.1 |
CONCLUSION:
Considering the above discussion it can be mentionable
that Indigenous isolates of G. lucidum mushroom has the excellent potential of free
radical scavenging activity against the DPPH and can be given as the folate supplement as it is considered as one of the
anti-oxidant also. Thus G. lucidum can be alternative for anti-oxidant drug.
ACKNOWLEDGMENTS:
The authors thank Shri.
A.M.M. Murugappa Chettiar
Research Center for providing laboratory facilities to carry out the research
activities.
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Received on 05.09.2014 Accepted on 18.10.2016
© Asian Pharma
Press All Right Reserved
Asian J. Pharm.
Ana. 2016; 6(4): 213-215.
DOI: 10.5958/2231-5675.2016.00032.6