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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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