Preliminary Screening of Endophytic Fungi from Medicinal Plants in India for Antimicrobial and Antitumor Activity

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Preliminary Screening of Endophytic Fungi from Medicinal Plants in India for Antimicrobial and Antitumor Activity Powered By Docstoc
					                                                 International Journal of Pharmaceutical Sciences and Nanotechnology
566     International Journal of Pharmaceutical Sciences and Nanotechnology Volume 2 • Issue 2 • July - September 2009
                                                                                 Volume 2 • Issue 2 • July – September 2009

Research Paper
Preliminary Screening of Endophytic Fungi from Medicinal Plants in
India for Antimicrobial and Antitumor Activity

Gani Sharmila Banu1, Ganesan Kumar2,*
 Centre for Biotechnology, Muthayammal College of Arts and Sciences, Kakkaveri, Rasipuram, Namakkal,
Tamilnadu, India-637408.
    Department of Biochemistry, Selvamm Arts and Science College, Namakkal (DT)-637 003. Tamilnadu, India.

ABSTRACT: The screening of antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria, yeast and
fungi was carried out on isopropanol extracts prepared from 121 isolates of endophytic fungi isolated from medicinal plants
in India. Sensitivity was found to vary among the microorganisms. Bacillus subtilis, Saccharomyces cerevisiae and
Alternaria sp. were susceptible to extracts from three, two and two isolates of endophytic fungi, respectively. None was
found effective against Salmonella typhimurium. Sixteen endophytic fungal isolates tested were also found to exhibit
antitumor activity in the yeast cell-based assay.

KEYWORDS: Medicinal plants; Endophytic fungi; Antimicrobial; Antitumor

Introduction                                                      Materials and Methods
The nature and biological role of endophytic fungi in their
plant host is variable. Endophytic fungi are known to have        Isolation of endophytic fungi
mutualistic relations to their isolation of endophytic fungi      A random sample from each plant consisting of
hosts, often protecting plants against herbivore, insect          asymptomatic leaves and branches was taken. The leaves
attack or tissue invading pathogens (Siegel et al., 1985;         and branches were thoroughly washed in running tap
Clay, 1986; Yang et al., 1994) and in some instances the          water, after which they were surface sterilized by
endophyte may survive as a latent pathogen, causing or            submerging them in 75% ethanol for 2 min. The branch
                                                                  portions were further sterilized sequentially in 5.3%
quiescent infections for a long period and symptoms
                                                                  sodium hypochlorite solution for 5 min, and 75% ethanol
appear only when physiological or ecological conditions           for 0.5 min. After drying, each leaf was divided into three
favors virulence (Carroll, 1986; Bettucci, and Saravay,           segments and placed on potato dextrose agar (PDA)
1993). In India, extract from many types of local plants are      supplemented with 50-mg/l chloramphenicol to suppress
used in traditional manner for treatments of various              bacterial growth. Branch portions were cut to expose their
ailments (Beulah Jerlin et al., 2004; Rajasekara Pandian et       inner tissue and placed on the same medium. All the plates
al., 2006). The question is whether they are produced by          were incubated at 270C for up to 3 weeks. Emerging fungi
the plant itself or as a consequence of a mutual relationship     were transferred to fresh PDA plates, incubated for 1 week
with beneficial organisms in their tissue. Many reports           and periodically checked for purity.
showed that in a microbe-plant relationship, endophytes
                                                                  Tests for Antimicrobial and antitumor activities
contribute substances that possess various types of
bioactivity, such as antibacterial and antifungal. Thus in        The endophytic fungi were grown at 270C with shaking in
this study, we focus on the isolation of endophytic fungi         5 ml F-4 medium (Glycerol 40 g/l, Soya bean meal 25 g/l,
                                                                  Yeast extract 5 g/l, Corn steep liquor 1 g/l, NaCl 0.5 g/l)
and screening them for bioactivity.
                                                                  and PD-Y medium (Potato dextrose broth 24 g/l, Yeast
                                                                  extract 2 g/l) for 5 days. For extraction, an equal volume of
* For correspondence: Ganesan Kumar,
                                                                  isopropanol was added to the culture broth and vortexed
                                                                  vigorously for 1 min followed by a centrifugation at 3000
                                                                  rpm for 10 min.

               Gani Sharmila Banu et al. : Preliminary Screening of Endophytic Fungi from Medicinal Plant in India for…     567

    About 80 ml of supernatant was applied per sterile            synthetic chemical also have long been used as active
paper disc (5 mm diameter). After drying, the extract             agents in reducing the incidence of plant, animal and
impregnated discs were used in a disc diffusion assays            human diseases, they are costly, have potentially harmful
using Alternaria sp. in potato dextrose agar (PDA),               effect on the environment and may induce pathogen
Bacillus substilis, Salmonella typhimurium in PMg agar,           resistance. Thus, biological controls or the use of
and Saccharomyces cerevisiae in YPG agar as test                  microorganisms or their secretions to prevent diseases
microorganisms for antimicrobial activity. Five millilitre of     offer an attractive alternative or supplement to disease
spore suspension of Alternaria sp. grown in vegetable juice       management without the negative impact of chemical
(tomato juice, 200 ml CaCO3, 4.5 g agar, 3.0 g in a total         control.
vol. of 300 ml) were used. S. cerevisiae was grown in YPG             In natural product discovery programs, typical
broth (yeast extract, 20 g/l; peptone 20 g/l; glucose, 20 g/l),   procedures included isolating microorganisms from
and B. subtilis and S. typhimurium were grown in PMg              samples, growing them at various temperatures in a variety
broth (peptone 10 g/l; MgSO4.7H2O, 2 g/l).
                                                                  of selective or nonselective media and testing the extracts
Chloramphenicol (50-mg/ ml) and nystatin (100 mg/ml)              in a spectrum of targeted screens for activity for potential
were used as positive controls.                                   industrial or pharmaceutical applications. For a successful
    To prepare assay plate in a 21.5 cm X 21.5 cm square          fungal screening, a varied and novel repertoire of either
plate, Yeast Nitrogen Base (YNB) broth (1.4 g) and Bacto          well-known or unexplored fungi is desirable. The most
agar (2.4 g) were dissolved in 150 ml of sterile distilled        promising trend in isolating new fungi is the move towards
water. The pH was adjusted to 6.5 prior to autoclaving at         investigating novel endophytes, with the idea that unusual
1210C for 15 min. When the agar is about 420C, the                endophytes may produce untapped natural products.
following components were added: 20 ml of 50%                         A total of 121 endophyte isolates were obtained from
galactose, 2 ml of 20% sucrose, 10 ml of 20X concentrated         62 of 72 (86.1%) different types of medicinal plants used
adenine (0.5 mg/ml), 2 ml of 100X concentrated histidine          by the local population in India (Table 1). The results of
(2 mg/ml), 2 ml of 100X concentrated tryptophan (2
                                                                  this study showed that endophyte fungi were more
mg/ml), 2 ml of 100X concentrated uracil (2 mg/ml) or 2           prevalent in the leaves (110/121 or 90.9%) than in the
ml of 100X concentrated leucine (2 mg/ml) when using the          branches. Further, more intensive samplings are necessary
test strain UCK or UCS, respectively; 10x concentrated 4
                                                                  to clarify the fungal assemblages of the leaves and
dropout amino acid (containing each at 10x concentrated           branches, as in traditional practice; the local population
of arginine at 240 mg, methionine at 240 mg, tyrosine at          used mostly the extract from the leaves of the plants (Ong
360 mg, isoleucine at 360 mg, lysine at 360 mg,
                                                                  and Nordiana, 1998; 1999).
phenylalanine at 600 mg, aspartic acid at 1000 mg, valine
at 1500 mg and thymine at 2000 mg per 1000 ml sterile                 Though there are still a lot of subjects to be explained
distilled water) and yeast glycerol (30-40%) stock of the         in the mutualistic association of endophytic fungi and their
yeast test strain W303-1AY18 containing either the                plant host, more reports indicated the occurrence of
plasmids pMR438-CyclinA D 24-62 (UCK) or Yep51-                   endophytes in plants especially in relation to the possible
SRX5 src (UCS) (Sikder et al., 1997), and poured into the         origin of the plant metabolites detected (Strobel et al.,
square plate on a horizontal place. The 5 mm paper discs          1996; Cacabuono and Pomilio, 1997; Rizzo et al., 1997).
impregnated with the supernatant described above were             The culture residue of the isopropanol extract of the
placed on the agar plate and incubated at 300C for 3-4            endophytes cultures 12L and 22L in F-4 medium and 1B,
days. The growth circle around the disk that indicates            19L, 21L2 and 27L1 in PD-Y medium yielded impressive
positive results for anti-tumor activity was measured.            anti-fungal, anti-bacterial or anti-yeast activities. However,
                                                                  extracts from endophytic fungal cultures of 5L, 24L2, 37L,
    Glucose (50%) was used as a positive control. All the         41L1, 50ML, b34L1, b53L, b20L, b69L2, b30L, b9l,
screening procedures were performed twice in duplicates.          b70L2, b14L, b49L and b29L in PD-Y broth showed
Inhibition zone (for antimicrobial test) or growth zone (for      positive activity for anti-tumor in the UCK/UCS yeast cell-
anti-tumor test) around the disk of 6 mm or more were             based assay (Table 2). The basis of the anti-tumor
defined as positive for biological activity.                      screening using a yeast cell-based assay was that the hyper
                                                                  activation of cylcin-dependent-kinase (CDK) resulted in
Results and Discussion                                            growth arrest of the yeast harboring the genetically
Plants have long provided mankind with a source of                engineered recombinant plasmids, and a compound from
medicinal agents, with natural products once serving as           the extract that can rescue the cyclinA1-induced growth
source of all drugs (Balandrin et al., 1993). Though              arrest is viewed as a potential anti-tumor candidate.
568    International Journal of Pharmaceutical Sciences and Nanotechnology     Volume 2 • Issue 2 • July - September 2009

                               Table 1 Endophytic fungi isolated from medicinal plants.

      No         Local name                Scientific name                   First isolation          Second isolation
       1.    Kuppai meni            Acalypha indica                                 -                         b1L
       2.    Vasambu                Acorus calamus                              2L, 2ML                       b2L
       3.    Nayuruvi               Acyranthes aspera                               -                         b3L
       4.    sirupulai              Aerva lanata                                   4L                         b4L
       5.    Vellai poondu          Alium sativum                                   -                          -
       6.    Kumari                 Aloe vera                                       -                          -
       7.    Malai vembu            Andrographis paniculata                        7L                          -
       8.    Karuvel                Areca catechu                                   -                          -
       9.    Kaduku                 Brassica juncea                              9L, 9B                       b9L
      10.    Usihthakarai           Cassia tora                                10L, 10ML                     b10L
      11.    Nithya kalyani         Catharantus roseus                              -                        b11L
      12.    Valuluvai              Celastrus paniculatus                         12L                   b12L1, b12L2
      13.    Ilavangam              Cinnamomum zeylanicum                         13L                        b13L
      14.    Athondai               Capparis zeylanica                              -                   b14L1, b14L2
      15.    Nallavelai             Cleome gynandra                                 -                   b15L1, b15L2
      16.    Kattukodi              Cocculus hirsutus                               -                        b16L
      17.    Omavalli               Coleus aromaticus                             17L                        b17L
      18.    Kanavalai              Commelina bengalensis                        18L/B                         -
      19.    Agasa karudan          Corallocarpus epigaeus                        19L                          -
      20.    Naruvili               Cordia oblique                                  -                          -
      21.    Kothamalli             Coriandrum sativum                              -                     b21L, b21B
      22.    Kottam                 Costus speciosus                                -                        b22L
      23.    Nilappanaik-kilangu    Curculigo orchioides                          23L                   b23L1, b23L2
      24.    Manjai inji            Curcuma amada                                   -                        b24L
      25.    Kua                    Curcuma angustifolia                            -                        b25L
      26.    Kasthuri manjal        Curcuma aromatica                          26L, 26B                 b26L1, b26L2
      27.    Karuumathai            Datura metal                                  27L                          -
      28.    Karisilankanni         Eclipta prostrata                          28L, 28ML                       -
      29.    Anashovadi             Elephantopus scaber                             -                        b29L
      30.    Kirambu                Eugenia caryophyllata                           -                   b30L1, b30L2
      31.    Palperukki             Euphorbia heterophylla              31L1, 31L2, 31ML                b31L1, b31L2
      32.    Amman pacharisi        Euphorbia hirta                                 -                          -
      33.    Athi                   Ficus racemosa                                  -                        b33L
      34.    Karunochi              Gendarussa vulgaris              34L1. 34L2, 34B1, 34B2                  b34L
      35.    athumathuram           Glycyrrhiza glabra                              -                   b35L1, b35L2
      36.    Nannari                Hemidesmus indicus                            36L                        b36L
      37.    Kasarai                Hibiscus cannabinus                             -                        b37L

                                                                                                                   Table 1 Contd…
            Gani Sharmila Banu et al. : Preliminary Screening of Endophytic Fungi from Medicinal Plant in India for…   569

 38.      Narunthali             Ipomoea obscura                              -                         b38L
 39.      Mallai                 Jasminum sambac                          39L, 39B
 40.      Adalai                 Jatropha glandulifera                       40L                        b40L
 41.      Kattamanakku           Jatropha gossypifolia                       41L                        b41L
 42.      Kacholum               Kaempferia angustifolia                      -                           -
 43.      Tindiyam               Lagerstroemia indica                    43L1, 43L2                 b43L1, b43L2
 44.      Eluthani poondu        Launaea sarmentosa                   44L1, 44L2, 44B                b44L, b44b
 45.      Maruthontri            Lawsonia inermis                             -                         b45B
 46.      Arai                   Marsilea quadrifolia                         -                         b46L
 47.      Elikathilai            Merremia emarginata                     47L1, 47L2                 b47L1, b47L2
 48.      Sathikkay              Myristica fragrans                           -                         b48L
 49.      Karuveppilai           Murraya koenigii                             -                      b49L, b49B
 50.      Sadamanjil             Nardostachys jatamansi                       -                         b50L
 51.      Thulasi                Ocimum basilicum                           51B                           -
 52.      Inbura                 Oldenlania umbellata                         -                         b52L
 53.      Anai nerunchil         Padalium murex                               -                         b53L
 54.      Uppilankodi            Pentatropis capensis                         -                      b54L, b54B
 55.      Sathakuppai            Peucedanum grande                                                      b55L
 56.      Thippili               Piper longum                         56L, 56B1, 56B2                   b56L
 57.      Milagu                 Piper nigrum                                 -                           -
 58.      Ilanthalari            Plumeria rubra                          58L1, 58L2                     b58L
 59.      Kodi veli              Plumbago zeylanica                      59L1, 59L2                 b59L1, b59L2
 60.      Narumunnai             Premna corymbosa                    60L1, 60L2, 60ML               b60L1, b60L2
 61.      Nagamalli              Rhinacanthus nasutus                         -                     b61L1, b61L2
 62.      Alingi                 Rhododendron arboreum                   62L1, 62L2                 b62L1, b62L2
 63.      Amanakku               Ricinus communis                           63ML                          -
 64.      Rosa                   Rosa centifolia                              -                     b64L1, b64L2
 65.      Asogam                 Saraca indica                               65L                        b65L
 66.      Nathai suri            Spermacoce hispida                          66L                    b66L1, b66L2
 67.      Puli                   Tamarindus indica                          67B                         b67L
 68.      Kadukkay               Termanalia chebula                           -                         b68L
 69.      Manakkarai             Vangueria spinosa                                                      b69L
 70.      Amukkiran              Withania somnifera                           -                         b70L
 71.      Iratchai               Zanthoxylum budrunga                    71L, 72ML                        -
 72.      Inji                   Zingziber officinale                         -                         b72L
 73.      Ilanthai               Zizyphus jujuba                              -                         b73L
 74.      Korgodi                Zizyphus nummularia                         74L                        b74L
 75.      Suraimullu             Zizyphusoenoplia                        75L, 75ML                      b75L
- No endophytic fungi isolated
570   International Journal of Pharmaceutical Sciences and Nanotechnology         Volume 2 • Issue 2 • July - September 2009

              Table 2 Endophytic fungal isolates showing biological-activity against test organisms.

                                 Antimicrobial activity (mm) a                            Antitumor activity (mm) b
            Endophytes             Bs            St             Sc               Al          UCS            UCK
                  12L              15             -              -               -             -              -
                  19L             19.5            -              -              20             -              -
                  22L             19.2            -              -               -             -              -
                  21L2              -             -             13.2             -             -              -
                  27L1              -             -             13.5             -             -              -
                  1B                -             -              -              21             -              -
                  5L               -              -              -               -            8              8.5
                  24L2             -              -              -               -            8               8
                  37L               -             -              -               -            9.2            9.1
                  41L1             -              -              -               -           8.3              8
                  50ML              -             -              -               -            10            10.3
                  b34L              -             -              -               -            7.2            7.1
                  b53L             -              -              -               -            8              8.1
                  b20L              -             -              -               -            7.5            7.5
                  b69L2             -             -              -               -           8.1              8
                  b9L               -             -              -               -            10            10.3
                  b30L             -              -              --              -            9               9
                  b7L               -             -              -               -            9.3            9.2
                  b70L2             -             -              -               -           8.6             8.3
                  b14L              -             -              -               -            8.2            8.2
                  b49L              -             -              -               -            8.7            8.5
                  b29L              -             -              -               -            8.3            8.1
                  Test microorganisms: Bs- Bacillus subtilis; St- Salmonella typhimurium;
                  Sc- Saccharomyces cerevisiae; and Al- Alternaria sp.
                  Yeast test strain W303-1AY18 containing: plasmid pMR438-CyclinA D 24-62 (UCK) or
                  Yep51-SRX5 src (UCS).
              (-) None detected.
    In this study, we demonstrated that crude extracts from               Also extracts which showed no anti-microbial or anti-
the culture broth of endophytic fungi grown, aerobically in           tumor activity in the disc-diffusion bioassay might be
PD-Y or F4 medium displayed anti-bacterial, anti-fungal,              active against other microbes, which were not tested.
anti-yeast or anti-tumor activity. These results suggest the          Looking at the differing activity of test results obtained,
presence of either good antimicrobial potency of the                  additional modes of action should be explored for those
extract or a high concentration of an active principle in the         isolates that do not have antimicrobial activity, as it is
extracts of strains showing positive biological activities.           possible that some of these endophytes may produce
Other endophytic fungal extracts, which showed low anti-              substances that may ward off microbial infections by
microbial or anti-tumor activity in the bioassay, may have            stimulating the host immune system rather than by
active compounds but probably in smaller amounts and/or               antimicrobial activity.
the screened crude extracts could yield more potent
                                                                         In addition, there is also the possibility that substances
compounds once they had undergone some purification
                                                                      present in the extract can stimulate the growth of the
(Fabry et al., 1998).
                                                                      microorganisms, as was evident by several isolates
               Gani Sharmila Banu et al. : Preliminary Screening of Endophytic Fungi from Medicinal Plant in India for…         571

showing good bacterial growth forming wide zone of                Carroll GC. The biology of endophytism in plants with particular
inhibition around the disk, thus counteracting the effect of         reference to woody perennials. In: Fokkema, N. and van den
inhibitory substances. The observation that antibacterial            Heuval, J., eds. Microbiology of the Phyllosphere. Cambridge
and antifungal activity, although in crude extract, were             University Press, Cambridge. pp 392 (1986).
detectable in several isolates, may indicate, but not prove,
                                                                  Clay K. Grass endophytes. In: Fokkema, N. and van den Heuval,
that these isolates produce bioactive substances. In
                                                                     J., eds. Microbiology of the Phyllosphere. Cambridge
traditional natural product screening programs extracts that
                                                                     University Press, Cambridge. Pp1395 (1986).
are ‘hits’ in a screen of interest require follow-up analysis,
typically involving analytical chemists.                          Fabry W, Okemo PO, and Ansorg R. Antibacterial activity of
                                                                     East African medicinal plants. J.Ethnopharmacol. 60: 79-84
    This aspect will be further investigated as in any
natural product screening, the “referm” problem (rare
cultures that produce an activity of interest the first time      Ong HC and Noralina J. Malay herbal medicine in Gemencheh,
they are grown often are unable to produce that activity             Negeri Sembilan, Malaysia. Fitoterapia 70: 10-14 (1998).
again when they are refermented) needs to be addressed to         Ong HC and Nordiana M. Malay ethno-medico botany in
enhance production of the secondary metabolites of                   Machang, Kelantan, Malaysia. Fitoterapia 70: 502-513
interest. Therefore, any information and/or research on              (1999).
endophyte-plant symbiosis, such as in this study is of            Rajasekara Pandian M, Sharmila Banu G, Kumar G, and Smila
value, especially taking into account the positive biological        KH. Medicinal plants of ethnobotanical importance curing
activity as anti-microbial and anti-tumor agents. Effective          diabetes from Namakkal district (Tamilnadu), India. Indian J.
extracts could provide potential leads towards the                   Environ. Eco-plan. 12: 201-205 (2006).
development of novel and environmental friendly
                                                                  Rizzo I, Varsavky E, Haidukososki M, and Frade H. Macrocyclic
biologically active agents.                                          trichothecence in Baccharis coridifolia plants and endophytes
                                                                     and Baccharis artemisioides plants. Toxicon 35: 753-757
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