Antifungal activity of some Himalayan medicinal plants using direct ...

Introduction
Journal of Cell and Molecular Biology 5: 95-98, 2006.
Haliç University, Printed in Turkey.
Antifungal activity of some Himalayan medicinal plants using direct
bioautography
Sanjay Guleria* and Ashok Kumar
CSK HP Agricultural University, Shivalik Agricultural Research and Extension Centre,
Kangra-176 001 (H.P.) India (*author for correspondence)
Received 15 May 2006; Accepted 12 July 2006
Abstract
In the search for bioactive compounds, direct bioautography of plant lipophilic leaf extracts was used in antifungal
screening. Thus, lipophilic (dichloromethane) leaf extract of medicinal plants used by Himalayan people were
investigated. Alternaria alternata and Curvularia lunata were used as test organism in bioautography. The results,
evaluated by the diameter of the inhibition zone of fungal growth, indicate that five plant species, among the 12
investigated, showed antifungal activity. CHCl 3 – CH 3OH (1:9, v/v) was used as a solvent to develop silica gel TLC
plates. Clear inhibition zones were observed for lipophilic extracts of Vitex negundo (R F value 0.85), Z a n t o x y l u m
a l a t u m (R F value 0.86), Ipomea carnea (R F value 0.86), Thuja orientalis (R F value 0.80) and Cinnamomum camphora
(R F value 0.89). The best antifungal activity was shown by lipophilic leaf extract of T. orientalis.
Key Words: Alternaria alternata, Curvularia lunata , Bioautography
Baz› Himalaya t›bbi bitkilerinin antifungal aktivitelerinin do¤rudan biyootografi
kullan›larak gösterilmesi
Özet
Biyoaktif bilefliklerin araflt›r›lmas›nda lipofilik yaprak ekstrelerinin do¤rudan biyootografisi antifungal tarama için
kullan›ld›. Bu nedenle Himalaya halk› taraf›ndan kullan›lan tibbi bitkilerin lipofilik (diklorometan) yaprak ekstreleri
araflt›r›ld›. Biyootografide araflt›rma organizmas› olarak Alternaria alternata ve Curvularia lunata kullan›ld›.
Sonuçlarda mantar büyümesinde inhibisyon bölgesinin çap› de¤erlendirildi, araflt›r›lan 12 bitki türünden beflinde
antifungal aktivite belirlendi. Silika gel G levhalar›n yürütülmesinde CHCl 3-CH 3OH (1:9, v/v) solvent olarak
kullan›ld›. Vitex negundo (Rf de¤eri 0.85), Zantoxylum alatum (Rf de¤eri 0.86), Ipomea carnea (Rf de¤eri 0.86),
Thuja orientalis (Rf de¤eri 0.80) ve Cinnamomum camphora (Rf de¤eri 0.89) lipofilik ekstreleri için belirgin
inhibisyon bölgeleri saptand›. En iyi antifungal aktivite lipofilik T. orientalis yaprak ekstrelerinde gösterildi.
Anahtar Sözcükler: Alternaria alternata, Curvularia lunata , biyootografi
Diseases of cultivated crops remain the principal
limitation to increased agricultural production.
Therefore, protection of plants from pathogens
remains a primary concern of agricultural scientists.
Despite serious environmental implications associated
with the excessive use, chemical fungicides remain the
first line of defense against fungal pathogens.
95

96 Sanjay Guleria and Ashok Kumar
Table 1. Plant species used in the study.
Scientific name Family Weight of lipophilic extracts (g)/100 g dry leaf
Murraya koenigii Rutaceae 1.10
Vitex negundo Verbanaceae 1.89
Adhatoda vasica Acanthaceae 1.18
Zantoxylum alatum Rutaceae 2.35
Agave americana Amaryllidaceae 0.52
Azadirachta indica Meliaceae 1.05
Eucalyptus globuluse Myrtaceae 3.05
Datura innoxia Solanaceae 1.17
Ipomea carnea Convolvulaceae 0.59
Thuja orientalis Cupressaceae 1.45
Cinnamomum camphora Lauraceae 1.40
Solanum xanthocarpum Solanaceae 1.73
Table 2. Antifungal activity of dichloromethane leaf extracts of the plants under study by direct bioautography.
Scientific name Diameter of inhibition zone (mm) R F Value
A. alternata C. lunata
V. negundo 28 14 0.85
Z. alatum 18 15 0.86
I. carnea 10 7 0.86
T. orientalis 30 22 0.80
C. camphora 9 12 0.79
Plants have supplied over 25% of prescription
drugs used in human medicine and such
pharmacologically active plants have also provided
leads to natural pesticides (Sener et al., 1998).
Himalayas has an extraordinarily rich flora and wide
knowledge of indigenous medicinal plants is well
documented. Accordingly, we are investigating the
potential of Himalayan medicinal plants as a resource
for new biofungicides. To investigate the biological
activity of Himalayan medicinal plants we have used
direct bioautography procedure (Lago et al., 2004) and
Alternaria alternata and Curvularia lunata as target
organisms.
Materials and methods
Extraction of plant material
Fresh leaves of test plants (Table 1) were air dried, and
the ground powder (100 g) was soaked in 500 ml of
dichloromethane for forty-eight hours. The solvent
was then removed under reduced pressure in a rotary
evaporator. Dark green mass obtained was dissolved
in dichloromethane to have 50 mg crude mass/ml.
P reparation of pathogen inoculum
Alternaria alternata was isolated from single spot
from infected leaves of sesame on potato dextrose
agar (potato 200 g, dextrose 20 g, agar 20 g and water
to make total volume of 1 L) and pure culture was
maintained on PDA at 26±2 o C. Similarly,
Curvularia lunata was isolated from the seed
mycoflora of mustard. Conidia were isolated from the
10 days old culture of the pathogens by flooding
culture plates with 5 mL of sterile distilled water and
conidia were dislodged by using a L-shaped glass rod.
Conidial suspension was filtered through sterile
double layered muslin cloth to remove bits of
mycelia. Spore suspension was then prepared in liquid
potato dextrose (potato 200 g, dextrose 20 g and water
to make total volume of 1 L) to obtain a
concentration of 3 x 10 5 conidia/mL.
Bioautography
20 µL of solutions corresponding to 1000 µg of crude

extracts were applied to precoated Si gel TLC plates,
developed with CHCl 3 - CH 3OH (1:9, v/v), and dried
for complete removal of solvents. Aliquots of 25-50
m L of inoculum spray solution (ca. 3 x 10 5
conidia/ml) were prepared for A. a l t e r n a t a or C. lunata
Antifungal activity of medicinal plants 97
with liquid potato dextrose (potato 200 g, dextrose 20
g and water to make total volume of 1 L). Using a
100 mLchromatographic sprayer, plates were sprayed
lightly (to a damp appearance) three times with the
spore suspension of A. alternata or C. lunata and
Figure 1. “Zones” of fungal growth inhibition, produced by distinct antifungal metabolites found in dichloromethane leaf extract
of Himalayan medicinal plants. Lanes A-L corresponds to the TLC “fingerprint” of different medicinal plants (A= M. koenigii,
B= V. negundo, C= A. vasica, D= Z. alatum, E= A. amiracana, F= A. indica, G= E. globuluse, H= D. innoxia, I= I. carnea , J= T.
o r i e n t a l i s, K= C. camphora and L= S. xanthocarpum) prepared from their dichloromethane leaf extract using CHCI 3 -CH 3 OH
(1:9, v/v) as developing solvent. Direct bioautography was performed by inoculating the plate (a) and (b) with spore suspension
of Alternaria alternata and C u r v u l a r i a l u n a t a respectively in potato dextrose broth.

98 Sanjay Guleria and Ashok Kumar
incubated for four days in darkness in a moist chamber
at 25 o C. Fungal growth inhibition appeared as clear
zones against a dark background. The experiment was
repeated twice and similar results were obtained.
Results and discussion
Out of the twelve plants tested, lipophilic extracts of
Vitex negundo, Zanthoxylum alatum, Ipomea carnea,
Thuja orientalis and Cinnamomum camphora
exhibited antifungal activity against C. lunata or A .
a l t e r n a t a by direct bioautography (Fig. 1). The zones
of inhibition (R F value 0.85) of V. negundo were 14
mm in diameter (Table 2) in case of C. lunata and 28
mm against A. alternata. Lipophilic leaf extract of Z .
a l a t u m against C. lunata produced inhibition zone (R F
value 0.86) of 15 mm in diameter. Against A. alternata
the zone of inhibition (R F value 0.86) was 18 mm in
diameter. In case of I. carnea zones of inhibition (R F
value 0.86) of 7 mm and 10 mm were detected against
C. lunata and A. alternata respectively. T. orientalis
showed zone of inhibition (R F value 0.80) of 22 mm
in diameter against C. lunata and 30 mm in diameter
against A. alternata. Similarly, C. camphora produced
inhibition zone (R F value 0.79) of 12 mm in diameter
against C. lunata and 9 mm in diameter against A .
a l t e r n a t a . Lipophilic leaf extract of M u r r a y a
k o e n i g i i, Adhatoda vasica, Agave americana,
Azadirachta indica, Eucalyptus globuluse, Datura
i n n o x i a and Solanum xanthocarpum did not show
inhibition on TLC plate against C. lunata or A .
a l t e r n a t a by direct bioautography.
Geraniol extracted from the essential oil of fruits of
Z. alatum was shown to have strong antifungal activity
against Colletotrichum falcatum and C e r a t o c y s t i s
p a r a d o x a fungal pathogens of sugar cane and was
more potent than commercial synthetic fungicides
(Rao and Singh, 1994). Essential oils from C .
c a m p h o r a have been reported to have antifungal
activity against Fusarium graminearum (Liu et al.,
2001). Mishra et al., (1992) reported antifungal
activity of aqueous leaf extract of T. orientalis against
C. lunata. Aqueous and ethanolic extracts of V.
n e g u n d o leaves has been shown to be inhibitory
against Pyricularia oryzae (Rajeswari and Mariappan,
1992). Similarly, chloroform leaf extract of I. carnea
has been reported to have strong antifungal activity
against Rhizoctonia solani (Kagale et al., 2004)
The best antifungal activity against C. lunata or A .
a l t e r n a t a was shown by lipophilic leaf extract of T.
o r i e n t a l i s. Further work is required to isolate the
bioactive constituents and test the antifungal
properties of these compounds, this may help to find
the compound(s) responsible for antifungal activity.
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