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Preclinical studies on the use of                              of the major activities reported for Ganoderma is its anti-
                                                               oxidant activity. Ganoderma lucidum extract (GLE) also
medicinal mushroom Ganoderma                                   contains ergosterols, complete proteins, unsaturated fatty
lucidum as an adjuvant in                                      acids, vitamins and minerals. It is the only known source
radiotherapy of cancer                                         of a group of triterpenes known as ganoderic acids, which
                                                               have a molecular structure similar to steroid hormones
                                                               and contains the most active polysaccharides among me-
G. Gopakumar, Femy Martin,                                     dicinal plant sources. Ganoderic acids may lower blood
Sherin K. Antony, Thulasi G. Pillai and                        pressure and decrease LDL cholesterol4. Anti-aging prop-
Cherupally Krishnan K. Nair*                                   erties have also been reported for this mushroom. In addi-
Amala Cancer Research Centre, Thrissur 680 555, India          tion, it is reported that the mushroom is used for the
                                                               preparation of an HIV tonic5. G. lucidum has been found
Our previous studies have demonstrated that an                 to play an important role in combination with radiother-
extract of Ganoderma lucidum occurring in South                apy and chemotherapy, to render complete regression of
India possesses significant radioprotective property ex        the tumours6–8. Since both polysaccharides and organic
vivo. The present study describes the in vivo radiopro-        germanium derived from G. lucidum are not cytotoxic
tection of normal cells in tumour-bearing mice                 to tumour cells, the anti-tumour effect is attributable to
exposed to gamma radiation. Oral administration of
                                                               induced immunopotentiation.
G. lucidum extract (GLE) to tumour-bearing Swiss
albino mice along with exposure to gamma radiation                Our earlier reports suggest that the aqueous extract of
resulted in tumour regression. Single-cell gel electro-        this mushroom has significant radioprotective activity ex
phoresis (comet assay) on cells of normal and tumour           vivo9. The present study describes the in vivo radioprotec-
tissues from tumour-bearing animals treated with               tion of normal cells in tumour-bearing mice exposed to
GLE and radiation, revealed that there was significant         whole-body gamma radiation and the sensitization of its
reduction in radiation-induced damage to cellular              tumour to radiation (as there was enhanced radiation-
DNA in normal tissues compared to the tumour, indi-            induced damage in cellular DNA in the tumour) by oral
cating preferential protection to normal tissues. The          administration of GLE. The work is also focussed on the
findings suggest the potential use of this mushroom            effect of GLE in tumour regression in tumour-bearing
extract as an adjuvant in radiotherapy, for tumour             Swiss albino mice when administered orally along with
regression and prevention of radiation-induced cellu-
                                                               radiotherapy.
lar damages in normal tissues.
                                                                  GLE was prepared from G. lucidum collected from the
                                                               outskirts of Thrissur, Kerala, India. Sporocarps of the
Keywords: Adjuvant, cancer, Ganoderma lucidum,
                                                               mushroom were dried at 40–50°C and powdered. Several
preclinical studies, radiotherapy.
                                                               batches of 100 g powder were extracted with 1 : 1 etha-
                                                               nol : distilled water mixture at 80°C for 8–10 h. The
IONIZING radiation is one of the well-established and
                                                               extracts were combined, filtered, concentrated and evapo-
widely used therapeutic modalities either for curative or
                                                               rated at low temperature. The residue thus obtained was
palliative treatment of tumours, but the major problem
                                                               used for the experiments. The yield was 10.6%.
associated with cancer radiotherapy is the severe side
                                                                  Swiss albino mice about 8–10 weeks old and weighing
effects and damage to normal tissues1. In radiotherapy of
                                                               22–25 g were purchased from the Small Animal Breeding
cancer, normal tissues need to be protected whereas cancer
cells are exposed to high radiation. Even though many
compounds have been studied for their radioprotecting
property, an agent producing differential radiation response
in the tumour and normal cells would be of importance in
effective treatment of cancer by radiation therapy2.
  Ganoderma (Figure 1), commonly known as reishi
mushroom is highly ranked in Oriental folklore. In Chi-
nese medicine reishi has been considered as a panacea for
all types of diseases. Reishi has attracted significant
attention in recent years due to its large number of phar-
macological properties. The fruiting bodies of this mush-
room contain a variety of chemical substances3. A
number of medicinal mushrooms have recently been
reported to possess significant antioxidant activity. One


*For correspondence. (e-mail: ckknair@yahoo.com)                              Figure 1.   Ganoderma lucidum.

1084                                                                   CURRENT SCIENCE, VOL. 99, NO. 8, 25 OCTOBER 2010
                                                                              RESEARCH COMMUNICATIONS
Section (SABS), Mannuthy, Thrissur. They were main-             Animals in groups I and II were orally administered
tained under standard conditions of temperature and          with distilled water, and groups III and IV with
humidity in the Animal House Facility at our Centre. The     200 mg/kg GLE. The animals in groups II and IV were
animals were provided with standard mouse chow (Sai          exposed to 4 Gy whole-body gamma radiation, 1 h after
Durga Feeds and Foods, Bangalore) and water ad libitum.      administration of distilled water or GLE. Immediately
All animal experiments in this study were carried out        after irradiation the animals were sacrificed and blood,
with the prior approval of the Institutional Animal Ethics   brain, bone marrow and tumour tissues were collected for
Committee (IAEC) and were conducted strictly adhering        performing alkaline single-cell gel electrophoresis (comet
to the guidelines of Committee for the Purpose of Control    assay)10,11.
and Supervision of Experiments on Animals (CPCSEA),             The DNA strand breaks in various tissues of mice were
constituted by the Animal Welfare Division, Government       estimated using alkaline single-cell gel electrophoresis
of India.                                                    which was performed using the method given by Singh10,
   Na2-EDTA was purchased from Sisco Research Labo-          with minor modifications11. Microscopic slides were
ratories Ltd, Mumbai. High melting point agarose, low        coated with normal melting point agarose (1% in PBS
melting point agarose, TritonX-100, DMSO and bovine          containing 0.8% NaCl, 0.02% KCl, 0.14% Na2HPO4,
serum albumin were purchased from Sigma Chemical             0.02% NaH2PO4), the coverslip was placed immediately
Company Inc, MO, USA. All other chemicals were of            and kept at 4°C for 10 min to solidify the agarose. After
analytical grade procured from reputed Indian manufac-       removal of the coverslip, 200 μl of 0.8% low melting
turers.                                                      point agarose containing 50 μl of treated cells was added
   Irradiation, i.e. exposure to gamma radiation was car-
ried out using a 60Co-Theatron Phoenix teletherapy unit
(Atomic Energy Ltd, Ottawa, Canada) at the Amala Can-
cer Hospital, Thrissur at a dose rate of 1.88 Gy/min.
   The combined effect of 4 Gy gamma radiation and
GLE on tumour growth in vivo was analysed as follows.
Solid tumour was produced by injecting Dalton’s Lym-
phoma Ascites (DLA) cells (1 × 106 cells/animal) subcu-
taneously into the right hind limb of Swiss albino mice
weighing 22–25 g. The animals were divided into four
groups, each consisting of six animals. The first group
was kept as untreated control, the second received 16
doses of GLE in 15 days (200 mg/kg body wt/dose), the
third was exposed to single dose of 4 Gy gamma radia-
tion and fourth group was administered with 200 mg/kg
body wt of GLE 1 h prior to and immediately after expo-
sure to 4 Gy gamma radiation and also daily for the next
14 days. The treatments were started on the 7th day after
transplanting tumour cells (when the tumour reached
a size of 1.0 cm3) and continued for 15 consecutive days.
The thickness of the hind leg was measured using a
vernier calliper once in three days from the 7th day of
tumour transplantation. The tumour volume was cal-
culated as follows: Tumour thickness = Thickness of
tumour-induced leg – Thickness of normal leg.
   Tumour volume = 4/3π r3, where r is the tumour
radius, the average of r1 and r2 which are the tumour
thicknesses.
   The effect of GLE on radiation-induced damage in cel-
lular DNA of cells of normal tissues and tumour in           Figure 2. Effect of Ganoderma lucidum extract (GLE) and 4 Gy
tumour-bearing mice exposed to whole-body gamma              gamma radiation on solid tumour in mice. Swiss albino mice bearing
radiation was studied as follows. Swiss albino mice were     solid tumour (Dalton’s Lymphoma Ascites) on hind limbs were orally
                                                             administered with GLE and exposed to 4 Gy gamma radiation. After
divided into four groups: Group I – 0.2 ml distilled water   radiation exposure, GLE administration was continued for 14 days and
(oral) + Sham irradiation; Group II – 0.2 ml distilled       the volume of tumour was monitored for 30 days. Photographs show an
water (oral) + 4 Gy 60Co-γ-rays; Group III – 200 mg/kg       animal and its hind limb bearing the tumour on the 1st, 7th and 30th
                                                             day following various treatments – day 1 at the start of the treatment,
GLE (oral) + Sham irradiation and Group IV – 200 mg/kg       1a and b – 0 Gy; 2a and b – 0 Gy + GLE; 3a and b – 4 Gy; 4a and b –
GLE (oral) + 4 Gy 60Co-γ-rays.                               4 Gy + GLE.

CURRENT SCIENCE, VOL. 99, NO. 8, 25 OCTOBER 2010                                                                               1085
RESEARCH COMMUNICATIONS




               Figure 3. Effect of GLE and gamma radiation on solid tumour in mice. Swiss albino mice bearing solid tumour
               (Dalton’s Lymphoma Ascites) in hind limb were administered with GLE and exposed to 4 Gy gamma radiation.
               After radiation exposure, GLE administration was continued for 14 days and the tumour volume monitored on
               alternate days, as described in the text.




       Figure 4. Photographs of silver-stained comets from cells of different tissues of tumour-bearing Swiss albino mice orally admin-
       istered with GLE and exposed to 4 Gy gamma radiation.

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Figure 5. Effect of oral administration of GLE on radiation-induced cellular DNA damage as assessed by comet assay in brain cells of mice-
bearing tumour on hind limbs, exposed to 4 Gy whole-body gamma radiation. Mean comet parameters like %DNA in tail, tail length, tail moment
and Olive tail moment of single cells subjected to alkaline single-cell gel electrophoresis are presented with ± SD. ns, Not significant; ***P < 0.001
when compared with respective control.



to the slides, the coverglasses were placed immediately                         The results of the study on the effect of gamma radia-
and the slides were kept at 4°C. After solidification, the                   tion (4 Gy) and GLE administration are presented in Fig-
coverglasses were removed and the slides were immersed                       ures 2 and 3. Figure 3 presents data on tumour volume,
in pre-chilled lysing solution containing 2.5 M NaCl;                        following the treatments and Figure 2 shows representa-
100 mM Na2EDTA, 10 mM Tris-HCl (pH-10), 1%                                   tive photographs of the animal and its tumour-bearing
DMSO and 1% TritonX, and kept for 1 h at 4°C. After                          limb. The growth of the tumour was found to be inhibited
lysis, the slides were drained properly and placed in a                      in animals exposed to 4 Gy gamma radiation compared to
horizontal electrophoretic apparatus filled with freshly                     untreated animals. The GLE itself reduced tumour growth
prepared electrophoresis buffer containing 300 mM                            to some extent. The tumour growth was found substan-
NaOH, 1 mM EDTA and 0.2% DMSO (pH ≥ 13). The                                 tially inhibited in the group of animals administered with
slides were equilibrated in the buffer for 20 min and elec-                  GLE and exposed to 4 Gy gamma radiation.
trophoresis was carried out for 30 min at 25 V and                              Alkaline comet assay was performed to analyse the
300 mA. After electrophoresis the slides were washed                         effect of administration of GLE on radiation-induced cel-
gently with 0.4 mM Tris-HCl buffer (pH 7.4) to remove                        lular DNA damage in normal and tumour tissues. Figure
the alkali. The slides were again washed with distilled                      4 shows representative photographs of the comets from
water and kept at 37°C for 2 h to dry the gel; then silver                   the cells of these tissues. It can be seen from Figures 5–8
staining was carried out11. The comets were visualized                       that the cellular DNA from various tissues such as blood,
using Olympus BX-41 microscope and the images were                           bone marrow, brain and tumour of the tumour-bearing
captured and analysed using the software ‘CASP’, which                       animals exposed to whole-body 4 Gy gamma radiation
directly gave the comet parameters such as %DNA in tail,                     shows increased comet parameters such as %DNA in tail,
tail length, tail moment (TM) and Olive tail moment                          tail length, TM and OTM. However, except in the tumour
(OTM)12. TM is the product of tail length and %DNA in                        tissues, these parameters were found to be lower in the
tail, and OTM is the product of the distance between the                     normal tissues of tumour-bearing animals administered
centre of gravity of the head and centre of gravity of the                   with GLE 1 h prior to radiation exposure (Figures 5–8).
tail and %DNA in the tail10–12. Data of the comet para-                      This would suggest that the administration of GLE
meters were presented as mean ± standard deviation.                          offered protection against radiation-induced damages to
CURRENT SCIENCE, VOL. 99, NO. 8, 25 OCTOBER 2010                                                                                                1087
RESEARCH COMMUNICATIONS




Figure 6. Effect of oral administration of GLE on radiation-induced cellular DNA damage as assessed by comet assay in bone marrow cells of
mice-bearing tumour on hind limbs, exposed to 4 Gy whole-body gamma radiation. Mean comet parameters like %DNA in tail, tail length, tail
moment and Olive tail moment of single cells subjected to alkaline single-cell gel electrophoresis are presented with ± SD. ns, Not significant;
***P < 0.001 when compared with respective control.




Figure 7. Effect of oral administration of GLE on radiation-induced cellular DNA damage as assessed by comet assay in blood leucocytes of
mice-bearing tumour on hind limbs, exposed to 4 Gy whole-body gamma radiation. Mean comet parameters like %DNA in tail, tail length, tail
moment and Olive tail moment of single cells subjected to alkaline single-cell gel electrophoresis are presented with ± SD. ns, Not significant;
***P < 0.001 when compared with respective control.

1088                                                                                CURRENT SCIENCE, VOL. 99, NO. 8, 25 OCTOBER 2010
                                                                                               RESEARCH COMMUNICATIONS




Figure 8. Effect of oral administration of GLE on radiation-induced cellular DNA damage as assessed by comet assay in tumour cells of mice-
bearing tumour on hind limbs, exposed to 4 Gy whole-body gamma radiation. Mean comet parameters like %DNA in tail, tail length, tail moment
and Olive tail moment of single cells subjected to alkaline single-cell gel electrophoresis are presented with ± SD. ns, Not significant; ***P < 0.001
when compared with respective control.

cellular DNA in normal tissues, and in the tumour tissues                    of GLE (200 mg/kg body wt) to the animals resulted in sig-
the extract offered no protection but helped enhance the                     nificant reduction in tumour volume, and this anti-tumour
radiation-induced cellular DNA damage, as can be evi-                        effect was more prominent in conjunction with gamma-
denced from the data in Figures 4–8.                                         radiation treatment (4 Gy). Studies on in vivo radiopro-
   The present study is focused on the radioprotective and                   tection efficiency by comet analysis demonstrated the
anti-cancer properties of G. lucidum under in vivo condi-                    efficiency of GLE to offer protection to normal tissues
tions using mouse as the model system. Radioprotective                       against gamma radiation-induced DNA damage, whereas
agents offer a possible solution to counteract the radiation                 sparing tumour tissues where the extract offered no pro-
damage to living systems1. The extracts of G. lucidum,                       tection against radiation-induced cellular DNA damage.
certain medicinal plants, vitamin derivatives and dietary                       The mushroom G. lucidum could be effectively used as
supplement formulations with good anti-oxidant activity                      a radioprotector for normal tissues in radiotherapy. It is to
can be considered as safe radioprotectors, whereas many                      be noted that there was an increased extent of cellular
of the synthetic drugs and chemicals prepared for radio-                     DNA damage in cells of the tumour tissues of animals
protection have limited application in the living systems                    administered with GLE prior to radiation exposure which
due to their toxicity and side effects13–17. Our previous                    could be due to induction of apoptosis in these cells. Fur-
studies have revealed that aqueous extract of G. lucidum                     ther studies are needed to support this possibility. The
possesses radioprotective activity9.                                         present results suggest the possibility of using this me-
   GLE has been reported to exhibit anti-tumour activity                     dicinal mushroom extract as an adjuvant in cancer radio-
and this has been attributed to immune-related mecha-                        therapy to protect normal tissues from radiation damage
nisms or cytotoxicity18–20. Some of the active ingredients                   and also to enhance the anti-tumour activity of gamma
in the extract have been identified as polysaccharides and                   radiation.
triterpenes21,22. The extract has also been shown to pre-
vent proliferation of cancer cells, mediated through inhi-                    1. Nair, C. K. K., Parida, D. K. and Nomura, T., Radiation protectors
bition of DNA synthesis23. Inhibition of DNA synthesis                           in radiotherapy. J. Radiat. Res., 2001, 42, 21–37.
following irradiation could bring about enhanced apop-                        2. Shueng, P. W., Hsu, W. L., Jen, Y. M., Wu, C. J. and Liu, H. S.,
tosis in the cells. This could be a possible mechanism by                        Neoadjuvant chemotherapy followed by radiotherapy should not
which GLE enhances the anti-tumour activity of gamma                             be a standard approach for locally advanced cervical cancer. Int. J.
                                                                                 Radiat. Oncol. Biol. Phys., 1998, 40, 889–896.
radiation.                                                                    3. Tim, L., Yihuai, G. and Shufeng, Z., Global marketing of medici-
   The tumour regression study carried out with solid                            nal Ling Zhi mushroom G. lucidum products and safety concerns.
tumour-bearing animals revealed that oral administration                         Int. J. Med. Mushroom, 2004, 6, 189–194.

CURRENT SCIENCE, VOL. 99, NO. 8, 25 OCTOBER 2010                                                                                                 1089
RESEARCH COMMUNICATIONS
 4. Maruyama, H. K. and Murofushi, Antitumour activity of Sarcodon        23. Gieni, R. S., Li, Y. and HayGlass, K. T., Comparison of [3H]
    aspratus. J. Pharmacobiodyn., 1989, 12(2), 118–123.                       thymidine incorporation with MTT- and MTS-based bioassays for
 5. Chang, R. T., Limitations and potential applications of Gano-             human and murine IL-2 and IL-4 analysis. Tetrazolium assays pro-
    derma and related fungal polyglycans in clinical oncology. In First       vide markedly enhanced sensitivity. J. Immunol. Methods, 1995,
    International Conference on Mushroom Biology and Mushroom                 187, 85–93.
    Products (eds Chang, R. T. and Nowel, T. P.), World Scientific,
    Washington, DC, 1993, pp. 96–98.                                      ACKNOWLEDGEMENT. We thank the DST, New Delhi for finan-
 6. Wang, D. H. and Weng, X. C., Antitumour activity of extracts of       cial support through research grant given to C.K.K.N.
    Ganoderma lucidum and their protective effects on damaged HL-
    7702 cells induced by radiotherapy and chemotherapy. Zhongguo         Received 17 August 2009; revised accepted 31 August 2010
    Zhong yao za zhi (China J. Chin. Mater. Med.), 2006, 31(19),
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 7. Jian, J., Slivova, V., Valachivocova, T., Harvey, K. and Sulva, D.,
    Ganoderma lucidum inhibits proliferation and induces apoptosis in
    human prostate cancer cells PC-3. Int. J. Oncol., 2004, 24, 1093–
    1099.                                                                 DNA microarray analysis targeting
 8. Stanley, G., Harvey, K., Slivova, V., Jiang, V. and Sulva, D.,        pmoA gene reveals diverse community
    Ganoderma lucidum suppresses angiogenesis through the inhibi-
    tion of secretion of VEGF and TGF-beta from prostate cancer           of methanotrophs in the rhizosphere of
    cells. Biochem. Biophys. Res. Commun., 2005, 330, 46–52.
 9. Pillai, T. G., Salvi, V. P., Maurya, D. K., Nair, C. K. K. and
                                                                          tropical rice soils
    Janardhanan, K. K., Prevention of radiation-induced damages by
    aqueous extract of Ganoderma lucidum occurring in southern parts      Pranjali Vishwakarma and Suresh K. Dubey*
    of India. Curr. Sci., 2006, 91(3), 341–344.
10. Singh, N. P., Microgels for estimation of DNA strand breaks,          Department of Botany, Banaras Hindu University,
    DNA – protein cross links and apoptosis. Mutat. Res., 2000, 455,      Varanasi 221 005, India
    111–127.
11. Chandrasekharan, D. K., Kagiya, V. T. and Nair, C. K. K., Radia-      The diversity of the methanotrophs community of two
    tion protection by 6-palmitoyl ascorbic acid-2-glucoside: studies     different rice fields of a typical tropical rice agroecosys-
    on DNA damage in vitro, ex vivo, in vivo and oxidative stress in      tem was assessed using microarray targeting pmoA
    vivo. J. Radiat. Res., 2009, 50, 203–212.                             gene-based approach. The presence of types I and II
12. Konca, K. et al., Across platform public domain PC image analy-
                                                                          methanotrophs was observed with the dominance of
    sis programme for the comet assay. Mutat. Res., 2003, 534, 15–20.
13. Weiss, J. F. and Landauer, M. R., Protection against ionizing
                                                                          Methylocystis in both the fields. The study revealed that
    radiation by antioxidant nutrients and phytochemicals. Toxicology,    the Barkachcha rice field harbours more diverse groups
    2003, 18, 1–20.                                                       of methanotrophs than the Ghazipur rice field. It was
14. Arora, R. et al., Radioprotection by plant products: present status   also observed that in some members of types I and II
    and future prospects. Phytother. Res., 2005, 19, 1–22.                methanotrophs, even the peat-associated group was
15. Maurya, D. K., Devasagayam, T. P. A. and Nair, C. K. K., Some         present in the enriched culture of the soils. The
    novel approaches for radioprotection and the beneficial effect of     Ghazipur soil and its enriched mixed methanotrophic
    natural products. Indian J. Exp. Biol., 2006, 44, 93–114.             culture showed higher methane oxidation potential
16. Nair, C. K. K., Devi, P. U., Shimanskaya, R., Kunugita, N.,           than the Barkachcha soil. These results suggest that the
    Murase, H., Gu, Y. H. and Kagiya, T. V., Water soluble vitamin E
                                                                          methanotrophs community and its potential for methane
    (TMG) as a radioprotector. Indian J. Exp. Biol., 2003, 41, 1365–
    1371.
                                                                          oxidation vary with change in soil type within the
17. Menon, A., Krishnan, C. V. and Nair, C. K. K., Protection from        same ecosystem.
    gamma-radiation insult to antioxidant defense and cellular DNA
    by POLY MVA, a dietary supplement containing palladium lipoic         Keywords. Methane oxidation, methanotrophs, micro-
    acid formulation. Int. J. Low Radiat, 2009, 6, 248–262.               array, pmoA gene, rice soil.
18. Lin, Z. B. and Zhang, N. H., Anti-tumour and immunoregulatory
    activities of Ganoderma lucidum and its possible mechanisms.
    Acta Pharmacol. Sin., 2004, 25, 1387–1395.
                                                                          DUE to their significant role in global CH4 cycling, meth-
19. Wang, Y. Y., Khoo, K. H., Chen, S. T., Lin, C. C., Wong, C. H.        ane-oxidizing bacteria (MOB; methanotrophs) have been
    and Lin, C. H., Studies on the immuno-modulating and antitumour       the focus of several scientific researchers. Methanotrophs,
    activities of Ganoderma lucidum (Reishi) polysaccharides: func-       abundantly found in the aerobic layer of the soil, the
    tional and proteomic analysis of a fucose-containing glycoprotein     rhizosphere1,2, oxidize significant amount of CH4 gener-
    fraction responsible for the activities. Bioorg. Med. Chem., 2002,
    10, 1057–1062.
                                                                          ated by methanogens. It is expected that methanotrophic
20. Lakshmi, B., Sheena, N. and Janardhanan, K. K., Prevention of         bacteria utilizing CH4 as substrate in the rhizosphere will
    mammary adenocarcinoma and skin tumour by Ganoderma luci-             vary in the population composition and density within the
    dum, a medicinal mushroom occurring in South India. Curr. Sci.,       rice rhizosphere3. The methanotrophic community is
    2009, 97, 1658–1664.                                                  complex and diverse, containing 10 genera which belong
21. Russel, M. and Paterson, R., Ganoderma – a therapeutic fungal
    biofactory. Phytochemistry, 2006, 67, 1985–2001.
                                                                          to type I (Gammaproteobacteria), and four genera to group
22. Boh, B., Berovic, M., Zhang, J. and Zhi-Bin, L., Ganoderma
    lucidum and its pharmacologically active compounds. Biotechnol.
    Annu. Rev., 2007, 13, 265–301.                                        *For correspondence. (e-mail: skdubey@bhu.ac.in)

1090                                                                                CURRENT SCIENCE, VOL. 99, NO. 8, 25 OCTOBER 2010

				
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